Industrial Biofouling

INDUSTRIAL BIOFOULING

T. REG. BOTT

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO

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Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-444-53224-4 For information on all Elsevier publications visit our web site at elsevierdirect.com Printed and bound in Great Britain 11 12 13 14

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PREFACE Astronomers when they are searching for life on other planets, look for the presence of water, which is the basis of all life as we know it. All living things on earthdplants, animals, fish, insects and microorganisms can live longer without food than without water. From earliest times, humans have gathered together in communities near sources of water, such as rivers or lakes. Not only has this water been necessary for their survival, it has been used for transport and the raising of food crops and in modern times for industrial purposes. There is an analogy here with microorganisms, which also exist in communities where there can be a wide variety of species attached to suitable submerged solid surfaces. A further analogy with humanity is that microorganisms tend to use flowing water to transport them to other locations for the formation of new communities. The flowing water also delivers nutrients and takes away waste products. Like humans too, different species may be supportive or aggressive towards other member species in their community. It is not surprising therefore, that solid surfaces in contact with water open to the atmosphere are likely to encourage the formation of communities of microorganisms (biofilms). In industrial operations such biofouling is generally unwanted since it is likely to introduce costly operational inefficiencies, which can also have implications in respect of global warming. It is for these reasons that steps must be taken to control the incidence of biofilm formation and the method adopted must, of course, be environmentally friendly. A common technique is the application of chemical biocides or dispersants. On the other hand, communities of microorganisms can be made to work for human communities in the treatment of waste water, sewage and for environmentally acceptable production of gaseous fuels. Despite widespread knowledge concerning the establishment and growth of microbial biofilms, there is still considerable opportunity for research and development into the control of unwanted microbial colonies and paradoxically, their utilisation for the benefit of humanity.

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ACKNOWLEDGEMENT The author would like to record his thanks to all the postgraduate students, Research Fellows, colleagues at the University of Birmingham and in industry, for their assistance in keeping him on the steep and winding road of biofouling research. Some of their names are referred to in the text.

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NOMENCLATURE A AH cA D DAB ED FW F g k l NA Q R r T v VD VT Vw x

Area Hamaker constant Concentration of A Diameter Diffusivity of A through B Eddy diffusion van der Waals force Friction factor Acceleration due to gravity Thermal conductivity Length Rate of diffusion of A Heat transferred Shear stress Radius Temperature Velocity Energy arising from double-layer interactions Total potential energy due to interactions Energy of van der Waals interactions Distance

Greek

F r m DH

Friction factor Density Viscosity Fluid head

Dimensionless number Re Reynolds number

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CHAPTER

1

Industrial Biofouling Contents 1. Introduction References

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1. INTRODUCTION In the natural world there are advantages for microorganisms; bacteria, fungi and algae if they are attached to a solid surface in contact with water, particularly if the water is moving across the surface. Under these conditions nutrients are brought to the microorganisms and waste products are carried away. These accumulations of living matter are generally referred to as ‘biofilms’ or ‘slimes’. When biofilms occur in industrial operations, they usually represent an impediment to efficient operation, particularly in respect of energy usage. As a result, there is a direct link to the problem of global warming. Effective control of ‘biofouling’ is of prime importance since it could make a substantial contribution to combating climate change in addition to reducing operating costs. In contrast, some biofilms are useful in industrial operations. Macroorganisms, as their collective name suggests, are much larger than microorganisms, for example, mussels, barnacles and hydroids. They too may be attached to submerged solid surfaces but they are far less likely to be impediments to industrial processes; because of their larger size they are more easily controlled. Costerton et al. (1994), in a review of biofilm activity entitled their paper ‘Biofilms Customized Microniche’. The title is an apt description of this natural phenomenon, particularly as applied to microorganisms, including bacteria, fungi and algae. In the natural environment, a surface exposed to water is likely to have several thousand bacteria attached to one square centimetre of surface, whereas the adjacent water will contain far fewer cells in a cubic centimetre. Clearly, attachment to a surface must be beneficial. It is not surprising therefore, to find microorganisms on rocks and stones in contact with water in the natural environment, such as rivers, canals, lakes and the sea. Similar observations may be made in respect of Industrial Biofouling ISBN 978-0-444-53224-4

Ó 2011 Elsevier B.V. All rights reserved.

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industrial operations, such as water distribution and treatment, coolingwater applications and even food processing. In the medical world, biofilms may be found on teeth, on implants and attached to urinary catheters. Although the accumulation of microorganisms on man-made structures such as bridge supports, sea defences, ships’ hulls and canal locks occurs, macroorganisms could also become attached. In industrial processes however, it is common practice for obvious reasons, to filter out macroorganisms before the water is put to the required use. Such a procedure for the control of biofilm-forming microorganisms is impossible on the scale required because of their small size and the associated cost that this procedure would entail. Other techniques therefore, have to be adopted in order to maintain process efficiency. Industrial problems associated with the accumulation of microorganisms from flowing water on the surfaces of industrial processing equipment increase operating costs. Some of the problems may be listed as: 1. Reduced heat transfer in heat exchangers that employ cooling water to reduce process-fluid temperatures. 2. Accumulation on surfaces in contact with fluids that are ultimately destined for human consumption, e.g., drinking water or food products associated with risk to health. 3. Increased pressure loss in water circuits, e.g., cooling-water systems. In lighted areas algae may produce slimy growths that may interfere with water distribution. Bacteria may produce a slime mass due to biofilm or waste production that can cause blockage and accelerate corrosion. 4. Interference with the function of chemical additives for the prevention of metal corrosion. 5. Surface corrosion of process equipment by bacteria, including pitting, corrosion through depolarisation of the oxygen released during microbial metabolism and the creation of potential differences on metal surfaces. Depending on the species of bacteria present, deposits of insoluble ferric hydroxide or insoluble ferrous sulphide may be produced. 6. The lack of oxygen near the solid surface and the associated encouragement of anaerobic microorganisms, including sulphate-reducing bacteria that utilise sulphates in their metabolic processes. In saltwater systems there may be severe problems at intake and discharge tunnels due to growth of macroorganisms. It has to be stated however, that in some processing operations the presence of biofilm is vital, for example, in the use of trickle filters for the treatment of waste water or the removal of unwanted components in

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drinking water to improve its quality to meet legal requirements. Under these conditions the associated costs will be acceptable. The cost of unwanted biofilm accumulation on the surface of industrial equipment can represent a large proportion of the total operating cost and the initial capital cost in making allowance for the likely problem of biofouling. At the equipment design stage to reduce operating problems, it might be thought that the equipment should be made larger to accommodate the fouling problem or a more expensive material of construction used to reduce the microbial accumulation and potential corrosion problems. These attempts at remediation will undoubtedly increase capital costs. Capital outlay may also be increased by the inclusion of ancillary equipment associated with counteracting the unwanted accumulation of biofilm on equipment surfaces. The presence of biofilm will reduce the flow area and generally present a rough surface to the fluid flow in equipment such as heat exchangers, so that for a given design throughput, the pumping energy requirement and the associated cost will be much greater than if there was no biofilm. If the pumping energy involved is electricity derived from a combustion process as it often is, then the carbon dioxide emission will be larger than necessary, thereby adding to the problem of global warming. Even though the potential problems due to biofouling may have been recognised at the design stage, it may still be necessary to take remedial action to prevent or restrict the development of biofilms on the surfaces of industrial equipment. The employment of biocides for instance, will involve increased capital cost for the equipment needed for dosing purposes. In addition, the purchase of biocides for dosing will add to operating costs. In order to protect the environment it may be necessary also, to treat the water after use and before discharge, with associated increased operating cost. It may be necessary to clean equipment affected by biofilm accumulation from time to time. Cleaning is likely to involve the halting of operations to allow access to the inner parts of the equipment. The additional costs will not only involve the cost of labour and materials for cleaning but also the cost of lost production. Furthermore it may be necessary to treat the ‘wash water’ before discharge into the public sewers or back into the environment. An unscheduled shutdown to deal with a sudden detrimental increase in biofilm accumulation could result in substantial costs in terms of lost production and emergency cleaning operations. In order to offset some of these costs it might be thought prudent to duplicate equipment, again with increased capital costs, so that during the

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cleaning of one equipment set, the other set can be operated and production can be maintained. Mu¨ller-Steinhagen (2000) provides data, admittedly based on rather old figures, which give an indication of the high cost of heat exchanger fouling alone, a substantial part of which will be due to biofouling. In industrial operations the cost of fouling is of the order of 0.20–0.25% of the Gross National Product (GNP). If the costs of all detrimental biofouling, in addition to those directly associated with heat transfer, are taken into account as a percentage of GNP, it would be substantially higher. Whitehouse, Khalanski, Saroglia, and Jenner (1986) stated that the presence of macroorganisms in marine and estuarine water used for cooling water in power stations could create problems. Macroinvertebrates usually colonise water intake tunnels and culverts, screens and possibly the tube plates of steam condensers. In certain circumstances they can colonise the discharge pipe work. The conditions that assist the growth of macroorganisms are the continuous flow of seawater that provides oxygen, food and reduced salt deposition. These conditions preclude competitive algae growth. The principal macroorganisms include: 1. Mussels. Growth is largely seasonal. During winter months with temperatures below 10  C there is no growth. Above 10  C, growth can take place and it can be rapid. 2. Barnacles. As part of a highly modified class of crustacean, they are able to settle in high water velocities of 3 m/s and provide surfaces on which mussels can settle. Barnacles can cause serious blocking of filter meshes but are not as serious a problem in Europe as in tropical areas. 3. Hydroids. These are primitive animals that may be mistaken for plants by the uninformed. They are pioneer organisms that assist the settlement of mussels by providing a better substrate than the underlying bare surface. 4. Serpulid worms. These are encrusting worms producing calcareous “tubes” on surfaces in cooling-water intakes. 5. Sea mats/sea squirts. These are soft bodied and are not thought to cause serious fouling problems. The control of macroorganism fouling falls into two groups of possibilities (chemical and physical) that are discussed in Chapter 4 on microorganism control. The discussion in this book will be centred principally on the accumulation of microorganisms on surfaces, particularly bacteria, but also to a lesser extent on fungi and algae that may be regarded as biofouling agents. Since these can present major problems in many industrial operations

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involving the use of water, techniques to counteract their adverse effects are discussed in detail. Industrial operations that employ biofilms to advantage are also covered. Macroorganisms in general, do not present problems in many industrial processing applications, since effective precautions, such as filtering, which cannot be employed for microorganism control, can be readily taken.

REFERENCES Costerton, J. W., Lewandowski, Z., De Beer, D., Caldwell, D., Korber, D., & James, G. (1994). Biofilms, the customised microniche. Journal of Bacteriology, 176, 21337. Mu¨ller-Steinhagen, H. (2000). Heat exchanger fouling mitigation and cleaning technologies. Essen, Germany: Publico Publications. Whitehouse, J. W., Khalanski, M., Saroglia, M. G., & Jenner, H. A. (1986). The control of biofouling in marine and estuarine power stations. CEGB-EDF-ENEL-N.KEMA, 1, 1–48.

CHAPTER

2

Fluid Flow, Mass and Heat Transfer Contents 2.1. Introduction 2.2. Fluid Flow 2.3. Mass Transfer 2.3.1. Deposition, Growth and Removal of Microorganisms 2.4. Heat Transfer 2.5. Concluding Remarks References

7 7 11 12 18 21 22

2.1. INTRODUCTION The colonisation and growth of living material on a surface in industrial equipment is greatly influenced by the fluid flow and mass transfer pertaining to the system. Because in general, fluids are contained in the pipes and vessels that constitute the industrial process plant, it is necessary to understand what happens when fluids flow across the surfaces of such equipment. An appreciation of the underlying principles of fluid flow, associated mass transfer and to some extent heat transfer are therefore, an aid to recognising the factors that contribute to the complexity of the formation of biofilms, their effect on the efficiency of many industrial processes and how the problems may be overcome.

2.2. FLUID FLOW In general the occurrence of biofilms in industrial equipment arises from a microbial-laden fluid, usually water, flowing across a surface. The conditions affecting deposition will depend principally on the flow rate of the fluid, its physical properties and the nature of the surface. The relationship between these variables was systematically examined by Reynolds (1883) and his discoveries concerning the flow of fluids are often used in the design and operation of process plants dependant on the movement of fluids. Industrial Biofouling ISBN 978-0-444-53224-4

Ó 2011 Elsevier B.V. All rights reserved.

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Two flow conditions exist: 1. Laminar flow often referred to as ‘streamline flow’, occurs at low velocities, where the movement of the fluid is parallel to the surface across which it is flowing. 2. Turbulent flow occurs at higher velocities, where in addition to the fluid movement parallel to the surface and close to it, there is bulk random movement remote from the surface. Due to the resistance to flow caused by friction between the fluid and the surface, the fluid layers in contact with the solid surface may be regarded as stationary. A drag force will be exerted at the solid surface by the moving fluid. Velocity will increase with distance from the retaining surface. Even under turbulent conditions a slow-moving layer of fluid will exist close to the surface with a stationary layer at the fluid/surface interface. The slowmoving fluid region close to the solid wall is often referred to as the ‘viscous sub-layer’ or the ‘laminar sub-layer’. In a tube for example, the thickness of the viscous sub-layer will depend on the bulk velocity through the tube. In general, the higher the bulk velocity the thinner the laminar sub-layer. Between the two conditions of streamline and turbulent flow, as might be expected, there exists a transition region. The change in flow rate between the turbulent region and viscous sub-layer is not abrupt and often referred to as the ‘boundary layer’. The situation is illustrated in Fig. 2.1, which also diagrammatically shows deposits of microorganisms and the removal of pieces of the biofilm which have become detached. As a result of his pioneering work, Reynolds devised a criterion to distinguish the difference between streamline and turbulent flow in tubes. A dimensionless number, the so-called Reynolds number, (Re), identifies the difference and is defined as:

Flow Bacteria

“Lumps” of Biofilm

Turbulent region

Viscous sub layer Biofouling Metal surface

Figure 2.1 Deposition and removal occurring at the same time. (Courtesy of Elsevier.)

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Fluid Flow, Mass and Heat Transfer

Re ¼

Dvr m

(2.1)

d D

Distance

Where D is tube diameter; v is bulk fluid velocity; r is fluid density; m is fluid viscosity; all in consistent units. Although the concept was originally devised for tubular flow, it can be adapted to flow systems other than tubes and pipes by the use of the socalled ‘equivalent diameter’. It has been shown that at an Re number below about 2000, the flow is streamline and above about Reynolds number 4000 the flow is turbulent. The transitional flow regime lies between these two Reynolds numbers, and is often referred to as the ‘boundary layer’. The profile of flow distribution across the diameter of the retaining tube depends on whether the flow, as defined by the Reynolds number, is streamline or turbulent. Under streamline conditions, the velocity profile is a parabola (see Fig. 2.2) and the mean velocity of flow is half the flow at the centre of the tube where the velocity is the maximum. Under turbulent conditions, the velocity profile is no longer a parabola as shown in Fig. 2.3. For turbulent conditions, the mean fluid velocity is 0.82 times the velocity at the centre of the tube.

C/L

Velocity

D

Distance

Figure 2.2 Velocity profile in a tube with laminar flow. (Courtesy of Elsevier.)

C/L

Velocity

Figure 2.3 Velocity profile in a tube with turbulent flow. (Courtesy of Elsevier.)

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The ‘drag’ force at the interface between a deposit on a solid surface and the flowing fluid will tend to remove the deposit from the surface. The extent of the removal process will depend on the tenacity of adhesion of the deposit to the surface and its intrinsic cohesive strength in relation to the force exerted by the flowing fluid. It is an extremely complex phenomenon, particularly in respect of accumulation of microorganisms and it is difficult if not impossible, to predict with any certainty except in very general terms. Of considerable importance in process plant operations is the pressure drop in the fluid flowing through the processing equipment, referred to earlier. Work by Stanton and Pannell (1914) made it possible to make reliable estimates of pressure loss under known conditions. They defined a dimensionless term called the ‘friction factor’ (F) and related it to the Reynolds number (Re). F ¼

R rn2

(2.2)

(where R is the shear stress). In the streamline region F ¼

8 Re

(2.3)

The friction factor F is a function of the Reynolds number in the turbulent and transition regions but in addition depends on the roughness of the surface across which the fluid is flowing. If ‘e’ is the height of the roughness, the dimensionless number e/D is used to take account of the roughness. The pressure loss in terms of head (height) DH of fluid is given by: DH ¼

4flv2 2g

f ¼ 2F

(2.4) (2.5)

(where ‘f ’ is the friction factor; ‘l’ is length; ‘g’ is the acceleration of gravity). In most industrial systems where biofouling occurs, the fluid involved is water, the natural environment in which both microorganisms and macroorganisms are found. Temperature affects both density and viscosity. The magnitude of these properties (density and viscosity) affects the numerical value of the Reynolds number and hence the condition may be laminar or

Fluid Flow, Mass and Heat Transfer

11

turbulent. The change in the density of water between 0  C and 100  C is not large. Over the same temperature range however, there is a significant reduction in the magnitude of viscosity as the temperature is raised. Since the Reynolds number therefore is dependent on temperature and it is a significant variable in the analysis of flow conditions, temperature is an important variable in the biofouling process. Quite apart from the effect of temperature on the flow conditions for a given volumetric flow rate and the deposition process together with the physical retention of biofilms on surfaces, temperature is also important for the survival and growth of biofilms in contact with water. There will be an optimum temperature at which growth is maximised. For microorganisms this is generally in the region of 25–45  C.

2.3. MASS TRANSFER The accumulation of unwanted living matter on a surface i.e., biofouling, involves the colonisation of the surface by organisms transferred from the water flow to the surface by the process of mass transfer. The ‘driving force’ for mass transfer is a concentration gradient i.e., the movement of mass being from the higher concentration towards the lower concentration. In biofouling, this involves both the concentration differences of microorganisms and nutrients between the bulk water flow and the receptive surface. Once the surface has been colonised, its sustained growth and development will depend on the availability of suitable nutrients and possibly of additional microorganisms. Furthermore, mass transfer provides the means by which the waste products from the microbial activity are removed from the region of the biofilm into the main bulk of the flowing water, thereby assisting further biofilm development. In stationary water or in water flowing under streamline conditions (e.g., the laminar sub-layer) with a concentration gradient of microbes between the bulk and the receptive surface, movement of the microbial cells to the surface is due to their random motion within the fluid, which is usually referred to as Brownian motion. In water that is turbulent, the movement of microbial cells or nutrients is very different. Eddy diffusion created by the turbulence is superimposed on the Brownian motion. What might be termed as ‘parcels of water’ containing the organisms, nutrient molecules or even solid particles will move down the concentration gradient from the bulk water towards the developing biofilm on the solid surface.

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If a component A is diffusing through a fluid B at right angles to the general flow of fluid B, the rate of diffusion may be obtained from Fick’s law (Fick, 1855): NA ¼ DAB

dcA dx

(2.6)

(where, NA is the rate of molecular diffusion of A; cA is the concentration of A at a distance x from the receptive surface; DAB is the molecular diffusivity of A through B). The diffusion of A through B will not only depend on the physical properties of A and B but also on the fluid flow conditions i.e., streamline or turbulent. If turbulence exists in the system, Equation 2.7 is modified by the inclusion of a term to account for the turbulence: NA ¼ ðDAB þ ED Þ

dcA dx

(2.7)

(where ED is the eddy diffusion; under turbulent flow conditions ED is very much greater than DAB).

2.3.1. Deposition, Growth and Removal of Microorganisms In Section 2.2 it was stated that even when the water is flowing under turbulent conditions, there exists a laminar sub-layer at the solid surface which impairs the transfer of molecules, particles, microorganisms and their required nutrients to the surface. Under these conditions, the movement towards the surface (and away from it) is due to diffusion on a molecular level i.e., Brownian motion. The thickness of this layer and hence its resistance to mass transfer will depend on the velocity of the bulk water. The higher the velocity of the bulk water the thinner the laminar sub-layer will be, other conditions remaining the same, thereby reducing the resistance to mass transfer. Hence there will be enhanced opportunity for colonisation and subsequent growth to occur. The removal of waste material from microbial activity will also depend in the same way, on whether the flow conditions are turbulent or streamline. The concentration gradient will however, be in the opposite direction from that for deposition i.e., highest in the biofilm to a low concentration in the bulk flow. Under flow conditions, the movement of a fluid stream across a deposit attached to a solid surface will exert a force on the deposit, as illustrated in Fig. 2.4. If the so-called drag force is strong enough, some deposit is likely to be removed from the solid surface. In very general terms the rate of growth

Fluid Flow, Mass and Heat Transfer

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Lift Fluid Flow Drag

Surface Attachment

Figure 2.4 Forces acting on a particle residing on a surface. (Courtesy of Elsevier.)

of a deposit will depend on the relative magnitude of the deposition, the removal forces and the inherent resistance of the deposit to removal. As the influence of the surface on velocity diminishes further away from the surface, the core fluid eventually becomes turbulent. Turbulent conditions ensure good mixing so that the concentration of dissolved nutrients or suspended cells in this region will be generally uniform. The colonising cells that initiate the creation of a biofilm and subsequently the nutrients that sustain biofilm growth, have to pass across the boundary layers. The driving force for mass transfer is the concentration gradient across the laminar sub-layer, i.e., the difference between the bulk concentration (in turbulent conditions) and the concentration at the fluid/ solid interface. As the laminar region offers a resistance to the transfer process, it could impose a limitation on the potential growth of the biofilm. However the greater the bulk velocity, the thinner will be the laminar sublayer and hence it will provide a lower resistance to mass transfer. On the other hand the higher the fluid velocity across a solid surface, the higher the shear forces at the fluid/solid interface. These shearing effects impose removal forces at the deposit interface with the fluid. There will be ‘survivors’ that resist the removal forces and these cells will initiate the development of biofilm. The idealised growth curve for the development of biofilm from a flowing water stream is illustrated on Fig. 2.5a. There are essentially three stages, although there is no clear demarcation between them. Initiation where the microorganisms colonise the surface, which depends on the ability to attach, is described in more detail in Chapter 3. Once colonisation has been established and the flowing water contains sufficient nutrients, there will be a rapid exponential growth of the biofilm till it reaches a roughly constant thickness dependent on the prevailing conditions of nutrient concentration and water velocity. Fig. 2.5b provides a more realistic

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Biofilm thickness

(a) Constant thickness

Rapid development Initiation

Sloughing

Plateau

Exponential growth

Lag phase

Biofilm thickness

(b)

Decreasing rate

Time

Time

Figure 2.5 (a). An idealised growth curve for a bioflim on a surface. (b). Growth curve as modified by local operating conditions. (Courtesy of Elsevier.)

picture of the development of a biofilm, of which every example will be a variation depending on the local conditions. As the velocity of flow in a given system is increased, the mass transfer towards the surface increases and the removal forces increase also. Under these circumstances there is an optimum velocity where the microbial biofilm development is maximum. Fig. 2.6 illustrates this effect. The data were obtained with water containing Pseudomonas fluorescens bacteria and nutrients (Nesaratnam, 1984). At low velocity, the extent of the biofilm is relatively small due to the high resistance to the transport of microorganisms but principally nutrients to the surface. As the velocity increases the resistance to nutrient transfer decreases but at the same time the removal forces increase. Eventually however, as the velocity is further increased, although the mass transfer is improved, the shear effect outweighs this advantage and the biofilm thickness is adversely affected. Biofilm growth usually occurs as a result of the availability of nutrients rather than by deposition of further microorganisms (Bott and Miller,

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Fluid Flow, Mass and Heat Transfer

Biofilm thicknes m

150 Nutrient: glucose 7 mg/ 1 100

50

0

0.5

1.0 1.5 Water velocity ms–1

2.0

2.5

Figure 2.6 The effect of velocity on plateau values of bioflim thickness. (Courtesy of Elsevier.)

Weight increase due to biofilm development 4 g/cm2 10

1983). Once the surface has been colonised by microorganisms, provided there is adequate mass transfer of nutrients, there is no need for further mass transfer of microorganisms as illustrated by Fig. 2.7. Without the availability of bacteria but with nutrients present in the flowing water and a colonised surface, the biofilm develops in the normal manner. Without a supply of nutrients however, even with bacteria in the water, the biofilm deteriorates as illustrated in Fig. 2.8. 700 Bacteria and 4 mg/l nutrient Temperature 25°C Velocity 0.5 m/s

600

Bacteria supply withdrawn

500 400 300 200 100 0 0

4

8

12

16

20

24

28

32

Time days

Figure 2.7 The effect of elimination of bacteria from the flowing stream. (Courtesy of Elsevier.)

T. Reg. Bott

Weight increase due to biofilm development gcm–1 104

16

700

Bacteria 4 mg/l nutrient Velocity 0.5 m/s Temperature 25°C

600

Nutrient supply withdrawn

500 400 300 200 100 0

4

0

8

12

16

20

24

32

28

Time days

Figure 2.8 The effect of elimination of nutrient from the flowing stream. (Courtesy of Elsevier.)

Weight increase of biofilm on tube g cm–1 104

Clearly, the concentration of nutrients will affect growth. The higher the concentration in the flowing water, the greater the ‘driving force’ and the mass transfer to the biofilm, thereby providing the opportunity for growth. Fig. 2.9 illustrates the effect of increasing the availability of nutrient (glucose) on the weight increase of a biofilm, other conditions remaining constant (Nesaratnam, 1984). 1000

Velocity 1.2 m/s Reynolds number 16,165

800

Glucose Concentration

600 4mg/l

7mg/l 10mg/l

400 200 0

0

2

4

6

8

10

12

14

16

18

20

22

Time days

Figure 2.9 The change in mass of bioflim on a surface at different nutrient concentrations.

Fluid Flow, Mass and Heat Transfer

17

Even with mature biofilms, due to the prevailing conditions, portions of a developing biofilm may detach and re-entrain into the bulk water flow. Biofilm growth is an extremely complex process. This detachment phenomenon is often referred to as ‘sloughing’. The principal cause of sloughing is the presence of the shear forces acting on the biofilm resulting from the flow, combined with any weakness in the newly-formed biofilm structure. The principal variables that affect the removal process include: 1. Biofilm thickness. As the thickness increases, the underlayers of microorganisms become vulnerable to the removal forces. However as time passes the structure becomes more resistant to these forces due to the removal of the weaker parts of the biofilm that has already taken place, to be replaced with the development of a more robust structure. 2. Biofilm density. The greater the density of the biofilm the greater the resistance to removal is likely to be, since the structure will be more compact. 3. Concentration of microorganisms The higher the concentration of microorganisms in the flowing water, the greater will be the mass transfer “driving force” towards the developing biofilm. As a result, additional cells can be introduced into the biofilm replacing cells that have been removed, with the opportunity to produce structures that are more resistant to the removal forces. 4. Concentration of nutrients As with the microorganisms, the higher the concentration of the available nutrients, the greater the mass transfer to the growing biofilm. Once the solid surface has been colonised, the mass transfer of nutrients is far more important than that of the cells, since it will enable the biofilm to develop through reproduction. 5. Fluid shear stress. The shear stress at the surface of the biofilm is dependent on the Reynolds number which for a given system, is dependent on the water velocity, its viscosity and density at the operating temperature and the characteristic dimension. There is an additional effect of velocity on the development of a biofilm. Some microorganisms will be better orientated to resist the removal forces. These cells, the survivors, may be arranged within the structure to reduce the effects of velocity. As a result, the structure of the ensuing biofilm may develop in a particular way and produce a dense biofilm.

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Figure 2.10 Pseudomonas fluorescens cells in-line with water flow. (Courtesy Harwood Academic Publishers.)

Fig. 2.10 demonstrates this phenomenon (Santos, 1993). The rod-like cells of Pseudomonas fluorescens are seen to be oriented in line with the water flow direction thereby reducing the removal forces imposed by the water flow. It was not clear whether or not the cells were able to position themselves in this way or if it was due to a natural selection process whereby the cells which were most likely to resist the removal forces remained attachedda further example of the ‘survival of the fittest’. There may be other implications that are detrimental to subsequent biofilm growth in that the diffusion of nutrients through the biofilm is restricted. This is due to the reduced flow channels in the biofilm resulting from the growth and orientation of individual cells.

2.4. HEAT TRANSFER Since microbial populations are influenced by the prevailing temperature, the extent of a biofilm will be dependent on the temperature of the transporting fluid (usually water). The bulk fluid temperature is itself affected by heat transfer. Just as mass transfer is influenced by concentration difference, heat transfer occurs as a result of a temperature difference. The slow-moving sub-layers of fluid also act as a resistance to heat transfer, so that at higher bulk velocities the laminar sub-layer thickness is reduced and heat

Fluid Flow, Mass and Heat Transfer

19

transfer is increased. If for instance, a biofilm occurs on the surface of a heat exchanger through which microbial-laden cooling water passes, the biofilm at the heat exchanger surface will be at a higher temperature than the cooling water and a temperature ‘driving force’ is provided across the biofouling layer between the heat exchanger surface and the water. Fig. 2.11 shows the effect of temperature, within the narrow temperature range suitable for microbial survival, on biofilm development (Bott and Pinheiro, 1977). In many industrial processes it is necessary to transfer heat between fluids to heat or cool as appropriate. The fluids will be separated by a wall, usually metal, through which heat is transferred by conduction. For a plane wall of thickness x with the two surfaces at temperatures T1 and T2, (T1 > T2) the heat transferred Q, is given by Equation 2.8: Q ¼

kAðT1  T2 Þ x

(2.8)

(where x is the thickness of the separating wall; A is the area of the separating wall; k is the thermal conductivity of the metal; all in consistent units.) Equation 2.8 can be rearranged and rewritten as: Q ¼

ðT1  T2 ÞA x=k

(2.9)

300

Biofilm thickness m

35 ºC

200 30 ºC

100

0 0

2

4

6 8 Time days

10

12

Figure 2.11 The effect of temperature change on the thickness of a bioflim of Escherichia coli. (Courtesy of Elsevier.)

20

T. Reg. Bott

(x/k is usually referred to as the ‘thermal resistance’). During industrial operations, the surfaces usually gather deposits referred to as ‘fouling’, which through their insulating effect restrict the flow of heat. The origins of these deposits are varied depending on the operation that is being undertaken in the equipment. They can include: 1. Corrosion of the metal wall. 2. Crystallisation of soluble material in the fluid onto the solid surface due to temperature reduction. 3. Deposition of particulate matter. 4. Accumulation of material resulting from chemical reaction. 5. The deposition and growth of microorganisms, usually referred to as ‘biofouling’dthe thrust of this book. The heat transfer situation is as illustrated in Fig. 2.12 with fouling on both sides of the dividing wall. The quantity of heat transferred must pass through each of the fouling layers that together with the metal make up a composite resistance to heat transfer. There is also the thermal resistance due to the laminar sub-layers discussed in Section 2.2 x1 Q k1 A

(2.10)

T2  T3 ¼

x2 Q k2 A

(2.11)

T3  T4 ¼

x3 Q k3 A

(2.12)

METAL

T3 T2 DEPOSIT

LAMINAR SUB LAYER

T1

Biofuling

WALL

HOT FLUID

X2

LAMINAR SUB LAYER

T1  T2 ¼

COLD FLUID

T4 X3

Figure 2.12 Temperature distribution across fouled heat exchange surface.

Fluid Flow, Mass and Heat Transfer

Therefore T1  T4 ¼

or

x1 Q x2 Q x3 Q þ þ k1 A k2 A k3 A

21

(2.13)

  Q x1 x2 x3 þ þ Rearranging ¼ A k1 k2 k3

(2.14)

ðT1  T4 ÞA i:e: Q ¼ x1 x2 x3 þ þ k1 k2 k3

(2.15)

Heat transferred ¼

Overall driving force x heat transfer area Total thermal resistance (2.16)

In the design of equipment that involves heat transfer subject to fouling, the basic equations concerning the flow of fluids and the thermal resistance of the metal wall are reasonably accurate but the extent of the effect of the fouling layers is difficult or virtually impossible to predict. There may be some empirical data that is helpful but the fact remains that often the allowance for the fouling is little more than an inspired guess based on previous practical experience. There are recommended fouling allowances for many different fluid streams, such as the one issued by the Tubular Heat Exchanger Manufacturers Association (TEMA, 1988). It is a useful guide for design purposes but it is the plant operator who has to make the final decisions regarding the minimisation of the fouling problem. Acquisition of data related to the particular plant is of considerable value in this respect. This is especially true where biofouling is concerned because of the wide variations that can exist, for instance, between cooling water systems in different locations.

2.5. CONCLUDING REMARKS The amount of resident biofilm depends on the magnitude of the competing deposition and removal forces. It has to be stated though, that the character of a biofilm residing on a surface is very different from the single or small groups of microorganisms that initially colonise the surface. After deposition, the individual cells form a matrix with the extra-cellular products of their metabolism. This development of the biofilm will resist the removal process. As the biofilm develops and thickens, it is likely that the cells close to the solid surface will become starved due to the consumption

22

T. Reg. Bott

of the nutrients by the outer layers of cells. The result may be the death (lysis) of cells near the surface or a change in the cells to a dormant state. In turn, this may weaken the cohesion of the biofilm and lead to a partial removal. In some species of microorganisms, there is the possibility of adaption to these changing conditions. It is possible for instance, that some aerobic bacteria (requiring oxygen) can revert to anaerobic (not requiring oxygen) status, if the oxygen level is drastically depleted by the activity of the cells in the outer layers of the biofilm. In addition to the effects of fluid flow and the associated mass transfer, there are other factors that can influence the laying down of a biofilm on industrial equipment. They include: 1. Quality of the surface on which the biofilm forms. 2. The material of the surface. Some metals such as copper are toxic to microorganisms. 3. Temperature and temperature gradients. As naturally occurring water is often used for cooling purposes in industrial operations, the season and hence the water temperature could influence biofilm growth. 4. The presence of soluble components that include suitable nutrients for the microorganisms. 5. The presence of toxic substances naturally occurring or deliberately applied, such as biocides. 6. The presence of particulate matter. The influence of these variables is discussed in some detail in Chapters 3 and 4.

REFERENCES Bott, T. R., & Miller, P. C. (1983). Mechanisms of biofilm formation on aluminium tubes. Journal of Chemical Technology Biotechnology, 33B, 177. Bott, T. R., & Pinheiro, M. M. V. P. S. (1977). Biological fouling – velocity and temperature effects. Canadian Journal of Chemical Engineering, 55, 473. Fick, A. (1855). Ueber diffusion. Annals of Physics, 94, 59. Nesaratnam, R. N., (1984). Biofilm formation and destruction on simulated heat transfer surfaces. Ph.D. thesis, University of Birmingham. Santos, R. C., (1993). Polymer coatings in relation to single and mixed population biofilms. Ph.D. thesis, University of Birmingham. Stanton, T., & Pannell, J. (1914). Similarity of motion in relation to the surface friction of fluids. Philosophical Transactions Royal Society of London, 214, 199. Reynolds, O. (1883). On the experimental investigation of the circumstances which determine whether the motion of water shall be direct or sinuous and the law of resistance in parallel channels. On the dynamic theory of incompressible viscous fluids and the determination of the criterion. Transactions Royal Society, 74, 935. TEMA. (1988). Standards of the Tubular Exchanger Manufacturers Association (7th ed.). TEMA, Inc.

CHAPTER

3

Biofilms Contents

3.1. Introduction 3.2. Microbiology 3.3. Biofilms 3.3.1. Fungi 3.3.2. Algae 3.3.3. Bacteria 3.4. Requirements for Microbial Growth 3.4.1. Nutrient Requirements 3.4.2. Metabolism 3.4.3. Matrix Formation 3.5. The Importance of Surfaces 3.6. Adhesion of Microbial Cells to Surfaces 3.7. Interacting Forces 3.7.1. van der Waals forces 3.7.2. DLVO and Wettability Theories 3.8. The Influence of Adsorbed Layers 3.9. The Physical Quality of the Surface 3.10. Industrial Conditions 3.11. The Establishment of Biofilms in Summary 3.12. The Influence of Other Fouling Mechanisms 3.13. Particle/Biofilm Interactions 3.14. The Growth of Biofilms 3.15. Structure and Stability of Biofilms 3.16. Biofouling Modelling 3.17. Biofilms and Corrosion 3.18. The Cost of Biofouling 3.19. Concluding Remarks References

23 26 29 34 35 36 38 41 41 44 45 46 49 50 51 55 58 61 62 64 64 67 69 73 73 74 76 76

3.1. INTRODUCTION Kemmer (1988) listed the problems associated with different microorganisms that may be encountered in industrial operations: Bacteria. 1. Slime-forming: Form dense, sticky slime with subsequent fouling. Water flows can be impeded and promotion of other organism growth occurs. Industrial Biofouling ISBN 978-0-444-53224-4

Ó 2011 Elsevier B.V. All rights reserved.

23

j

24

T. Reg. Bott

2. Spore-forming: Spores become inert when their environment becomes hostile to them, however, growth recurs whenever the environment becomes suitable again; difficult to control if complete kill is required. Most processes are not affected by spore formers when the organism is in the spore form. 3. Iron-depositing Causes the oxidation and subsequent deposition of insoluble iron from its soluble form. 4. Nitrifying: Generates nitric acid from ammonia contamination. Can cause severe corrosion. 5. Sulphate reducing: Generates sulphides from sulphates and can cause serious localised corrosion. 6. Anaerobic corrosive: Creates corrosive localised environments by secreting corrosive wastes. They are always found underneath other deposits in oxygen deficient locations. Fungi and moulds: Cause the degradation of wood in contact with the water system. Cause spots on paper products. Algae: Grow in sunlit areas in dense fibrous mats. Can cause plugging of water inlet ports, impair effective distribution in cooling towers or create dense growths on reservoirs and evaporation ponds. Protozoa: Grow in almost any water that is contaminated with bacteria. They indicate poor disinfection. Bacteria are the microorganisms that are likely to be the major cause of operating difficulties in industrial equipment, since they are more adaptable than algae that require sunlight and fungi that require a consumable substrate. In a water environment, planktonic (unattached and drifting) bacteria are attracted to any solid surface that is in contact with the water where they attach in a relatively short time. Attached to a surface they are described as “sessile” organisms. Paulson (2007) presents a useful summary of basic cell types and their properties; they include eukaryotes and prokaryotes. Eukaryotes are the basic unit structures of plants, animals, fungi, many algae cells and some single-celled organisms such as protozoa.

Biofilms

25

Ribosomes

Flagellum Granular inclusion

Cytoplasm Cytoplasmic membrane

Nucleus Cell wall Mesosome

Capsule Pili

Figure 3.1 Diagram of microbial cell structure.

Prokaryote cells are generally simpler in structure and represent the form of bacteria and blue-green algae. They do not contain nuclei bounded within membranes but nuclear filaments. Basic cell structure is shown on Fig. 3.1. Bacteria can be divided into gram-positive and gram-negative, based on the extremely useful staining method devised by Christian Gram (Davis, Dulbecco, Eisen, et al 1980), which distinguishes between bacteria that have similar morphologydi.e., gram-positive cells stain purple while gramnegative cells stain red. With staining it is possible to discern the shape and the size of bacteria, which display three basic morphologies; coccus (spherical), bacillus (rod-like) or spiral. Paulson (2007) asserted that possibly all bacteria found in the environment are surrounded by layers of gelatinous, poorly defined material that has been termed ‘the capsule’, ‘the slime layer’, ‘glycocalyx’, or ‘biofilm’. The term capsule is generally applied to the material that surrounds the cell. ‘Slime layer’ is often used to describe the matrix that envelopes a microcolony clump of cells but the choice of description usually depends on the context. In industrial operations, ‘biofouling’, ‘slime layer’ and ‘biofilm’ are common descriptions. When attached to a surface they are then described as sessile microorganisms. Indeed speed of attachment is part of the strategy for survival (Watnick & Kolter, 2000). The formation of a biofilm therefore, depends on the presence of these colonising microorganisms, the availability of a suitable surface, the continued presence of water in contact with the surface and an adequate continuous source of nutrients, even if only available in relatively low concentration. It will be recognised that these conditions are often present in industrial environments. In contrast to inert particles that may form a rather loose deposit, microorganisms have a natural tendency to resist removal once they have

26

T. Reg. Bott

become attached to a surface as part of a biofilm. Furthermore, because they are living entities they reproduce within the biofilm, which may also provide resistance to removal. As briefly discussed earlier (Chapter 2), there may be orientation in the biofilm in respect of the direction of water flow across it and hence of the direction of the thrust of the associated removal forces, so that there is less opportunity for removal to occur. This is a good example of Darwin’s theory of survival of the fittest. On the other hand, there may be foreign bodies such as solid particles deposited within the biofilm that weaken its structure, thereby facilitating removal. As time passes and the biofilm thickness increases, the availability of nutrients to the layers of microorganisms close to the surface to which they are attached, is greatly reduced or eliminated altogether by their consumption in the outer layers of organisms. This may lead to the death of these organisms or at least lead to some form of dormancy. In turn, this may weaken the resilience of the biofilm to the removal forces. In industrial operations, water is the usual transport medium for microorganisms and macroorganisms, for instance, cooling water taken from natural sources. Its qualities, such as the presence of soluble components, particulate matter and its temperature, will affect the relationship between deposition, growth and removal. The brief discussion of the formation and removal of biofilms in Chapters 1 and 2 indicates that it is an extremely complex mechanism. It is probably true to say that each example is unique, making it difficult to come to a full understanding of the precise effects of the prevailing variables. As a result, it is often necessary to revert to empiricism in the solution of a particular problem involving biofouling. The same principles apply, although in modified form because of their size, to the accumulation of macroorganisms on surfaces. A principal component of transport to the surface is the energy of momentum due to the mass of the organism and its velocity. If the momentum is high enough, its kinetic energy will overcome the drag forces and the organism will pass through the 1aminar sub-layers to the surface. The nutrients may arrive at the biofouling layer by a similar mechanism or by the eddy diffusion process referred to in connection with the transport of nutrients for microbial biofilm growth (see Chapter 2).

3.2. MICROBIOLOGY It is not necessary to discuss all the detailed aspects of microbiology for an appreciation of the occurrence and consequent effects of biofilms in

Biofilms

27

industrial operations. The following brief review however, of the underlying features of microbiology, provides a background to biofilm technology. Microscopic organisms were not seen, till in the seventeenth century, microscopes with sufficient magnification power became available. The variety of different shapes and structures of microorganisms were then discovered, including spheres, rods, and spiralsdthe latter being less common. Investigations also revealed the larger microbes including protozoa, fungi and algae. Since that time there has been a steady growth of knowledge concerning the physiology and behaviour of microbial cells, with of course, an explosion of understanding with the invention of the scanning electron microscope and associated equipment in more recent times. Some cells have surface appendages that may include: 1. Flagella that consist of very fine threads of protein with a helical structure protruding from the cell cytoplasm that aid motility. 2. Pili or fimbriae that consist of very fine threads much shorter than flagella. A single cell may have several such appendages appearing as tufts on the cell surface. 3. Stalks that consist essentially, of blunt extensions of the cell wall and membrane in one or more places. The bacterial capsule is a loose gel-like structure, which varies widely between many different strains of bacteria in thickness, density and adherence to the cell wall. The capsule often consists of simple polysaccharides containing two or three sugars that are not present in the cell wall. The purpose of the capsule is to protect the cell from environmental damage and to act as a source of energy if the availability of nutrients becomes diminished. The cell wall itself is rigid, retains the cytoplasmic membrane and may be of the order of 10–25% of the cell mass. The cell wall topography can have wide variation in structural detail, ranging from a regular structure to a matted irregular fibrous structure. The permeability of the cell wall is qualitatively expressed in terms of “gram-positive” or “gram-negative” referred to in the introduction to this chapter. The cell wall of gram-positive organisms is relatively thick; whereas the cell wall of gram-negative bacteria is thin, at the same time the entire wall is more complex. Clearly these differences will impact on the vulnerability of cells to biocide treatment as a means of control. The membrane has several functions. It is generally accepted that it controls the permeability through the wall, e.g., the passage of nutrients,

28

T. Reg. Bott

which may be various mineral ions, sugars and amino acids vital for the well-being of the cell. The membrane has the ability to scavenge nutrients effectively from very dilute media and maintain a relatively constant intracellular ionic environment through a very large range of external concentrations. The semi-permeable character of the membrane allows water to pass in and out of the cell by osmosis. In addition the membrane must transport outward not only small molecular waste materials but also larger polymeric materials that are incorporated into wall polymers. In certain bacteria, this is accompanied by the excretion of ‘exoenzymes’ to assist in the assimilation of macromolecular nutrients. The availability of nutrients and the associated metabolism reactions that produce macro-molecules means that cells must grow in size and ultimately must divide. Ribosomes are present to synthesise proteins, and they are usually grouped in chains called polysomes. In many species of bacteria, the cytoplasm contains relatively large granules, composed of cellular storage materials in amounts that vary widely with the prevailing nutritional conditions. Though the nucleus is well defined and regularly occupies a predominantly central position, its shape is often quite irregular. It may be regarded as the life centre of the cell, which dictates the cell’s activity and metabolism. Mesosomes are large irregular agglomerates of the cytoplasmic membrane that are usually attached to the membrane. It is anticipated that they simply increase the membrane surface area. They may also be attached to the body of the nucleus. Extracellular polymers are produced by many microorganisms which facilitate attachment to a surface and afford a measure of protection in adverse conditions. It is possible that these polymers can interact with solid surfaces to produce ‘polymer bridging’ that assists attachment. Granular or particulate material may be randomly included during formation of the cell structure. Certain gram-positive rods (aerobes of the genus Bacillus and anaerobic Clostridia) have developed a powerful, specialised mechanism for survival in times when nutrients are not available or conditions are hostile, i.e., the formation of spores (Greek for ‘seed’). In simple terms, the spore consists of a double layer of cell membrane, which closes around a chromosome and a small amount of cytoplasm, to provide protection. These cells may be compared with the seeds of higher plants or the cysts of protozoa that are in a state of cryptobiosis (latent life) with no metabolic activity. They exhibit remarkable resistance to the lethal effect of changes to environmental factors including the effect of heat, drying, freezing and poisonous chemicals.

Biofilms

29

Some spores may be converted to vegetative cells stimulated by a suitable environment. Other spores may remain dormant unless they are first activated by some traumatic agency such as a short exposure to heat. Ageing, with its multiple ‘difficult to define’ characteristics, is probably the most important stimulus in nature. The process of germination probably depends on damage to the outer impermeable layer with consequent rupture followed by an uptake of water and a loss of solid content. It is common knowledge that some microorganisms are responsible for disease in plants or animals (including humans). They include prions and protozoa. It is quite possible that they might create problems associated with industrial biofilms.

3.3. BIOFILMS The accumulation of microorganisms on surfaces is quite a natural occurrence. It happens when suitable conditions of moisture and temperature exist with the availability of suitable nutrients and surfaces on which to become attached, along with the absence of poisons or predators. Mozes (1994) citing Rijnaarts (1994) set out in simple form, the sequence of events that lead to the formation of biofilms; starting with the transport of cells towards a hosting surface, initial adhesion, and polymer production by the cells lead to the formation of biofilms. These steps are illustrated in Fig. 3.2. All accumulations of living organic matter such as biofilms, represent an assembly of subunits usually referred to as ‘cells’. The structure is very dependent on the totality of the various factors and their interactions that contribute to establishment and growth (see Section 3.14). In general terms, the chemical composition is similar with common chemical reactions that represent the metabolic activity. The cell wall represents a barrier between

transport adhesion

polymer production

biofilm formation

Figure 3.2 Succeeding steps in microbial deposition and bioflim formation on solid surfaces. (Courtesy of Technique & Documentation.)

30

T. Reg. Bott

the cell itself and the environment in which it exists. The rigid outside wall affords some measure of protection for the cell membrane against potential chemical and physical damage but the basic characteristic of the cell wall is associated with permeability and regulates the level of nutrients, intermediate metabolites, inorganic salts and the pH within the cell. It controls the passage of macromolecules. For many species the cell wall is often rigid and determines the shape of the cell as spherical, rod like or filamentous. It has to be stated however, that some cells do not have rigid cell walls and require a way of controlling osmotic pressure brought about by differences in the concentration of soluble substances, which could damage or even destroy the cells. The presence of a rigid cell wall means in general, that the cell relies on soluble nutrients. The components making up the cell wall will of course, determine its surface qualities and ultimately determine the ability of the cells to form a biofilm, e.g., ion bonding, electrical charge with resistance to infection from viruses and immunisation. Some organisms can produce extracellular layers on the outside of the cell wall, such as slimes and mucilages, which may aid attachment to a solid surface and give protection against adverse conditions. These substances may also act as food sources, should the usual source of nutrient be curtailed for any reason. Very little if any, non-microorganism mass is involved in the formation of the slime deposit. Microorganisms that cause biofilm accumulation are usually those that are heavily–encapsulated, fast-growing bacteria. They include species such as Aerobacter, Arthrobacter, Proteus, Bacillus, Pseudomonas and Nitrobacter. The biofilm may contain many different species thereby forming a complex community that can exist in the particular environment in which it finds itself, involving for instance, temperature, pH and suitable nutrient sources. Biofilms formed under turbulent conditions tend to be dense with strong matrixes of extracellular polysaccharides. In contrast formation under laminar conditions tends to give a scattered accumulation over the surface, from which the cells are easily removed (Vieira, 1992). The presence or otherwise, of suitable nutrients in a particular environment will determine the survival of the microorganisms and their consequent growth. In general microorganisms are capable of utilising a variety of nutrients. As would be expected, the elements that are found in the make-up of microorganisms are those that are to be found in association with organic chemistry, carbon, hydrogen and oxygen, in association with elements such as nitrogen, sulphur, phosphorus and other inorganic mineral elements. The very basic needs are sources of energy, carbon and reducing

31

Biofilms

equivalents. The response to external stimuli may be different in biofilm structures as compared to the response by a single cell. Work by Keen and Prosser (1988) with Nitrobacter demonstrated that the specific growth rate of attached cells was higher than bacteria that were not attached to a surface. Microorganisms can be classified in terms of their nutritional requirements: 1. Phototrophs acquire their energy directly from the sun. 2. Chemotrophs obtain their energy from the oxidation of organic, and in some cases, from inorganic compounds. 3. Autotrophs recover their carbon requirements from simple carbon containing molecules such as CO2. 4. Hetrotrophs require fixed organic sources of carbon. 5. Lithotrophs require inorganic molecules such as H2S and ferrous iron for cell synthesis. 6. Organotrophs need organic molecules for oxidation. Microbial growth will depend on the quality of the nutrients that are available. A medium successfully used as a basis for experimentation with Pseudomonas fluorescens is presented in Table 3.1, together with glucose (5.0 g/l) as a source of carbon, It demonstrates the complexity of the nutrient requirements of microorganisms. It will be seen that in addition to the glucose the medium includes trace elements and mineral salts. Nitrogen is included as ammonium chloride but in the natural world sources of nitrogen are more likely to be amino acids, nucleotides, uric acid and urea The source of sulphur is exclusively the sulphate listed in Table 3.1, but a wide range of sulphur-containing compounds can be utilised by microorganisms. The metabolism of some microorganisms, for instance sulphur reducing bacteria, can use sulphides including hydrogen sulphide. Organic sulphur compounds may also provide a source of sulphur. The origin of phosphorous for microbial nutrition is usually from soluble inorganic compounds such as hypophosphate included Table 3.1 Medium used in the growth of Pseudomonas fluorescens Mineral Salts g/l Trace Elements 3

NaH2PO42H2O  l0 Na2 HPO4  103 K2SO4  l03 MgSO47H2O  103 Na2EDTA 2H2O 0.025  103 NH4Cl 0.024  l03

1.01 5.50 1.75 0.10 0.83 3.82

MnSO4 4H2O H2BO3 ZnSO4 7H2O Na2MoO42H2O CuSO45H2O CoCl2 6H2O

mg/l

13.30 3.00 2.00 0.24 0.025 0.024

32

T. Reg. Bott

Absorbance measure of biofilm thickness

0.16

0.12

Different surfaces

Velocity 0.5 m/s

0.08 Trace elements added

0.04

0

200

400

600 Time h

800

1000

1200

Figure 3.3 The effect of trace elements on bioflim (Pseudomonas fluorescens) development. (Courtesy of Elsevier.)

in Table 3.1. Trace elements are vital for microbial survival. A number of inorganic salts that provide trace elements are given in Table 3.1 Exclusion of these salts in the nutrient supply in some experimental work carried out by Santos (1992) on the growth of Pseudomonas fluorescens demonstrated quite clearly the essential nature of at least some of the trace elements. See Fig. 3.3. “Absorbance” refers to the absorbance of infrared radiation by the biofilm; the greater the thickness of biofilm, the greater the infrared absorbance (Bott, 2003). The following lists some of the qualities of raw water that affect its utilisation for cooling purposes including the possibility of the occurrence of biofouling; 1. Mud and silt that will be dependent on the origin of the water and the local climatic conditions e.g., rainfall and season (winter or summer). 2. Dissolved solids such as calcium salts and soluble iron, which are very dependent on the soil through which the water has percolated before collection. 3. Biological material that could be living or deaddi.e., living flora or fauna and decomposing dead material. 4. If the water is recycled through a cooling tower or spray pond, it is likely to have picked up dust particles that may be dependent on the local conditions such as grinding plant and combustion operations. Wind direction is likely to play a part in this form of contamination so that the

Biofilms

33

presence of these contaminants is likely to fluctuate and any methods of control need to be adaptable. Other factors that can influence water quality include: 1. Differences in temperature due to seasonal changes. 2. Changes in flow rate due to altered demand. 3. Changes in water treatment. 4. Changes in pH. All the factors present in a biofouling process could in some measure affect cell metabolism and hence the need to reside on a surface. In addition to the quality, the availability of nutrients will be vital to survival as a biofilm. Depending on their requirements, some cells have the ability to respond to the environment in which they exist. Some bacteria for instance, can move towards a more favourable nutrient source. Such a facility is not found in fungi or algae. It is possible for other bacteria to demonstrate the ability to move towards a greater oxygen concentration if they are aerobic in character or away from it if they are anaerobic. Some microorganisms have the incentive to move towards or away from a light source, depending on their need for light energy to allow photokinetic reactions with nutrients to take place. Yet other microorganisms respond to pressure or contact and others to temperature, so that they are capable of moving to a place where the temperature is optimum for growth. Some algae can counter the effects of gravity by producing gas vacuoles in order to resist the force of gravity, enabling them to take advantage of a source of daylight. It is probably true to say that most microorganisms have the capacity of being able to place themselves in the most favourable location for growth in the environment in which they exist, with implications for the development of biofilms. Whether in an industrial plant or in a more natural habitat, biofilms consist of communities of different organisms in a state of dynamic interaction. The composition of the biofilm in respect of the species present will probably change with time, with particular dependence on the nutrient composition and availability. Various forms of microbial dependence may be cited: 1. Mutualism. Each microorganism presents benefits in some way from the presence of other species. 2. Commensalism. One organism benefits while others do not. 3. Amensalism. One organism benefits at the expense of others, by imposing a change on the local environment. 4. Parasitism. One organism lives off others in some way.

34

T. Reg. Bott

5. Predation. One organism consumes another. 6. Neutralism. All species live together without interaction. In general, a biofilm will have water content of the order of 85–95%. Specific differences between fungi, algae and bacteria biofilms are likely to be important in the consideration of different industrial operations for control.

3.3.1. Fungi Fungi include yeasts and moulds and are ‘plant-like’ in character. Most grow by extension and branching. Furthermore, they are immobile and the cell wall is similar to that of plants. There exists two forms; yeasts that are single cells, oval or spherical, 3-5mm in diameter; and moulds that are filamentous, multi-cellular colonies, with branched tubular structures 2-10mm in diameter. The cell wall of a fungus, like a bacterium, lies immediately external to the cytoplasmic membrane. In some yeasts it is surrounded by an external capsular polysaccharide. Sometimes yeast cells and their progeny adhere to each other and form chains. The principal element of the growing form of a mould is the hypha. As an accumulation of mould cells grows, the hyphae form an intertwined strand. Hyphae grow by lengthening at their tips and the production of side branches. The hyphae penetrate into the medium or substrate, where they absorb the available nutrients. Most colonies grow at the surface of a solid or liquid, as an irregular dry, filamentous mat. Because of the interlocking of the filamentous hyphae, the colony is difficult to dislodge. Inside the mat, it is likely that the cells are dead due to the depletion of nutrients and oxygen, and the accumulation of waste products. Fungi, unlike algae, lack chlorophyll. All fungi are heterotrophic in metabolism; they grow on organic substrates that are found in the natural environment, in or on plants and animals. Some fungi can hydrolyze even the most complex organic substances including wood, bone and wax. As a result, they are able to attack wooden structures in contact with water, which was a serious problem when cooling tower packing was generally fabricated from wood. They continue to be a problem in paper manufacture by causing ‘spotting’ of the product. Fungal growth is usually external to the bulk water associated with bacterial biofilms. Suitable growth conditions involve pH in the range 2–8 (though neutral is preferred) and a temperature generally in the range 0–30  C. Some characteristics of typical fungi are given in the following Table 3.2 taken from ‘Principles of Industrial Water Treatment’ (Drew, 1994).

Biofilms

Table 3.2 Examples of fungal species. Type fungi Examples

Filamentous mould

Yeast-like Basidiomycetes

Aspergillus Penicillium Mucor Fusarium Alternaria Torula Saccharomyces Poria Lenzites

35

Characteristics

Black, tan, blue Yellow, green White, grey Brown, tan Pink, brown Leathery or rubbery Usually pigmented White or brown

3.3.2. Algae Algae are larger than bacteria and have a more complex structure. Their existence may be in the form of single cells or colonies in strands with a filamentous structure that may be partially attached to a solid surface, allowing oscillatory movement within a flowing stream. In this way, the acquisition of nutrients is facilitated. Provided there is access to sunlight and moist conditions, algal biofilms can develop on solid surfaces. As a result, they can cause serious blockage problems in open, cooling water circuits. These microorganisms can be found submerged or floating in water, or exposed on surfaces, provided there is moisture in the surrounding atmosphere. Algae contain chlorophyll and in the presence of light, by a process of photosynthesis, produce carbohydrates from carbon dioxide and water. Restriction of any of these requirements will reduce their growth potential. Under ideal conditions with no restrictions, extensive and prolific blooms of algae can be produced. Large accumulations of algae (living or dead) on metal surfaces can facilitate corrosion. There are three basic types of algae and they are diatoms, green and bluegreen algae. Diatoms are unicellular and characterised by the presence of an elaborate silica frustule (shell). A pigment that gives rise to a golden-brown appearance can mask the basic green colour. Green algae exist as multicellular entities or multicellular colonies. The blue-green strains are generally filamentous with cells submerged in chains. Blue-green algae can extract and react with nitrogen from the air to produce nitrogen compounds. All types of algae can release oxygen, which can encourage the growth of aerobic microorganisms. The biofouling problems with algae are principally associated with water intakes and the distribution and discharge back to source, of water used for

36

T. Reg. Bott

cooling purposes or for human consumption after suitable purification. The formation of large colonies of algae in the region of the water intake from which it is possible for mats of algae to break away, can cause extensive blockage of the filter system. In salt water sources, the problems can be exasperated by the presence of sponges and barnacles.

3.3.3. Bacteria Bacteria are generally the microorganisms that reside on surfaces in industrial operations, usually carried out in closed equipment fabricated from a metal of some sort. Where there is no light algae cannot thrive and the surfaces do not encourage the growth of fungi that require an organic substrate. In natural and industrial environments bacteria have a great ability to adapt to different conditions and furthermore, many of the different species produce exopolymers that facilitate their attachment to a solid surface. The advantages of residing on a surface (sessile) as opposed to having a free swimming existence (planktonic) can be readily appreciated, although difficult to prove experimentally. A planktonic organism only has access to the nutrients in close proximity, where the movement of nutrients to the cell is likely to be by Brownian movement. On the other hand, cells residing on a surface in a flowing water system have nutrients brought to them through turbulence-created mass transfer towards the biofilm/water interface (see Chapter 2). The nutrients are able to diffuse through the biofilm towards the regions nearer the solid surface, with the potential to sustain the viability of the biofilm. Furthermore, it would appear also that attachment to a surface would offer some protection from detrimental pH fluctuations and biocide concentration shock. Kemmer (1988) listed some typical bacteria in relation to their different activities and useful background information has also been provided by the Ashland Chemical Co. (1997). Examples of the species and the potential problems that are caused by bacteria, are: 1. Slime-forming: (e.g., Aerobacter and Pseudomonas) form a dense sticky slime with subsequent biofouling. Water flows can be impeded and other microorganism growth promoted. 2. Spore-forming: (e.g., Bacillus subtilis and Bacillus mycoides) become inert when their environment becomes hostile in some way but growth reoccurs whenever the environment becomes suitable again; difficult to control completely. Most industrial processes are not affected when the organism is in the spore form.

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3. Aerobic sulphur bacteria: (e.g., Thiobacillus) can oxidise sulphur and sulphides to sulphates and sulphuric acid, The consequent fall in pH can give rise to corrosion problems in process plant. 4. Iron bacteria: (e.g., Gallionella) utilise iron in their metabolism with the production of ferric hydroxide that may interfere with certain industrial processes. Iron-depositing bacteria for instance, cause the oxidation and subsequent deposition of insoluble iron from soluble iron. 5. Anaerobic Sulphate reducing: (e.g., Desulfovibrio desulfuricans) Generate sulphides from sulphates and can cause serious localised corrosion. 6. Nitrifying: (e.g. Nitrosomonas generate nitric acid from ammonia.) Contamination can cause serious corrosion. 7. Corrosive: (e.g. Clostridium aceticum) Create localised, corrosive environments by the secretion of corrosive wastes. Bacteria may be classified in several different ways related to shape, nutritional requirements and optimum growth temperature. The simplest classification is probably based on shape as in Table 3.3. It is usual to divide bacteria rather arbitrarily in terms of their preferred temperature range for growth, as defined in Table 3.4, to provide an alternative method of classification. The need for oxygen (or air) or not, is also a way of differentiating between different species of bacteria as in Table 3.5. Nutritional requirements may also provide a basis for classification as demonstrated by Table 3.6.

Table 3.3 Classification based on shape Shape Distinguishing Name

Rod Sphere Curved

Bacillus Coccus Spirillum

Table 3.4 Classification based on optimum growth temperature Optimum temperature Definition range  C

Mesophiles Psychrophiles Thermophiles

35e45 45e50 50e55

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Table 3.5 Classification based on oxygen requirement Oxygen Requirement

Oxygen required Small availability of oxygen required Can live with or without oxygen Oxygen must be absent

Table 3.6 Classification based on nutritional requirements Nutritional Requirements

Distinguishing Name

Aerobe Microaerobe Facultative aerobe Anaerobe

Distinguishing Name

Energy derived from oxidation of inorganic compounds Energy derived from oxidation of organic compounds Nutrition obtained from living organic matter Nutrition obtained from dead or decaying organic matter

Autotrophic Heterotrophic Parasitic Saprophytic

Although bacteria are small in size there is a large variation in that small size, in addition to variation in shape. Among the smallest are the spherical mycoplasmas species with a diameter of around 0.125 mm, whereas one of the largest is the rod-like Bacillus megaterium with dimensions of 1.35–3.0 mm. A feature of the growth of bacterial biofilms is the production of extracellular materials that surround the cells and constitute a major proportion of the biofilm. As a result, a complex matrix of cells in amongst strands of different polymers, usually containing polysaccharides and/or polypeptides. The term that is usually applied to define this accumulation is ‘extra polysaccharides’ or ‘extra polymeric substances’ (EPS). Cooksey (1992) however, suggested that there may be other substances present in biofilms originating from other microorganisms, in addition to bacteria. It is generally agreed that the ‘glue-like’ character of EPS helps the biofilm to stick to a surface and it is also likely to assist the retention of particulate matter such as mineral solids and corrosion products present in the water containing the colonising bacteria. In addition, the presence of EPS is likely to afford a measure of protection for the embedded cells from predators, biocides and from removal forces that may be present.

3.4. REQUIREMENTS FOR MICROBIAL GROWTH The discussion so far has indicated that the growth of microorganisms depends on physical conditions and the availability of suitable nutrients in

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the environment in which they reside. As with all living things, the temperature in which microorganisms exist is fundamental to growth. Most bacteria for instance, grow over a relatively narrow range around 30  C. In general, this narrow range is, depending on the species, in the region of 25–45  C. The range is even narrower where growth is regarded as substantial. It is usual to divide bacteria rather arbitrarily in terms of their preferred temperature range for growth, as defined in Table 3.4. The metabolism and growth of microorganisms are very dependent on the quality and components of the water in which they exist including dissolved and particulate matter and particularly, its pH. The pH range of tolerance is around neutral, i.e., pH ¼ 5–8. In addition, there is likely to be some dependence on the quality of the nutrients. The pH of the bulk water not only affects microbial physiological processes but also affects the physiochemical properties of the water in relation to the solid surface (See Section 3.6). The effect of pH on the adhesion of Pseudomonas fluorescens has been studied by Chen, Zhang, and Bott (2005). The mean adhesive strength for three different pH values of 5.5, 7.0 and 8.5 (i.e., around neutral pH), at three different water flow velocities of 0.6, 1.0 and 1.6 m/s are presented in Fig. 3.4. It shows the mean adhesive strength of biofilms under various conditions over a period of 10–20 days. It would appear from the data obtained, that for a given water velocity, the adhesive strength was little affected by pH change over the range studied. However, it is evident from Fig. 3.5 that the mean biofilm thickness was greatly influenced by pH. The maximum thickness, as might be expected, occurred at a pH of 7.0. It is likely that the internal pH of the biofilm will be different from the bulk water flowing across it. The data confirmed earlier studies that maximum biofilm thickness of Pseudomonas fluorescens occurred at pH 7. The biofilm accumulation at the acid conditions of pH ¼ 5.5 appeared more suppressed than at the alkaline conditions of pH ¼ 8.5. Vaisanen, Nurmiaho-lassila, Marmo, and Salkinoja-Salomen (1994) and Vrom et al (1999) suggest that indeed the pH profile within biofilms, is very different from the surrounding water. The research of Chen, Zhang, and Bott (2005) demonstrated that at a pH of 8.5, the biofilm had a loose structure with more filamentation compared with biofilms grown at pH 7.0 and at acid conditions at pH 5.5. A rather patchy biofilm slowly developed under the acid conditions, whereas at neutral pH conditions a rather thick uniform biofilm was produced. It was also observed that a difference in water velocity seemed to

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1.4 0.6 m/s 1.0 m/s 1.6 m/s

Adhesive strength (J/m2)

1.2

1.0

0.8

0.6

0.4

0.2

0.0 5.0

5.5

6.0

6.5

7.0 pH

7.5

8.0

8.5

9.0

Figure 3.4 The influences of pH of bulk liquid on the adhesive strength of bioflim under different fluid velocities. Error bars indicate the standard error of the mean. (Courtesy of Elsevier.)

0.7 pH - 5.5 pH - 7.0 pH - 8.5

Mean biofilm absorbance

0.6 0.5 0.4 0.3 0.2 0.1 0.0 0

3

6

9 12 Time (day)

15

18

21

Figure 3.5 The influence of pH of bulk liquid on the growth profile of bioflim developed under a fluid velocity of 1.6 m/s. (Courtesy of Elsevier.)

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have more influence on the viability of the biofilm than the other environmental factors. This phenomenon could be due to the profound changes in mass transfer brought about by the changes in velocity.

3.4.1. Nutrient Requirements The basic nutrient requirements may be summarised as: 1. Water. The metabolism and growth of microorganisms are very dependent on the presence of ambient water. The range and tolerance of pH as already discussed, is around neutral i.e. 5–8. Furthermore, there will be some dependence on the quality of the nutrients associated with the particular water in which they exist. 2. Oxygen. Two classifications of microorganisms in respect of oxygen have been mentioneddthe term ‘aerobic’ identifies microorganisms that require oxygen for their metabolic processes and ‘anaerobic’ microorganisms are not dependent on the presence of oxygen. 3. Carbon dioxide. Carbon dioxide is a product of microbial activity and therefore forms a component of the environment in which microorganisms exist. 4. Inorganic ions These are essential nutrients and include PO4, Kþ, Mgþþ, together with sources of nitrogen and sulphur. Trace elements that are required depending on the species include; iron, calcium, potassium, sodium, molybdenum and nitrogen.

3.4.2. Metabolism The physiology and metabolic activity may change through the biofilm as Mozes and Rouxhet (1992) suggested. Three regions could be distinguished, although there were no definite lines of demarcation between the zones: 1. The layer of cells actually attached to the surface. 2. The bulk of the biofilm. 3. The layer of cells in direct contact with the flowing water. The activity of the cells in each of these regions, will be affected by the local mass transfer of nutrients, including oxygen for aerobic cells that is directly dependent on the bulk flow conditions, the concentration driving force and the mass transfer resistance (See Chapter 2) from the bulk water and that offered by the biofilm itself. The change in concentration of

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Turbulent

Distance

flow

Viscous sub layer Biofilm Metal surface

Nutrient concentration

Figure 3.6 Idealised interfacial diffusion after a period of time. (Courtesy of Elsevier.)

nutrients between the bulk water and the biofilm/solid interface is schematically illustrated on Fig. 3.6. The amount of nutrients including oxygen (and further organisms) at the surface of a growing biofilm will depend on the flow conditions as indicated in Chapter 2. Of great influence is the resistance to mass transfer from the flowing liquid phase to the interface between the solid surface and the growing biofilm and through the biofilm itself. The transport of nutrients through a biofilm towards the microorganisms closer to the solid surface will depend on the ‘porosity’ of the existing biofilm and the concentration gradient across the biofilm. The latter will depend on the concentration at the biofilm/water interface, itself affected by the mass transfer from the bulk flow. In addition, the concentration gradient through the biofilm will be affected with the consumption of nutrients by the organisms contained in the biofilm, which will in turn, be affected by the biofilm thickness. The degree to which there is nutrient penetration through the biofilm will affect its viability, remote from the water flow and close to the solid surface. If for instance, the oxygen level reaches zero at a point within the biofilm as a result of consumption by aerobic microorganisms, the growth of anaerobic species may be encouraged. For species that can exist as aerobic or anaerobic entities, a change in activity is made. Fletcher (1992) suggested that bacteria attached to surfaces appear to have a different metabolic activity compared to their free swimming counterparts. She suggested two reasons for this difference:

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1. The physiochemical conditions at the solid surface/liquid interface are not identical to those in the bulk water phase due to the different local environmental conditions. 2. Biofilms usually contain more than one species of microorganism. In addition, under natural conditions, a biofilm may contain fungi and algae in addition to bacteria and may even have macroorganisms in close proximity. As a result, there could be a range of effects due to the proximity of different organisms such as: (a) Symbiosisda mutually beneficial partnership between organisms of different kinds, where the waste products from one species is a suitable nutrient for another species. (b) The utilisation of less readily degradable substances by specialised organisms, e.g., the breakdown of cellulose. (c) Competitiondrivalry between different species of organism for the nutrients that are available. (d) Antagonismdinterference in growth between organisms. There may also be predators grazing on the biofilm. Within the biofilm, ecological niches may be created e.g., anaerobic zones beneath regions where oxygen is readily available. In mature biofilms, it is more than likely that the nutrient level at the biofilm/surface interface is very low or even non-existent. The result may be death of the cells close to the surface, which might affect the stability of the biofilm. Much will depend on the quality of the structure and content (e.g., particle inclusion) of the biofilm, which in turn, will depend on the rate at which the biofilm originally developed. Where there is a range of microorganisms, the concentration gradients between the water/biofilm interface and the solid surface/biofilm interface of oxygen, essential inorganic and trace nutrients, and dissolved organic carbon may produce ‘cross-feeding’ by waste products and cell death (lysis). In summary, it is probably true to state that the cells within the bulk of a biofilm have the opportunity to survive and reproduce. The cells in the outer layers are vulnerable to the effects of the shear stress forces of removal, acting on those close to the water flow across the biofilm. As already suggested, the cells near the solid surface may be seriously affected by lack of nutrient availability. Accumulations of cells forming microcolonies on a surface, generally surround themselves with complex extracellular matrix structures principally comprised of polymeric material.

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3.4.3. Matrix Formation Presumably because of the extremely complex and often unique, nature of biofilms and the scientific difficulty of establishing chemical detail, there is some confusion over the identification of the material forming the extracellular matrix that constitutes a major proportion of a biofilm structure. A paper by Allison (2003) has clarified matrix terminology. The principal feature of extracellular material forming the matrix is that it consists of biosynthetic microbial polymers lying outside the integral cellsurface components of the bacteria within the biofilm. These extracellular biopolymers include exopolysaccharides, nucleic acids, proteins, glycoproteins and phospholipids. Allison (2003) pointed out that since glycoproteins were identified as being a common component, the term ‘glycocalyx’ has been used to describe the gelatinous mass surrounding attached cells. The word ‘glycocalyx’ could suggest a definite structure, which is not appropriate to describe microbial systems where the extracellular biopolymers are subject to change. It is suggested that ‘biofilm matrix’ provides a better description implying a multi-component, dynamic, heterogeneous system. The abbreviation EPS (exopolysaccharide) has been used to describe exopolysaccharides, exopolymers and extracellular polymeric substances (Wingender, New, & Fleming, 1999). Because of the complex nature of the biofilm dependent on the microorganism present, nutrients available and the effects of operating conditions, the composition of the extracellular matrix is likely to be variable. Allison (2003) presented data on composition reproduced as Table 3.7 and at the same time pointed out that a detailed chemical description of a biofilm matrix is only accurate at the point at which it was measured, since its composition is far from uniform. Table 3.7 General composition of biofilm matrices Component Matrix%

Cells Water Biosynthetic microbial polymers EPS Nucleic acids Proteins (including enzymes and regulatory proteins) Glycoproteins Phospholipids Adsobed species

2e5 c.98 1e2 1e2 1e2

1e2 1e2 ?

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45

Whitfield (1988) stated that bacterial extracellular polymeric material occurs in two forms: 1. A capsule that is intimately associated with the cell surface 2. A slime that is only loosely bound to the cell wall but covers the solid host surface between the cell clusters. A1lison and Sutherland (1987) suggest that polymers are not directly involved with the initial attachment process but principally in the subsequent development of the biofilm. The initiation of its production will depend on the operating conditions and the microbial species present. Of importance to the production of EPS, is the presence of cations particularly Mgþþ and Caþ. It is interesting in this context, that Abu, Weiner, Rice and Colwell (1991) discovered that a marine bacterium associated with oyster beds (Shewanella colwelliana), produces an unusual extracellular polymer that facilitates irreversible adhesion to surfaces.

3.5. THE IMPORTANCE OF SURFACES In order for a biofilm to be formed, clearly a surface to which the microorganisms can attach is required. It is probably true to say that bacteria can attach to the many different surfaces with which they come into contact. The fact that bacterial microorganisms in the biosphere prefer attachment to a surface rather than ‘free-swimming’, is clearly the reason for microbial attachment to man-made structures and equipment in contact with water i.e., biofouling. The advantages can be readily perceived, particularly where there is a flow of water across the colonised surface, including a likely continuous supply of nutrients and the continuous removal of waste products resulting from the microbial metabolism. In respect of aerobic bacteria that require oxygen, the continuous movement of flowing water, which is more than likely to have an interface somewhere with the atmosphere, will be at least, near-saturated with oxygen. These advantages for sessile microorganisms may be compared with the situation in respect of suspended (planktonic) microorganisms, where the bacteria are carried in a flowing stream. They only have access to nutrients in their proximity, the availability largely being dependent on Brownian movement. Flemming (1991) has provided a more detailed list of ecological opportunities for microorganisms residing on a surface. Apart from the continuous availability of nutrients, the biofilm provides protection from short term pH fluctuations and shock exposure to biocides and other detrimental substances. The compactness of the biofilm itself offers

46

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resistance to the effects of shear forces that result from the flowing water. The continuous contact with flowing water also prevents dehydration. Further advantages accrue in the possibility of symbiosis with other living matter and the creation of ecological niches that provide anaerobic regions below aerobic species. Breakdown products as result of microbial activity within a biofilm, may provide nutrients for other species. The degree of activity of the cells in a biofilm attached to a surface will be affected by mass transfer of nutrients from the bulk water through the viscous sub-layer and the passages and voids in the biofilm itself (see Chapter 2). In very general terms, three regions can be visualised within a biofilm: 1. The primary layer that is attached to the solid surface. 2. The bulk of the biofilm. 3. The surface layer that is in direct contact with flowing water. This ideal concept is useful in providing an appreciation of factors that could influence activity and stability of a biofilm, whereas an actual biofilm is far more complex. The microbial activity in each of these regions will be influenced by differences in nutrient availability, by physicochemical interactions with the solid surface and the forces applied to the biofilm due to the liquid flow external to the biofilm. In many respects, it could be argued that each biofilm is a unique entity. Mozes and Rouxhet (1992) made pertinent observations on how solid surfaces may affect the attached cells through a number of different mechanisms: 1. Solid surfaces may initiate an internal physiological response that will affect cell morphology and consequent behaviour. 2. Surface and mass transfer limitations could create an environment where local concentrations of ions, small molecules or polymers are different from the bulk concentrations in the flowing water. The chemical species concerned may be substrates for cells or toxic agents. The effect may lead to changes in cell morphology and activity: 1. Numbers of appendages e.g. flagella, due to restrictions to movement. 2. Dewatering due to restricted nutrient availability. 3. Gliding motility due to the presence of the solid surface.

3.6. ADHESION OF MICROBIAL CELLS TO SURFACES The attachment of microorganisms might at first sight, be thought of as being related to two smooth surfaces: the surface of the microorganism itself

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and the solid surface it encounters. However the adhesion of microorganisms to a surface is clearly a complex phenomenon being the result of interaction between physical and chemical phenomena. Fletcher (1992) in connection with bacterial attachment to surfaces under static rather than flowing conditions, listed the factors that influence adhesion. She stated that the numbers of bacteria that attach to a surface and the rate of attachment depend on the species and strains of the bacteria and on the nutritional status, particularly whether they are starved of specific nutrients. Attachment will also depend on: 1. Environmental conditions, which include the flux, concentration and source of nutrients. 2. Electrolyte concentration. 3. pH 4. Temperature. It is interesting to note that some microorganisms at least, produce extracellular polymers that facilitate adhesion to surfaces. Abu, Weiner, Rice, and Colwell (1991) reported that Shewanella colwelliana, a marine bacterium that grows in association with oysters, produces an unusual extracellular polymer, which mediates irreversible adhesion to surfaces. There are two further important variables concerning the solid/liquid interface that affect the metabolism of the microorganisms in a biofilm. They are the prevailing hydrodynamic conditions at the interface that are very different from the conditions in the bulk fluid (as described in Chapter 2) and the interaction of dissolved solutes and particulate matter with the receptive surface. Denyer, Hanlon, and Davies (1993) pointed out that two forms of interaction between microorganisms and surfaces may be required i.e. reversible and irreversible (See Table 3.8). Table 3.8 Forces involved in microbial adhesion to surfaces. Type of interaction Forces involved

Reversible Long range, weak Low specificity Irreversible Short range High specificity

van der Waals electrostatic Dipole-dipole Dipole-induced dipole Ion-dipole Ionic Hydrogen bonds Hydrophobic

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The complexity of the mechanism of adhesion was indicated by Chen, Zhang and Bott (2005) who studied the effects of some parameters. Using a novel micromanipulation technique they were able to demonstrate the effect of different conditions on the adhesive strength of Pseudomonas fluorescens. The results confirm the concept that the adhesive strength depends on the conditions under which the biofilm was established. The adhesive strength of a biofilm may be defined as the work required per unit area of surface to which it is attached, to remove the biofilm. Table 3.9 based on results from the experiments carried by Chen, Zhang, and Bott (2005) provided data on the adhesion of Pseudomonas fluorescens, by changing the nutrient availability in terms of the glucose concentration at three different water velocities across the receiving surface. Table 3.9 Adhesive strength: at different nutrient availabilities and water velocities. Velocity m/s Glucose Concentration mg/l Adhesive Strength J/m2

0.6

1.0

1.6

15 30 45 15 30 45 15 30 45

0.3 0.6 0.07 0.28 0.79 0.28 0.51 1.00 0.30

The results indicate that adhesive strength initially increases at all velocities as the glucose concentration increases up to 30 mg/1; but a further increase to 45 mg/l appears to reduce the adhesive strength. It is difficult to provide a precise explanation for this phenomenon but it is possible that higher glucose availability leads to the formation of a more open and therefore a weaker structure. It is interesting to note that there was only a modest increase of about 10% in the mean adhesive strength over the fluid velocities studied (06, 1.0 and 1.6 m/s) and as the cell concentration was raised from 4  l06 to 6  106 cells/ml. The effect may be due to the ‘blocking’ of the surface by attached bacteria, resulting from the increased mass transfer towards the surface at the higher concentration. Over the relatively small pH range of 5.5–8.5 in the circulating water, an increase in bulk water velocity over the range 0.6–1.6 m/s, (as demonstrated in Fig. 3.4), produced an increase in adhesive strength of the biofilm that may be attributed to a greater compactness of the biofilm

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1.0 0.6 m/s 1.0 m/s 1.6 m/s

Adhesive strength (J/m2)

0.8

0.6

0.4

0.2

0.0 0

3

6

9

12 15 18 21 Biofilm age (day)

24

27

30

Figure 3.7 The adhesive strength of bioflims developed under different fluid velocities vs. their age. Error bars indicate the standard error of the mean. (Courtesy of Elsevier.)

produced under the higher velocities. The age of the biofilm is likely also, to affect compactness and associated adhesive strength as indicated in Fig. 3.7. Roughness of the surface will also facilitate adhesion (see Section 3.8) but data on the effects of roughness tend to be empirical and extremely difficult to explain from a theological standpoint.

3.7. INTERACTING FORCES Although microorganisms in suspension have been described simply as ‘living colloids’ by Marshall (1976), and Daniels (1980) described them as macroscopic ions having a multiplicity of electrostatically charged surfaces, they remain complex structures. Nevertheless microorganisms can be influenced to a lesser or greater extent by the interactions experienced by conventional colloid particles in suspension, close to a collecting surface. The interaction of solid particles that may be regarded as representative of microorganisms and the surface that is being colonised, can fall into a number of different groups of forces (Bott, 1995): 1. Long-range forces determine whether or not microorganisms are able to approach a solid surface to interact with it; once they are in

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proximity to the surface, it is possible for short-range forces to come into play. 2. The forces of adhesion include van der Waals, electrostatic forces and hydrophobic interactions. Hydrophobic interactions are of polar origin and may be very much larger than the van der Waals or electrostatic forces. In addition, chemical bonding is possible once contact has been made, including ionic, covalent and hydrogen bonding. 3. Long range attractive forces are responsible for bringing the particles to the surface that allows the colonisation to begin and continue to take place. The forces involved may include the van der Waals forces and electrostatic and magnetic forces. 4. Bridging effects may result from mutual diffusion, an effect that might be regarded as an alloying process between the substance of the surface and the particle. Liquid/solid bridging may be involved at the interface that facilitates the involvement of capillary forces. Once intimate contact between the particle and the surface has been attained through the combination of long-range forces and bridging effects, it is possible for short-range forces that could include chemical bonding and hydrogen bonding, to take effect.

3.7.1. van der Waals forces The van der Waals forces result from the polarisation of atoms and molecules in a material that exhibits dipoles and quadrapoles, which may occur naturally or by induced polarity. Other van der Waals non-polar forces may be referred to as the London van der Waals forces in recognition that London (1930) associated these forces with the production of optical dispersion. These dispersion forces will have a significant effect on the intermolecular forces but where there is little opportunity to polarise and the dipole moment is large, the effect is negligible. In order to better understand the arrival and retention of particles on surfaces, it is usual to consider them in the simple terms of a solid sphere residing on a surface. Krupp (1967) and Hamaker (1937) extended the London concept to the interaction between atoms and molecules, allowing the attraction between the layer bodies to be estimated by a process of integration. It involves the physical and chemical nature of the interacting bodies. For a sphere close to a flat plate (Fig. 3.8): Fw ¼

AH r 6x2

(3.1)

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Sphere

r

X Plate

Figure 3.8 Sphere resting on a flat plate. (Courtesy of Elsevier.)

where Fw is the van der Waals force; r is the radius of the sphere; x is the distance separating the sphere from the plate; AH is the Hamaker constant. The Hamaker approach however, is considered to be too simplistic and therefore inaccurate; the reason being that the interactions between molecules at close range, screen the effects on molecules with larger separations. For this reason, a layer of material resting on a surface can influence the interaction occurring between two separate macroscopic bodies within range. It would be expected that surface layers or microscopic bodies would affect their interaction due to the properties of the layer rather than that of the underlying material. In general, solid materials immersed in an aqueous medium often acquire a surface charge, which arises from the adsorption of specific ions in the medium or the dissociation of molecules or surface groupings. The pH of the aqueous medium will have a strong influence on this process, particularly in biological adhesion to surfaces (Oliveira 1992). At usual pH conditions in a natural environment, bacteria possess a net negative charge. A charged surface in an aqueous system containing charged particles or ions will change their distribution. Particles of similar charge (co-ions) near the surface will be repelled away from the surface, while those of an opposite charge will be attracted to the surface. The overall effect of combination and redistribution in conjunction with Brownian motion gives rise to the Poisson–Boltzmann distribution of ions and particles throughout the extent of the aqueous phase. Fig. 3.9 is a simplified illustration of the effects in relation to a charged solid sphere immersed in an aqueous medium, in close proximity to a charged solid surface. In ‘real’ systems in nature and in processing equipment, there will be considerable interaction.

3.7.2. DLVO and Wettability Theories The theory developed by Derjauin and Landau (1941) and Verwey and Overbeck (1948) in conjunction with the wettability theory has been used to

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solid surface

diffuse layer

Figure 3.9 Electrostatic double–layer around a flat surface and a spherical particle. (Courtesy of Kluwer Academic Publishers.)

explain the attachment of particles on surfaces that can be interpreted in terms of biofilm development. The net force of interaction arises from a balance of the van der Waals forces and the forces attributed to electrostatic double layers. The majority of solids immersed in an aqueous fluid acquire an electrical charge as indicated on Fig. 3.9. This charge is often due to the pH of the solution. At pH near neutral, most bacteria possess a net negative charge; in the majority of examples only the van der Waals forces are attractive. Fig. 3.10 provides a profile of the total potential energy of interaction. Attraction occurs at the primary minimum, whose adhesion is deemed to be irreversible at a small distance of separation. At a larger distance of separation at the secondary minimum, the adhesion is reversible. Once reversible adhesion has been established other forces, such as polymer bridging, can reinforce the adhesion. It is unlikely that individual microorganisms can approach the substratum close enough to overcome the repulsive barrier that exists between the primary and secondary minima. Oliveira (1992) reported experimental evidence that other forces act in short or intermediate distances, being attractive or repulsive, which can play an important role in microbial adhesion to surfaces. They include: 1. Hydrophobic interactions. These interactions can be attractive or repulsive in character being up to two orders of magnitude higher than the forces based on the DLVO theory. 2. Steric forces These forces are considered to be due to the effects between polymer coated surfaces. It is anticipated that the forces may be relevant in systems

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Potential Energy of Interaction (VT)

Energy barrier

distance (d) Secondary minimum

Primary minimum

Figure 3.10 Total potential energy profile according to DLVO theory. (Courtesy of Kluwer Academic Publishers.)

of microorganisms where macromolecules such as glycoproteins and polysaccharides are present. The adhesion of these macromolecules to solid surfaces represents a conditioning layer (see Section 3.7). 3. Surface wettability. Initially at least, biofilm formation is usually in connection with microbial suspension in water. The degree of wettability of a surface is variable, depending on the physicochemical nature of the surface. It can be high or low and leads to definitions of “hydrophobic” or “hydrophilic” surfaces, i.e., low or high wettability respectively. It would be expected that wettability would influence biofilm formation but the effects are difficult to quantify and this may be due to the complex character of microbial cells. Ionic bonding may also play a part in the attachment of microbial cells to a surface, although the mechanism is not fully understood. It could possibly depend on some sort of bridging effect. Furthermore, as discussed earlier, many microbial cells have surface appendages that must influence the bonding process occurring between the cell itself and the surface. The production of extra cellular polymers may also aid attachment through interaction with the solid surface to provide ‘polymer bridging’. According to the DLVO theory the total potential energy of interaction VT is the sum of the energy due to van der Waals interactions VW and the energy arising from double-layer interactions VD, i.e.,

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VT ¼ VW þ VD

(3.2)

Potential Energy of Interaction (VT)

The repulsive forces are conventionally given a positive sign and the attractive forces a negative sign. It is therefore possible to think in terms of the ‘height’ of an energy barrier and the ‘depth’ of a minimum energy. Fig. 3.11 indicates the effects of the interaction where only the van der Waals forces are attractive.

energy barrier

distance (d) secondary minimum

primary minimum

Figure 3.11 Total potential energy profile considering the effect of Born repulsion. (Courtesy of Kluwer Academic Publishers.)

The interactive bodies attain maximum stability at the primary minimum. The existence of two energy minima is possible, which enables an explanation of the reversible and irreversible adhesions to be made. Where reversible adhesion is present in a system, stabilisation occurs at the secondary minimum. Under this condition particles such as microorganisms are still capable of Brownian motion with the possibility of removal by washing. Irreversible adhesion occurs at the primary minimum and simple washing is not capable of removing deposited materials. Mozes (1994) gives an indication of the active distance involved with the different forces in the deposition process, by the thickness of the lines on Fig. 3.12. Although the DLVO theory is effective in the description of conditions pertaining to the behaviour of colloid particles, its application to the accumulation of microorganisms on surfaces is not so reliable, due essentially, to the complex and variable quality of living cells. For simplicity, the DLVO theory

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conditioning film

Chemical Interactions

Al3+

Fe3+ Cu2+ Mg++

polymers

support

receptor fibril adhesion receptor

Van der Waals forces (attractive) electrical forces (generally repulsive) H-bonding, solvation

5

10

15

20

distance (nm)

Figure 3.12 Cells approaching a surface. (Courtesy of Technique & Documentation.)

assumes spherical particles but as the discussion has revealed, microorganisms are generally not spherical and do not have smooth surfaces. Often there are appendages on the cell surface (see Sections 3.1 and 3.2). The quality of the aqueous environment such as pH may influence the force distribution and hence the strength of the attachment. The DLVO approach assumes movement due to Brownian motion but some microorganisms are capable of ‘swimming’ with random or even directed movement. In addition, industrial surfaces are not smooth with variable roughness. Attachment is also influenced by adsorbed material on the solid surface as discussed in the following Section. Furthermore Mozes (1994) points out that living cells are entities that contain coded informationdthey produce and consume energy, they can ‘read’ various signals and respond to stimuli and they are continually changing systems.

3.8. THE INFLUENCE OF ADSORBED LAYERS Chamberlain (1992) drew attention to the large amount of evidence in support of the concept that the adherence of microorganisms to a surface,

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(notably bacteria) in an aqueous environment, is facilitated by the presence of an adsorbed layer on the receptive surface. It is generally accepted that this layer is organic in character. When a clean solid surface first comes into contact with for instance, water from natural sources, such as a river, a lake or the sea containing low concentrations of organic macromolecules, an organic monolayer is adsorbed that changes the nature of the surface in contact with the water. The adsorbed material becomes a conditioning layer by its modification of the surface wettability, i.e., whether it is either hydrophilic or hydrophobic. It is suggested that the tendency is to bring the surface to a narrow range of hydrophilic values. It would appear that in natural systems the conditioning film is characterised as primarily glycoproteins, protoglycans and humic residues (Baier, 1980). Grant (1999) considered that there was no evidence to support the concept that microorganisms can only attach to surfaces that have been conditioned. Cooksey (1981) suggested that the diatom Amphora coffeaeformis may not need a conditioning layer to attach to a surface. Fletcher (1976) on the other hand, found that adsorbed layers of albumen, gelatine, fibrinogen and pepsin, actually prevented the adhesion of a marine Pseudomonas to polystyrene. Chamberlain (1992) also pointed out that the onset of the adsorbed layer formation is rapid compared to the arrival of bacteria. In general these adsorbed layers are made up of organic macromolecules but may also include inorganic particles, depending on the origin of the water in which the microorganisms exist and in contact with the receptive surface. In fresh water it is likely that the adsorbed layer will include humic acids and complex polysaccharides. In water of marine origin the compounds that aid attachment will be different, comprising of complex condensation molecules with polysaccharides, peptides and lipid moieties that are aliphatic in character (Chamberlain and van Woerkom, 1986). Railkin (2004) suggested that the attachment of E. Coli for instance, is facilitated by the presence of sugars, galactose, glucose and vibose and the amino acids seine, aspartate and glutamate. It is quite possible that the nature of the adsorbed layer attracts microorganisms through chemotaxis to the surface, thereby facilitating the colonisation process. However it is difficult to distinguish between chemotaxis and random colonisation. An attribute of some microorganisms in this context is motility, which could be an important aspect of surface colonisation. Microorganisms that do not possess motility can only arrive at the surface through the influence of flow conditions and consequent mass transfer in the bulk water and near to the receptive surface (see Section 2.3.1). Static electric charge distribution may also affect the colonisation process.

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More details relating to the attributes of the adsorbed layer were provided by Chamberlain (1992). They include; 1. Modification of physicochemical properties of the solid surface. 2. Action as a concentrated nutrient source. 3. Suppression of the release of toxic metal ion release. 4. Adsorption and detoxification of dissolved substances. 5. Supply of trace metal ions that may be involved in metabolism. 6. Initiation of sloughing (removal) processes. 7. Suppression of inhibitory surface polymer effects. Detailed knowledge of the extent of some of these variables is very limited but the following comments on some aspects are helpful: 1. Surface physicochemical properties. As indicated earlier, key physicochemical properties of the surface may undergo alteration due to the presence of the adsorbed layer, including surface charge and wettability. These effects will become apparent after a period of time, presumably due to the time required to ‘coat’ the surface with the adsorbed layer. The work at the University of Birmingham over many years, in connection with the microbial fouling of ‘clean’ surfaces in a flowing environment, clearly demonstrates that it is delayed for a period of time (the induction period) ranging from a few hours to around 100 hours. It is apparent that once deposition and growth are initiated, the rate of accumulation of biofilm is quite rapid. It is also apparent with surfaces that have previously been exposed to the possibility of adsorbed conditioning layers, the initiation of microbial attachment and growth of biofilm is almost instantaneous. 2. Nutrient availability It is possible that the increase in microbial activity is due to an increase in nutrient material being adsorbed onto the surface that encourages attachment. 3. Toxic metal ion release Although the quantities involved would be quite small, it is to be expected that in the presence of metal surfaces, metallic ions toxic to microorganisms could be released, thereby affecting the adhesion of the microorganisms to the surface. Vieira, Oliviera, Melo, Pinheiro, and Martins (1993) demonstrated that metallic ions such as Cu2þ and Zn2þ seem to interfere with the initial adhesion of Pseudomonas fluorescens, whereas Al3þ had no such effect. It is possible that the presence of the adsorbed layer restricts the release of these toxic ions and so allows microbial growth to take place.

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3.9. THE PHYSICAL QUALITY OF THE SURFACE The discussion so far has in general, assumed that the surface to which the microorganisms attach is smooth; but as mentioned earlier, industrial plant surfaces are unlikely to be smooth except in very special circumstances possibly for instance, to reduce biofilm formation. Metal continues to be the preferred material of construction for industrial plant although there is a tendency to substitute polymers or polymer coated metals, whenever this is feasible. Here again though, there is the distinct possibility that the surface will not be perfectly smooth. Furthermore other materials of construction such as glass, brick and wood may be employed. In general, the roughness that occurs on the surfaces of the metal created by its manufacturing process is much larger than the size of individual microorganisms that constitute the biofilm. As a consequence, the cells are able to ‘hide’ within the roughness to be protected from the removal forces introduced by the effects of flowing water. The roughness and its morphology also affect the rate of slime build-up on a surface (Gibbons and Denton 1981). Mozes and Rouxhet (1992) in a discussion regarding cell immobilisation, presented a very instructive figure (reproduced as Fig. 3.13) with regard to the relative size of crevices in stainless steel and microbes. Quoting Messing and Oppermunn (1979) they suggested that the optimum pore diameter of the support for the accumulation of biomass should be in the range of 4–5 times the length of the microorganism. The orientation of the roughness will also influence at least, the initial accumulation of biofilm, i.e., whether the roughness ‘grooves’ are in line or at an angle to the waterflow direction. Using stainless steel tank reactors, Reid, Bott, and Millar (1992) investigated the effect of surface roughness on biofilm accumulation for three different microbial species, so that a direct comparison could be made between Bacillus subtilis, Penicillium chrysogenum and Pseudomonas fluorescens. The effect of three different surface-roughness heights on biofilm accumulation was also studied. The surface roughness carefully created, was measured in accordance with British Standard 1134 (1988), which defines roughness height (Ra). The three roughness heights were: rough (Ra ¼ 3.8 mm), intermediate (Ra ¼ 2.3 mm) and smooth (Ra ¼ 0.06 mm). The amount of biofilm was estimated from the quantity of protein retained at particular positions on the surface in contact with the microbial-laden water. Although there is scatter in the data, Table 3.10 illustrates the general trend of the results obtained with the surfaces in contact with the water.

MILL FINISH DESIGNATIONS FOR COLD ROLL STAINLESS STEEL SHEET mm 5

# 2D

# 2B

#7

Surface profile height

4 3 TYPICAL SIZE BACTERIUM

2 1 MEAN SURFACE

0 1 2 3

Profile Height

4 5

COLD ROLLANNEAL - PICKLE

2D PLUS COLD ROLL

2B PLUS 400 GRIT POLISH

Biofilms

Figure 3.13 Diagrammatic comparison of the size of roughness on stainless steel tubing with the size of a microbial cell. (Adapted from Characklis, 1990; Courtesy of Kluwer Academic Publishers.)

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Table 3.10 Biofilm accumulation on roughened stainless steel surfaces. Organism Mean protein value mg Surface (Ra) Bacillus subtilis Penicillium chrysogenum Pseudomonas fluorescens

0.06 2.37 3.38

240 305 356

209 256 299

220 151 217

In very general terms it would appear that the roughest surface retained the largest microbial deposit. No explanation of the variation of the data relating to Pseudomonas fluorescens could be offered. It is possible that the Pseudomonas fluorescens was less robustly attached and therefore more susceptible to the removal forces generated by the water movement in response to the stirring motion created by the rotor. Other work with Pseudomonas fluorescens by Mott and Bott (1990), using 316 stainless steel ‘as received’ and electropolished, demonstrated that the electropolished surface was much less fouled than the ‘as received’ stainless steel, under identical operating conditions. Some of the data are presented in Fig. 3.14 together with data on biofilm accumulation on glass and fluorinated ethylene polypropylene. It can be seen that there is appreciable

Mass of biofilm on test section g/m2

300

200

100

Glass Fluorinated ethylene polypropylene Electropolished stainless steel Stainless steel 316 Velocity 1 m/s Temperature 30 ºC

0 0

200

400

600

800

Time hours

Figure 3.14 Bioflim (Pseudomonas fluorescens) development on various surfaces.

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Friction Factor, f

0.12

.90

.60

.30

0

0

300 Biofilm Thickness, mm

600

Figure 3.15 Change in friction factor with bioflim thickness in the constant pressure drop system. (Courtesy of EPRI.)

additional biofilm on the ‘as received’ stainless steel. Coating a recipient surface may restrict biofilm growth (See Section 4.39). As a biofilm develops, the roughness of the surface in contact with the flowing water will change due to the deposition and growth of the biofilm. The change in the morphology of the outer layers of the deposit is likely to affect the quality of the water flow. Work that illustrated these effects was published as early as 1980 (EPRI, 1980). Figure 3.15 reproduces some of the data based on the friction factor that depends on surface roughness (see Section 2.2). It demonstrates that over a period of time, the surface in contact with the flowing water moves from being smooth to fully rough at a critical biofilm thickness due to the presence of an irregular surface produced by the growing biofilm in contact with the flowing water. The critical biofilm thickness can be explained as the stage in biofilm development when surface irregularities protrude through the laminar sub layer. The critical biofilm thickness for the data on which Fig. 3.15 is based is in the region of 30–35 mm. In addition, it has to be remembered that microorganisms are living entities, so they can adapt to changing conditions by mutation, rapidly responding to the conditions in which they currently exist.

3.10. INDUSTRIAL CONDITIONS The overview of the conditions that lead to the accumulation of microorganisms on surfaces is largely based on simplified ideal conditions and

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laboratory data gathered under similar constraints. In reality, the surfaces of industrial equipment will be far from ideal, possibly undergoing corrosion that fundamentally changes its morphology. The microorganisms involved may arrive singly or in small agglomerates. In addition industrial equipment involves flow-round bends through junctions, together with changes in the cross section through which the water flows that may affect the retention and thickness of any associated biofilm, through the effect these disturbances have on velocity and turbulence. Other factors that will modify the theoretical and laboratory approach, is the range of likely components in the aqueous phase. It may contain complex organic and inorganic molecules, dissolved or particulate matter, together with a potentially wide range of microbial species that may change in the long or short term. It is certain that for water taken from natural sources that the composition and amount of microorganisms will change over a period of time, particularly in response to the prevailing season. Some industrial processes such as water purification, involve porous surfaces. The biofilm formation on these surfaces will be more complex than with non-porous surfaces, because of the accumulation of microorganisms within the pores. The presence of these microorganisms will considerably reduce the water flow and in consequence, the efficiency of the process. Frequent cleaning may be required to restore process efficiency (See Chapter 5).

3.11. THE ESTABLISHMENT OF BIOFILMS IN SUMMARY It will be apparent from the foregoing discussion that the establishment of a biofilm on a surface is a complex multi-stage process, the result of interactions between fluids and surfaces of one sort or another and the effect this has on the transport of microorganisms to and away from a surface. The accumulation of bacteria may be thought of as taking place in three distinct stages, originally suggested by Marshall, Stout and Mitchell (1971): 1. Initial reversible stage or the attraction of cells to the surface. It is generally accepted that this involves electrostatic forces. 2. Attachment. 3. Development of the biofilm by growth. The last two stages are likely to be accompanied by the production of the biofilm matrix.

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Furthermore the maintenance of the biofilm will depend on the effect the flowing fluid (water) has on the transport of nutrients (and the biocidal agents that may be used for control) to and through the biofilm itself. In addition, the conditions established by the flowing fluid at the fluid/solid surface or fluid/biofilm interfaces, will also affect temperature distribution. If heat transfer across the biofilm is involved for instance, where cooling water containing microorganisms is employed, the temperature of the biofilm and hence the rate of metabolism in the biofilm will be affected. The consequent increase in biofilm thickness will in turn increase resistance to heat transfer, thereby reducing cooling efficiency. From the foregoing discussion the steps that lead to the establishment of a biofilm are as presented by Bryers and Characklis (1992). They include: 1. Conditioning of the surface that facilitates the attachment of microorganisms. The conditioning may be achieved either by macromolecules present in the bulk liquid or initially by some sort of coating process. 2. Transport of planktonic (free swimming) cells from the bulk liquid to the receiving solid surface. 3. Reversible attachment of cells to the surface. It is recognised that after a time the attachment may become irreversible through some kind of bonding between the macromolecules of the conditioning layer and the cells. 4. Release of reversibly attached cells to the surface. 5. Metabolism and reproduction of cells attached to the surface, with extracellular material production. Metabolism will depend on the availability of nutrients dependent both on the concentration of the ‘driving force’ in the liquid and the mass transfer conditions in the region of the liquid/ biofilm interface that apply to the nutrients. 6. Detachment or sloughing of biofilm material resulting from the prevailing conditions, erosion by solid particles carried in the liquid and high sheer forces due to the fluid flow conditions, particularly if relatively high velocities are involved. The overall result of the various interactions is difficult if not impossible, to predict in a particular example, because of the complexity of the interaction between the various components in the biofouling process. Nevertheless, knowledge of the overall mechanism does provide some assistance in controlling the incidence of biofouling or in applying biofilms to useful industrial applications.

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3.12. THE INFLUENCE OF OTHER FOULING MECHANISMS The fouling of surfaces can result from mechanisms apart from the deposition and growth of living organisms. They include: 1. Particulate accumulation. 2. Crystallisation and scale formation. 3. Corrosion of the surface material, particularly metals. 4. Chemical reaction accompanied with product attachment. 5. Freezing of the process liquid, usually water, due to low temperature. In many industrial operations where unwanted solids accumulate on surfaces, the overall fouling process often results from a combination of different mechanisms. Biofouling can be influenced by the presence of particles and their deposition, crystallisation and corrosion. Indeed some microbial activity can give rise to corrosion due to changes in the local pH, caused by cell metabolism (see earlier). Such changes in pH can also influence chemical reactions and possibly as a consequence, associated crystal formation. Particulate deposition may also be affected by pH, so that localised changes in pH could affect the overall fouling process. Particles that reach a biofilm may become embedded in it, thereby adding to the accumulation. The two mechanisms, chemical reaction and freezing fouling are in general, not likely to influence biofouling to any extent. It is possible however, that particles may be created by a chemical reaction that may affect the stability of the biofilm. It is very unlikely that freezing will occur since in general, temperatures will not be sufficiently low. Localised changes in temperature distribution brought about by the presence of the biofilm may modify the extent of deposit formation by these mechanisms, should they occur.

3.13. PARTICLE/BIOFILM INTERACTIONS In contrast to laboratory studies, in industrial systems that involve biofilms, where the water is taken from natural sources it will contain so-called detritusdsolids such as clay, sand or rock particles or decaying pieces of formally living matter (vegetable or animal in origin). In addition the quantity of particulate matter, the size, shape and consistency of composition and what might be termed the ‘quality of the water’ may vary. Variations may be due to changes in season or exceptional circumstances, such as flooding or changes in human activity near the source of the water. Particles

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65

emerging as the products of chemical reactions and particles of corrosion products from the particular process plant, may also find their way into the developing biofilm. It would be expected, particularly on account of the physicochemical properties of biofilms, that these solids would be incorporated into the biofilms in contact with the water. The inclusion of these particles will affect the consistency of the biofilm and may produce weaknesses in the structure. It is also possible that some of these particles could be toxic to the microorganisms making up the biofilm. In some situations, the particulate matter may abrade the biofilm. Indeed, in principle, it may be made the basis of a technique for biofouling control (see Chapter 4). There are reports that cooling water taken from locations such as a river estuary that contained suspended sand or clay particles, has kept power station steam condensers free of biofouling. This may be an advantage where the biofilm is unwanted, but the presence of particulate matter in the water could be detrimental where the maintenance of a viable biofilm is required, such as in trickle filters. Furthermore, the presence of particulate matter in fastflowing water may cause erosion of the material from which the equipment is made. In contrast to these suggestions, Vieira and Melo (1995) considered that the presence of clay particles could contribute to a more expanded and mechanically stronger structure. Clearly, with so many variables involved, there could be a variety of resulting biofilms possible with a wide range of different qualities. The character of the particulate matter is likely to influence its effect on the development of the biofilm. Lowe (1988) conducted experiments on the effect of suspensions of particles of kaolin and sand on the stability of established biofilms in comparison with the shearing effects of water alone. Surprisingly, he demonstrated that there was little difference between removal as a result of circulating water containing kaolin (50, 100 and 500 mg/l) and water alone, across established biofilms. At a shear stress of about 75 N/m2, about 75% of the biofilm was removed. On the other hand, for sand suspensions of 5000 mg/1 and above, the indications were that there was enhanced removal of biofilm compared with water alone. At a shear stress of 75 N/m2, almost 90% of the biofilm was removed for fine sand suspensions of 5000–20,000 mg/l. A removal of 95% was obtained with coarse sand at a concentration of 20,000 mg/l. Earlier work (Lowe, Dudderidge, Pritchard, & Bott, 1984) on the effect of kaolin particles on the formation and development of Pseudomonas fluorescens biofilm revealed some interesting results. The incorporation of the particulate matter into the biofilm naturally increased the mass of attached

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Table 3.11 Asymptotic thermal resistance of biofilm Maximum thermal resistance after ten days m2 k/W  104 Kaolin in suspension Fluid velocity m/s 150 mg/l None

0.28 0.34 0.54 0.59 0.82 0.87 1.24

42.0 e e 32.0 e 2.00 7.4

e 42.0 31.0 e 10.0 e 3.6

biofilms but the increasing mass was attributed to the combined deposition of cells and particulate matter and the natural development of the biofilm with the production of extracellular products. It was also demonstrated that the biofilm development was delayed in the presence of 30 mg/l of kaolin suspended in the circulating water. It was suggested that this extension of the lag phase could have been caused by two possible mechanisms dependent on the presence of the kaolin particles: 1. Physical interaction, such as abrasion of a weak developing biofilm. 2. Reduction in the planktonic microorganisms in the water due to adsorption onto the suspended particulate matter, thereby reducing the mass transfer of cells to the receptor surface. On the other hand, it is quite feasible that this effect could be offset by the transport of particles, with attached bacteria on their surface, to the hosting surface. Other factors such as pH, the prevailing temperature and the quality of the available nutrients can, as already discussed, affect the character of the biofilm and hence its susceptibility to the effects of particulate matter. Experiments on biofilm formation were carried out by Vieira (1992), with and without kaolin. Data obtained are presented in Table 3.11. Bott and Melo (1992) made two observations in respect of this work: 1. The maximum thermal resistance of the biofilm decreases with velocity. 2. The presence of the kaolin enhances biofilm development. It has to be borne in mind that the thermal conductivity of the kaolin particles is less than the substance of the biofilm itself, which could explain the higher thermal resistance of the biofilm containing kaolin. Additional experiments were carried out to investigate the effects of removing nutrients from the water once the deposit thermal resistance reached a maximum

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value. Sometime after the exclusion of the nutrients the amount of deposit started to decrease. The deposit containing both kaolin and bacteria retained its structure for a longer period than the biofilm containing only bacteria. Three explanations are possible for these differences: 1. The deposit containing the kaolin particles has a higher nutrient availability possibly emanating from the kaolin particles that act as carriers, i.e., resources of nutrients. 2. The presence of the kaolin particles protruding from the biomass disturbs the laminar sub-layers of the flowing water, thereby improving the mass transfer of nutrients towards the deposit. As a result, there would have been the potential to raise the amount of nutrients available to the bacteria in the deposit, which was formed when the nutrients were excluded from the flowing water. 3. The structure of the deposit is more robust due to the presence of the kaolin particles. There are probably other explanations for the differences between the two sets of experiments, which emphasises the empiricism that has to be applied to understanding biofilm accumulation. Other factors that are likely to affect biofilm structure, which have been discussed, include temperature, pH, and the character of the available nutrients.

3.14. THE GROWTH OF BIOFILMS The general pattern of biofilm development with time is illustrated on Fig. 2.5. It is exponential in form with three characteristic featuresdconditioning, exponential growth and a reasonably stable thickness. The time span of the conditioning period may only be a few hours or much longer (Santos, 1992). Much depends on the quality of the water from which the biofilm is formed, particularly the occurrence of macromolecules that condition the surface. Where a surface has been in contact with the flowing water for some time, even where the water is ‘cleaned’ from time to time, it is evident that biofilm growth is established quite rapidly. It was concluded that although difficult to prove, this was due to the retention of at least, some parts of the original conditioning layer that allows attachment of colonising microorganisms to occur quite rapidly. It is often the case that a ‘virgin’surface takes a longer time to establish a biofilm because of the time required to condition the surface. Once colonisation has been established, a fairly rapid period of steady growth is initiated. The rapidity of the growth will depend on the interaction of the variables previously discussed, particularly water velocity and

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nutrient quality and availability. For a given nutrient concentration, a higher water velocity in a given geometry, increases the Reynolds number with an attendant increase in turbulence that in turn, increases the mass transfer. Ultimately however, the cohesive forces within the biofilm are unable to resist the removal forces created by the flowing water. As a result, a plateau is reached where deposition and growth become offset by the fluid shear forces. Under these circumstances, it is likely that the biofilm becomes more robust by internal strengthening of the structure, through the destruction and subsequent removal of the weaker parts. It was noted earlier (Section 2.3), that at higher velocities, the rod-like cells of Pseudomonas fluorescens in a biofilm tended to be oriented in line with the water flow direction thus reducing the effectiveness of the removal forces created by the water velocity. With any living thing, the quality of its nutrients will affect growth and development. It is so for microorganisms. Since the nutrient represents a carbon source, cell metabolism will be affected by the carbon:nitrogen (C:N) ratio, which is in a crude sense, a measure of the nutrient quality. It was shown some years ago (Bott and Gunatillake, 1983) that although there was scatter in the data, when the C:N ratio in the nutrient was raised from 1:1 to 250:1, with a water velocity in a circular duct of 1.61 m/s, the biofilm thickness was increased approximately threefold over a period of five days. It is interesting to note that in these experiments, the concentrations of carbon and nitrogen in the biofilm remained at around 37% and 3.7% respectively. Some sloughing of the biofilm occurred during the period of the tests. For aerobic bacteria such as Pseudomonas fluorescens oxygen availability is vital. Some data on the penetration of oxygen through a biofilm of that bacterium were shown to be very dependent on the bulk water-flow velocity as illustrated by Table 3.12 (Patel & Bott, 1991). It shows the effect of bulk water velocity on oxygen concentration at the biofilm interface with the solid surface for a biofilm thickness of 250 mm. It is assumed that the

Table 3.12 Effect of velocity on oxygen availability at the biofilm/surface interface. Oxygen concentration at Bulk water velocity m/s the biofilm/solid interface%

0.7 1.0 2.0

5 32 43

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consumption of oxygen by the bacteria in the biofilm is constant for a given biofilm thickness. At the low bulk water velocity, the amount of oxygen at the biofilm/solid interface is quite small. At the highest velocity, the oxygen level is much greater. When the biofilm thickness had reached 500 mm the oxygen concentration at the biofilm/solid interface was virtually zero, with special significance for the bacteria close to the surface. These data illustrate the effects of velocity on the mass transfer of oxygen through the laminar layers to the biofilm/water interface (see Section 2.3). At the lower velocities mass transfer is restricted by the presence of the laminar sub-layers so that the oxygen concentration at the water/biofilm interface is relatively low. As a result the “driving force” to transfer oxygen through a given thickness of biofilm towards the solid surface is reduced. Furthermore some of the oxygen as it passes through the biofilm, will be assimilated by the microorganisms in the biofilm.

3.15. STRUCTURE AND STABILITY OF BIOFILMS Where a biofilm is an asset, clearly the structure of a biofilm impinges on the application for which the biofilm is intended. The control of the structure however is difficult to achieve, since many of the influencing factors, such as availability and quality of nutrients, the pH and temperature, cannot be suitably changed as they constitute part of the inception of the particular process. In addition, the fact that the biofilm is a living entity must impose a further input that influences biofilm development. It must also be appreciated that the nature of the living biofilm may change with corresponding difference to the structure. Due to the resistance to mass transfer of nutrients imposed by the biofilm thickness, the cell metabolism may change. The obvious example is the restriction of oxygen diffusion through the biofilm (discussed in Section 3.14) that can cause a change from aerobic to anaerobic metabolism of the cells. Some cells are capable of functioning in either mode but if this is not possible because the dominant species present is not capable, cells near the solid surface may die with consequences for the stability of the biofilm. Clearly the form and structure of a biofilm will be extremely complex and widely variable due to the microorganisms present, the availability and quality of nutrients, water flow velocity, the presence of predators and any biocidal material. Figs. 3.16 and 3.17 show scanning electron micrographs of biofilms of a single species of bacteria, Pseudomonas fluorescens, residing on

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Figure 3.16 Bioflim of Pseudomonas fluorescens illustrating its irregularity of structure.

Figure 3.17 Close-up of Pseudomonas fluorescens bioflim showing its open structure.

glass plates and grown under the effects of a bulk water velocity of 0.5 m/s (Grant, 1999). They illustrate the complexity and the difficulty of visualising the structure of biofilms.

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Water flow

Biofilm

Surface

Figure 3.18 The simple traditional concept of a bioflim.

For many years the concept of the biofilm was as shown in Fig. 3.18, i.e., a continuum of cells in close proximity, surrounded by extracellular material, usually polysaccharides, in which channels and interstices exist. Bott and co-workers in the 1970s observed that biofilms formed on a surface in contact with water flowing at a high velocity (greater than 1 m/s) were much denser than those produced under lower velocities. It was assumed that in order to resist the shear forces at the higher velocities the biofilm would become compact, possibly with an orientation of the cells to reduce the drag force imposed by the flowing water. Work by Costerton, Geesey, and Cheng (1978) at Montana State University, using confocal scanning laser microscopy, has produced interesting facts regarding biofilm structure. Fig. 3.19 is an attempt to illustrate the kind of structure observed. Examination of fully hydrated biofilms under flow conditions has revealed a heterogeneous structure consisting of cell clusters separated by interstices, voids and channels. Perhaps even more revealing is real time imagery of biofilms growing on surfaces in contact with fast flowing water that has revealed the presence of ‘streamers’ attached to the cell clusters that oscillate rapidly in response to the turbulence in the flowing water. Streamers as long as 3 mm have been found in a seven-day-old biofilm grown under turbulent flow conditions (Stoodley, de Beer, and Lappin-Scott 1997). It has also been demonstrated that the flow of water around cell clusters and through the channels between the cell clusters has implications for the mass transfer of nutrients and control-biocides to the cells. The cell clusters may be composed predominantly of a single species but may also be mixtures of different species. As might be expected, extracellular polysaccharide tends to be concentrated around the clusters, although some may be found in the spaces between the clusters. Stoodley, de Beer, and

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‘Mushroom’ colonies of bacteria Sphere colonies

Grazing protozoa

Surface

Thick slime: mainly polysaccaharides & water

Water channels carrying nutrients, enzymes oxygen & waste

Figure 3.19 A modern concept of a bioflim based on scanning confocal laser microscopy. (Courtesy New Scientist.)

Lappin-Scott (1997) found that the largest clusters in a five-day-old mixed population biofilm were nearly hemispherical in shape and ranged in height from 175–225 mm. The cluster base diameter ranged from 10–500 mm. It is likely that mature biofilms will present a modified structure, due to the continuing effects of the flow conditions in the system. Massol-Deya, Whallon, Hickey, and Tiedje (1995) described the development of a mixed population biofilm on granular activated carbon. They discovered that initially the entire surface was colonised by a thin film (10–20 mm thick), with no structural consistency. After 63 days however, the biofilm was much thicker and a mosaic of lobes of cells was apparent. This structure was more pronounced after 77 days with channels between the lobes having widths in the range of 2–25 mm. Many of the channels extended from the outer regions of the biofilm deep into the structure. The network of channels was clearly an aid to the availability of nutrients to the cells remote from the flowing water. It would appear that this structure was not unique since Massol-Deya et al. (1995) found that naturally occurring biofilms had a similar structure, with a continuous layer attached to the surface with thickness in the range 10–75 mm and a superimposed network of channels with depths in the range 20–300 mm

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3.16. BIOFOULING MODELLING The discussion so far has revealed the complexity of the biofouling process, with the interaction of many variables that often change over long or short intervals of time. The range of variables includes the metabolism of the microorganisms, their distribution that is likely to change with time and the interaction between species, nutrient availability and quality, mass transfer through the biofouling layer and its porosity with changing thicknesses. The quality of the receptor surface may also affect attachment. In addition to these variables, are the basic variables of temperature and its distribution, water velocity and pH discussed earlier. In the light of so many variables and their interaction, the common simplifying assumption in modelling of steady state conditions is clearly unlikely to be applicable to the problem of biofilm formation. Despite these difficulties however, De Gooijer, Wijffels, and Tramper (1992) suggested that steady-state models for growing cells can quite satisfactorily be used for design purposes but may fail to describe the start-up events and response to changing conditionsdchanges that are generally difficult to appreciate and therefore difficult to define effectively. It is probably true to admit that the development of a comprehensive and reliable model to explain the accumulation of microorganisms on surfaces is very remote. The use of empirical relationships based on experience with particular systems may be possible but attempts to apply such models widely could be risky.

3.17. BIOFILMS AND CORROSION Corrosion in process plant is an ever-present problem and the engineers responsible for design, operation and maintenance need to be vigilant in the control of corrosion. (Bott, 1995) The potential influence of biofilms on the corrosion process therefore, has to be taken into account. The corrosion of iron gives an indication of the basic chemistry involved in the corrosion process. Equations 3.1 and 3.2 represent the electro-chemical processes that take place with the production of electrons. Fe/Fe2þ þ 2e

(3.1)

and Fe/Fe3þ þ 3e

(3.2)

The result is the removal of a metal atom from an anodic site on the metal surface giving rise to an anion in solution and an excess of electrons on the

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Fe - 2e – Fe2+

Ionic

Metal ions

migration O2 + 2H2O + 4e – 40H– Corrosion deposits

Anode

Cathode

Electron flow

Figure 3.20 The electrochemical corrosion cell. (Courtesy of Elsevier.)

metal surface. Utilisation of these electrons released by the reactions at a nearby cathodic site, gives a balancing reaction, which in solutions that have a pH of around 7, usually involves the release of dissolved oxygen to OH ions. These processes are symbolically illustrated on Fig. 3.20. The metal itself provides a path for electron flow resulting from the potential created between the anode and cathode. Chamberlain (1992), lists the ways in which biofilms assist corrosion in process plants. They include; 1. Physical effects a. Diffusion barrier. b. Reduction of turbulence and surface flow. c. Reduction of heat transfer where applicable. 2. Chemical effects These can be considered as acting on the anodic or the cathodic reaction or influencing both. a. Anodic reaction. Prevention of anodic polarisation through metal ion complexation and/or binding. b. Modulation of reducible species. Supply of reducible species 3. Dissolution of protective oxide/hydroxide layers. 4. Selectivity of ion permeability. 5. Effect on pH. Chamberlain (1992), however, points out that the most important aspect is the creation of differential concentration cells producing galvanic corrosion cells. Clearly the influence of a biofilm is extremely complex and as the biofilm develops and the conditions change the corrosion rate is likely to be affected.

3.18. THE COST OF BIOFOULING Although in some industrial processes, such as the treatment of waste water for instance, a biofilm is used to advantage, in many industrial operations the presence of a biofilm is an impediment with serious effects on capital and

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operating costs. In respect of the use of cooling water the following contributions to costs can be envisaged (Bott, 1992): 1. Increased capital cost. In anticipation of a biofouling problem it is usual to over-design for instance, the heat exchangers, such as condensers using cooling water, to allow for the heat transfer resistance imposed by the biofouling. Equipment to control biofouling is very likely to be required. The overdesign and control equipment represent additional capital investment. 2. Additional operating costs. A number of additional costs resulting directly from the growth of biofilm can be identified: a. Additional energy. Inefficient heat transfer usually means that extra energy is required to offset the energy that could not be recovered. In power station steam condensers for example, more energy is required to produce 1kWh of electricity in the fouled condition compared with the clean condition. In addition the increased pressure drop means that increased pumping energy will be required. Doubling the velocity represents a fourfold increase in the pumping energy, in addition to the increase due to the biofilm roughness. b. Additional maintenance. The need to clean the heat exchangers represents additional costs. Furthermore, the presence of biofilm can lead to other problems such as corrosion, leaking flanges or pump malfunction that will require additional maintenance. If the fouling problem is protracted for any reason and difficult to solve with existing equipment, it may be necessary to install additional plant to overcome the problem. c. The cost of additives. The need to apply biocides to control biofouling and possibly other chemical additives represents a charge to operating costs. d. Lost production. If heat exchangers or other equipment have to be removed from service to be maintained or cleaned, it represents lost production with an attendant loss of income from the sale of the product. These losses may be even more unacceptable if an emergency shutdown is involved. e. Loss of employee morale. Continual problems with the operation of plant can affect the morale of employees, particularly if there is a bonus scheme that is tied to production. There can also be additional problems, such as general lack of operating efficiency as a result, thereby adding to the problems.

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f. Hidden costs. The use of chemicals (e.g., biocides) that persist in the environment can lead to other costs when the water is discharged back to its source. These costs might involve surveillance and subsequent treatment. Increasingly rigorous legislation on the disposal of contaminated water is likely to increase treatment costs. The use of an alternative source to avoid these problems could be expensive.

3.19. CONCLUDING REMARKS The discussion of the creation of biofilms on the surfaces of industrial processing equipment demonstrates the complexity of the biofouling process. It is difficult theoretically to take into account all the interacting chemical, physical and biological processes that occur. Furthermore, in a particular situation these processes are likely to change with time. As discussed in the following chapters, in order to counteract the detrimental effects that are brought about by the occurrence of unwanted biofouling and the utilisation of biofilms in process applications, it is necessary to revert to empiricism. The costs associated with biofouling can be high.

REFERENCES Abu, G. O., Weiner, R. M., Rice, J., & Colwell, R. R. (1991). Properties of an extracellular adhesive polymer from the marine bacterium. Shewanella colwelliana. Biofouling, 3, 69. Allison, D. G., & Sutherland, J. W. (1987). The role of exopolysaccharides in adhesion of freshwater bacteria. Journal of General Microbiology, 133, 1319. Allison, D. G. (2003). Molecular architecture of the biofilm matrix. In P. Lens, A. P. Moran, & T. Mahony, et al. (Eds.), Biofilms in medicine, industry and environmental biotechnology (pp. 81). London: IWA publishing. Ambasht, J., & Bott, T. R. (1998). The effect of some operating variables on the action of ozone as a biocide, Conference Ozonation and AOP’s in water treatment. Poitiers: International Ozone Association. Ashland Chem. Co. (1997). Drew industrial division, Ajax Ontario, L1S 2ES. Baier, R. E. (1980). Substrata influences on the adhesion of microorganisms and their resultant new surface properties. In G. Britton, & K. C. Marshall (Eds.), Adsorption of microorganisms to surfaces, 59. New York: Wiley. Bartlett, R., Santos, R., Bott, T. R., & Grant, D. (1999). Measurement of biofilm development within flowing water using infrared absorbance. In T. R. Bott, L. F. Melo, C. B. Panchal, & E. F. C. Somerscales (Eds.), Understanding heat exchanger fouling and its mitigation. New York: Begell House Inc. Bott, T. R. (1992). Introduction to the problem of biofouling in industrial equipment. In L. F. Melo, T. R. Bott, M. Fletcher, & B. Capdeville (Eds.), Biofilms science and technology. Dordrecht: Kluwer Academic Publishers. Bott, T. R. & Gunatillaka, M. (1983). Nutrient composition and biofilm thickness. In R. W. Bryers (ed). Fouling of heat exchange surfaces, Proceedings of England Federal Conference., White Haven, Pennsylvania. United Engineering Trustees Inc.

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Bott, T. R. (1995). Fouling of heat exchangers. Amsterdam: Elsevier. Bott, T. R. (2003). Monitoring biofouling using infrared absorbance. In P. Lens, A. P. Moran, & T. Mahony, et al. (Eds.), Biofilms in Medicine, Industry and Environmental Biotechnology. U.K.: IWA Publishing. Bott, T. R., & Melo, L. F. (1992). Particle-bacteria interactions in biofilms. In L. F. Melo, T. R. Bott, M. Fletcher, & B. Capdeville (Eds.), Biofilms science and technology (pp. 207). Dordrecht: Kluwer Academic Publishers. Bryers, J. D., & Characklis, W. G. (1992). Biofilm accumulation and activity ; a process analysis approach. In L. F. Melo, T. R. Bott, M. Fletcher, & B. Capdeville (Eds.), Biofilms - science and technology (pp. 221). Dordrecht: Kluwer Academic Publishers. Chamberlain, A. H. L. (1992). The role of adsorbed layers in bacterial adhesion. In L. F. Melo, et al. (Eds.), Biofilms - science and technology (pp. 59). Dordrecht: Kluwer Academic Publishers. Chamberlain, A. H. L. (1992). Biofilms and corrosion. In L. F. Melo, T. R. Bott, M. Fletcher, & B. Capdeville (Eds.), Biofilm science and technology (pp. 207). Dordrecht: Kluwer Academic Publishers. Chamberlain, A. H. L., & von Woerkam, R. (1986). The effects of adsorbed marine organics on microbial fouling, Proceedings 6th International. Biodeterioration Symposium, Washington, 556. Characklis, W. G. (1990). Biofilm processes. In W. G. Characklis, & K. C. Marshall (Eds.), Biofilms. New York: John Wiley and Sons Inc. Chen, M. J., Zhang, Z., & Bott, T. R. (2005). Effects of operating conditions on the adhesive strength of Pseudomonas fluorescens biofilms in tubes, colloids and surfaces. Biointerfaces, 43, 61. Cogllan, A. (1996). Slime city. New Scientist, 51, 32, Interview with J.W. Costerton. Cooksey, K. E. (1981). Requirement of calcium in adhesion of a fouling diatom to glass. Applied and Environmental Microbiology, 41, 1378. Cooksey, K. E. (1992). Bacterial and algal interactions in biofilms. In L. F. Melo, T. R. Bott, M. Fletcher, & B. Capdeville (Eds.), Biofilms-science and technology (pp. 163). Dordrecht: Kluwer Academic Publishers. Cooksey, K. E. (1992). Extracellular polymers in biofilms. In L. F. Melo, T. R. Bott, M. Fletcher, & B. Capdeville (Eds.), Biofilm-science and technology (pp. 137). Dordrecht: Kluwer Academic Publishers. Costerton, J. W., Geesey, G. G., & Cheng, K. J., (1978). How bacteria stick, Scientific American, 228, No.1. Daniels, S. L. (1980). In G. Bitton, & K. C. Marshall (Eds.), Adsorption of microorganisms to surfaces, 8. New York: Wiley. Davis, B. D., Dulbecco, R., Eisen, H. N., & Ginsberg, H. S. (1980). Microbiology (3rd ed.). Cambridge: Harper and Row. De Gooijer, C. D., Wijffels, R. H., & Tramper, J. (1992). Dynamic modelling the growth of immobilized nitrifying bacteria biofilm development. In L. F. Melo, T. R. Bott, M. Fletcher, & B. Capdeville (Eds.), Biofilms science and technology. Dordrecht: Kluwer Academic Publishers. Denyer, S. P., Hanlon, G. W., & Davies, M. C. (1993). Mechanisms of microbial adherence. In S. P. Denyer, et al. (Eds.), Microbial biofilms-formation and control. Oxford: Blackwell Scientific Publishers. Derjaguin, B. V., & Landau. (1941). Theory of the stability of strongly charged lyophobic solutions and adhesion of strongly charged particles in solutions of electrolytes Acta Physicochim. USSR, 14, 633. Drew, Principles of industrial water treatment, 11th ed, (1994), Drew Industrial Division, Ashland Chemical Co. 1, Drew Plaza, Boonton, U.S.A. EPRI. (1980). Biofilm development and destruction, CS, 1554. Palo Alto, CA: EPRI.

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Flemming, H. C. (1991). Biofilms as a particular form of microbial life. In H. C. Flemming, & G. G. Gessey (Eds.), Biofouling and biocorrosion in industrial water systems. Berlin: Springer – Verlag. Fletcher, M. (1976). The effects of proteins on bacterial attachment to polystyrene. Journal of General Microbiology, 94, 400. Fletcher, M. (1992). The measurement of bacterial attachment to surfaces in static systems. In L. F. Melo, T. R. Bott, M. Fletcher, & B. Capdeville (Eds.), Biofilms science and technology (pp. 603). Dordrecht: Kluwer Academic Publishers. Gibbons, D. B., & Denton, P. R. (1981). Surface roughness and biofouling, Private internal report. University of Birmingham. Grant, D. M. (1999), Biofilm control through optimised biocide dosing, Ph.D. Thesis, University of Birmingham. Hamaker, H. C. (1937). The London–van der Waals attraction between spherical particles, Physica (Utrecht), 4, 1058. Keen, G. A., & Prosser, J. I. (1988). The surface growth and activity of Nitrobacter. Microbial Ecology, 15, 21. Kemmer, F. N. (Ed.). (1988), The Nalco water handbook (2nd ed.), 22.2. New York: McGraw-Hill Book Co. Krupp, H. (1967). Ad. Colloid Interface Science, 1(2), 111. Lowe, M. J., (1988). The effect of inorganic particulate materials on the development of biological films, Ph.D. Thesis, University of Birmingham. Lowe, M. J., Dudderidge J. E., Pritchard A. M., & Bott T. R. (1984), Biologicalparticulate fouling interactions: effects of suspended particles on biofilm development, Proceedings 1st U. K. Nat. Heat Trans. Conf., Vol. 2. I Chem. E. Symposium Series 86, 391–400. Marshall, K. C., Stout, R., & Mitchell, R. (1971). Mechanisms of initial events in the sorption of marine bacteria to surfaces. Microbiology, 68, 337. Marshall, K. C. (1976). Interaction in microbial ecology. Cambridge: Harvard University press. Massol-Deya, A., Whallon, J., Hickey, R. F., & Tiedje, J. M. (1995). Channel structures in aerobic biofilms of fixed-film reactors treating contaminated groundwater. Applied and Environmental Microbiology, 61, 769. Messing, A., & Oppermann, R. A. (1979). Pore dimensions for accumulating biomass. 1. Microbes that reproduce by fission or by budding. Biotechnology and Bioengineering, 21, 49. Mott, I. E. C. & Bott, T. R. (1990). The adhesion of biofilms to selected materials of construction for heat exchangers, In: G. Hetsroni, (Ed.) Pro. 9th, Intl. Heat Trans. Conf., Jerusalem, 5, 21. Mozes, N. (1994). The ways we study interfacial phenomena of living cells. In M. N. Bellon-fontaine, & J. Fourniat, (Eds), Adhe´sion des micro-organismes aux surfaces. Paris Technique and Documentation Chaˆtenay-Malabry, 27. Mozes, N., & Rouxhet, P. O. (1992). Modification of surfaces for promoting cell immobilization. In L. F. Melo, T. R. Bott, M. Fletcher, & B. Capdeville (Eds.), Biofilms science and technology (pp. 125). Dordrecht: Kluwer Academic Publishers. Oliveira, D. R. (1992). Physico-chemical aspects of adhesion. In L. F. Melo, T. R. Bott, M. Fletcher, & B. Capdeville (Eds.), Biofilms - science and technology, Vol. 223 (pp. 45). NATO ASI Series E, Applied Sciences. Patel, T. D., & Bott, T. R. (1991). Oxygen diffusion through a developing biofilm of Pseudomonas fluorescens. Journal of Chemical Technology Biotechnology, 52, 187. Paulson, D. S. (2007). Topical antimicrobials. In P. C. Zhu (Ed.), New biocide development, the combined approach of chemistry and microbiology (pp. 124). Washington: American Chemical Society. Railkin, A. I. (2004). Marine biofouling. Washington: CRC Press. 103.

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Reid, D. C., Bott, T. R., & Millar, R. (1992). Biofouling in stirred tank reactors-effect of surface finish. In L. F. Melo, T. R. Bott, M. Fletcher, & B. Capdeville (Eds.), Biofilmsscience and technology (Vol. 521). Dordrecht: Kluwer Academic Publishers. Rijnaarts, (1994). Doctoral thesis. Santos, R., Callow, M. E., & Bott, T. R. (1991). The structure of Pseudomonas fluorescens in contact with flowing systems. Biofouling, 4, 319. Santos, R. (1992). Polymer coatings in relation to single and mixed population biofilms. Ph. D. thesis, University of Birmingham. Vaisanen, O. M., Nurmiaho-Lassila, E., Marmo, S. A., & Salkinoja-Salomen, M. S. (1994). Applied and Environmental Microbiology, 60, 641. Vieira, M. J. (1992). Ph.D. Thesis, University of Minho. Vieira, M. J., Oliveira, R., Melo, L., Pinheiro, M. M., & Martins, V. (1993). Effect of metallic ions on the adhesion of biofilms formed by Pseudomonas fluorescens. Colloids and Surfaces B: Biointerfaces, 7(1), 119. Vieira, M. J., Melo, L. F., & Pinheiro, M. M. (1993). Biofilm formation – hydrodynamic effects on internal diffusion and structure. Biofouling, 7(1), 67. Vieira, M. J., & Melo, L. F. (1995). Effect of clay particles on the behaviour of biofilms formed by Pseudomonas fluorescens. Water Science and Technology, 32(8), 45. Verwey, E. J. W., & Overbeck, J. T. G. (1948). Theory of the stability of lyophobic colloids. Amsterdam: Elsevier. Vrom, J., de Grauw, K. I., Bradshaw, D. J., Marsh, P. D., Watson, G. K., Birmingham, J. J. et al. (1999). Applied Environmental Microbiology, 65, 3502. Watnick, P., & Kolter, R. (2000). Biofilm, city of microbes. Journal of Bacteriology, 182(10), 2675. Whitfield, C. (1988). Bacterial extracellular polysaccharides. Canadian Journal of Microbiology, 34, 415. Wingender, J., New, T. R., & Flemming, H. L. (1999). What are bacterial extrapolymeric substances? In J. Wingender, T. R. New, & H. L. Flemming (Eds.), Microbial extracellular polymeric substances (pp. 1–9) Berlin: Springer.

CHAPTER

4

Biofouling Control Contents 4.1. Introduction 4.2. Chemical Control 4.2.1. Biocides 4.2.2. Operation and Biocide Effectiveness 4.2.3. Classification of Biocides

82 84 87 95 99

4.2.3.1. Oxidising Biocides 4.2.3.2. Non-oxidising Biocides

99 100

4.2.4. Common Biocides in More Detail

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Chlorine Chlorine Dioxide Ozone Glutaraldehyde Hydrogen Peroxide 2,2eDibromo-3-nitrilopropionamide (DBNPA) Amines Organo-sulphur Compounds

101 105 107 114 118 118 120 120

4.2.5. Immobilised Biocides 4.2.6. Microparticle Transport of Biocides 4.2.7. Removal of Biofouling with Biocides

121 122 123

Chlorine Hydrogen Peroxide Peracetic Acid Potassium Permanganate

124 125 126 126

4.2.8. Biodispersants 4.2.9. Concluding Remarks on Chemical Control 4.3. Physical Methods of Control 4.3.1. Introduction 4.3.2. Ultraviolet Light and Gamma Rays 4.3.3. Sponge Rubber Ball Circulation 4.3.4. Brush and Cage System 4.3.5. Air or Gas Injection 4.3.6. Use of Inserts

127 128 129 129 129 130 133 133 134

1. Static Inserts 2. Oscillating Inserts

134 136

4.3.7. Flexible Tubes 4.3.8. Fluidised Beds 4.3.9. Coating of Surfaces 4.4. Potential physical techniques for biofouling control

Industrial Biofouling ISBN 978-0-4445-3224-4

137 137 138 141

Ó 2011 Elsevier B.V. All rights reserved.

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4.4.1. Circulation of Polymer Fibres 4.4.2. The Use of Ice Crystals 4.4.3. Biofouling Control Using Ultrasound 4.4.4. High Voltage-capacitance Technology 4.4.5. Reciprocating Scraped Surface Heat Exchanger 4.4.6. Physical Controldconcluding Remarks 4.5. Combined Use of Physical and Chemical Techniques 4.5.1. Potential Combination of Ultrasound and a Biocide 4.5.2. Combination of Inserts and Biocides 4.6. Potential Combined Physical and Chemical Controldconcluding Remarks References

141 143 143 146 146 147 147 147 149 150 150

4.1. INTRODUCTION At the beginning of this chapter it is necessary to state that there is some confusion in the literature between the words ‘clean’ and ‘control’. In general, the former refers to off-line removal of deposits, whereas ‘control’ refers to attempts to prevent or restrict deposit formation, such as biofouling. This will be the terminology used in this book. Sometimes however, the term ‘to clean’ is used to mean ‘to control’. Confusion can arise since some of the control technologies discussed in this chapter can be adapted for cleaning purposes. Control of biofilm growth can occur due to the removal forces created by water flow across the biofilm. In addition, control can be evident through natural agencies. Rather obvious control mechanisms include lack of nutrients and temperatures outside the range for sustained growth. As is common throughout the whole natural world, the presence of predators can be a controlling influence. In this context, the biofilm can be the host that supports the growth of a range of protozoa that may be observed grazing on an established biofilm. Protozoa represent a disruptive effect on the biofilm weakening its structure and thereby facilitating removal through the ‘sloughing’ of lumps of the biofilm into the flowing water. They may selectively graze on the biofilm population consisting of a range of different microbial species and in turn, affect the balance of microorganisms in the biofilm by the removal of specific species. The ‘quality’ of the biofilm may be so affected that it could possibly jeopardise its retention on the surface. Pathogens such as Legionella pneumophilia may be so affected. Although the employment of natural control of unwanted deposits of microorganisms sounds attractive and has been investigated, it is not really an option since it is likely to cause severe operational difficulties to maintain effectiveness.

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Microorganisms, such as bacteria in biofilms, demonstrate some protection against removal techniques; nevertheless, control of unwanted biofouling in industrial operations where it is likely to occur is imperative so that operating costs and other associated problems are kept to a minimum. Not only does effective control reduce operating costs but invariably good control will reduce process energy requirements thereby making a potential positive contribution to energy management and reducing greenhouse gas emissions. For instance, the presence of a biofilm on the inside of a pipe through which a process fluid is passing at a required volumetric rate will increase the pressure loss through the tube and hence the pumping energy demand will be increased. Process fluid pumps are usually powered electrically, the electrical energy being derived from the combustion of fossil fuels. Biofouling control therefore offers a double benefitdimproved competitiveness by the minimisation of operating costs and at the same time, a helpful contribution to human society through reduced carbon dioxide (CO2) emission. In order to be able to control the incidence of biofouling additional capital investment will be inevitable. For instance, high-quality materials of construction to restrict microbial accumulation may be employed. Additional subsidiary equipment may also be required to facilitate biocide application. A potential difficulty where biofouling is a serious problem is that unscheduled shutdown for cleaning may become necessary to maintain product quality and output. Not only is this an additional operating charge, it could also represent a loss of production and associated loss of profit. Under these circumstances, it might be prudent to duplicate equipment that is likely to be affected by biofouling even though a control procedure has been put in place, so that one set of equipment can be taken out of service for cleaning while full-scale operation is maintained in the duplicate equipment. Even if equipment design has been directed towards the minimisation of biofouling, taking into account the factors that influence the accumulation of microorganisms on surfaces as discussed in Chapters 2 and 3, it is more than likely that some form of biofouling control will also be required. Of particular importance is the variability of the biofilm in terms of composition and structure, which may influence the control technology. There are two basic methodsdchemical control and physical control and in certain situations benefits accrue from a combination of these two methods. In addition, it would be desirable to monitor closely the extent of biofouling so that effective steps may be taken to prevent serious consequences occurring, such as unscheduled interruptions in production.

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The sequence of events that leads to the accumulation of biofilm may influence the method of control that is adopted. If deposition of microorganisms is the primary factor in the development of a biofilm, it would be important to concentrate the control technology in the liquid phase. On the other hand, if it is reproduction and growth of the biofilm itself that is the essence of the problem, it would be imperative that this is where the control technique should be applied. Microorganisms in suspension in water for instance, may be controlled by the application of ultraviolet light and under certain conditions this can be very effective. In general though, the effectiveness of ultraviolet light on a biofilm residing on a surface is generally unsatisfactory, as it is necessary for the ultraviolet light ‘to see’ the microorganisms in order to be successful. In much industrial equipment this would not be feasible so that an alternative method for control must be employed, such as biocide application (see later). Some preliminary studies by Lowe (1988) and Bott and Miller (1983) demonstrated that once a surface had been colonised, the presence of microorganisms in the flowing water was not necessary for the continuance of the biofouling process, provided nutrients were available in the water. Any microorganisms that escaped the effects of the ultraviolet radiation could thrive on a surface so that additional control technology would have to be applied. Usually it is possible that the control method adopted is directly applicable to both components of the fouling process i.e., deposition and growth and the comments concerning the use of ultraviolet light do not apply because the biofilm itself may not ‘see’ the radiation. The discussion of control will largely be related to cooling water systems since this is the principal occurrence of biofouling in industrial operations. It is a problem that has attracted much research activity in recent years. Application to other industrial systems, such as paper making and food processing, will be apparent but they are discussed in more detail in Chapter 6. An important aspect of all methods of control is good housekeeping, i.e., maintaining the process plant’s operating efficiency so that its performance is consistent, thereby allowing the control technique to be applied effectively. Where the plant design and proposed method of control have been carefully considered, the related operating conditions should be adhered to, unless development work demonstrates the possibility of improving control.

4.2. CHEMICAL CONTROL The use of chemicals to control microbial activity has been employed for many years but particularly over the recent past, considerable research has

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been carried out in the development of techniques to meet the changing needs of industry and society at large. The assessment of the efficacy of biocides was originally measured in a laboratory, in terms of the effects on planktonic microorganisms, often under static conditions. The accumulation of microorganisms and attendant EPS in a biofilm however, afford a measure of protection against the use of chemicals so the effects of operating conditions must be taken into account to provide a realistic assessment of a particular biocide. The term biocide is often loosely used to include all chemicals that are employed to control biofilm formation and growth. The word ‘biocide’ is derived from the Latin ‘bio’ (which means life) and ‘cida’ (which means killer). In strict terms therefore, ‘biocide’ refers to a substance that is toxic to microbial life forms. An alternative term that is sometimes used to describe chemicals that are employed to control populations of microorganisms is ‘antimicrobial’. In industrial terminology the word ‘slimicide’ is often used. Although a biocide may be used on its own in the control of microbial life forms, it is often a component of a group of chemicals that are used to control the incidence of biofilms. Chemical control technology may be grouped into three broad sectors: biocides that kill microorganisms, biostats that interfere with metabolism and surface active agents that restrict adhesion to surfaces. A proprietary chemical product may contain all three of these features. It will be readily understood that a biofilm will have a greater resistance to the effects of a biocide than planktonic microorganisms. In an industrial situation, the application of a biocide may kill all the planktonic organisms but the biofilm population may very well be still active unless the quantity of biocide added to the system is substantially increased or the method of application is modified. There is a need therefore, that the biocide effectively reaches the biofilm. In order to achieve this requirement, it is necessary to ensure that the flow is turbulent so that mass transfer of biocide across the boundary layer to the biofilm is maximised. Crevices and other ‘hide-aways’ for microorganisms may not be penetrated by a biocide and these pockets of activity represent in effect, inocula for regrowth. Biocidal treatment therefore, must take account of this possibility. Mass transfer across the boundary layers between the bulk flow (e.g., cooling water) and the biofilm must be effective to ensure that the biocide is effective. Due to concern for the environment, a complication that arises is the ultimate destination of the chemical additive. Operating conditions therefore,

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must facilitate effective use so that the quantities of biocide that may be discharged to the environment meet the legal requirements. This could involve the choice of biocide and the method of maximising its effectiveness. It may also mean pre-treatment before discharge of the carrier e.g., cooling water. The purpose of chemical control is not to establish and maintain sterile conditions since this would be technically difficult to sustain in a large scale industrial operation with prohibitive cost. The principal objective of chemical control or any method of control is to maintain manageable and acceptable operations at minimum cost. The interactions of biocides with microorganisms can be considered to be the result of a sequence of three events: 1. Adsorption on the cell wall. 2. Penetration of the biocide into the cell. 3. Damage to the cell structure and its functions by the combination of the biocidal agent with the cell membrane or the cytoplasm of the cell that leads to cell lysis. A rather obvious generalisation is that planktonic microorganisms are more susceptible to the detrimental action of biocides than the microorganisms residing in a biofilm on a surfacedafter all, this is one of the natural survival techniques! The extracellular polymeric materials will constitute a barrier to the ingress of the biocide into the biofilm, thereby restricting effectiveness to some extent at least. Presumably, by a process of natural selection it is possible that some microorganisms may be able to change their sensitivity to certain biocides and certain disinfectants, depending on the local conditions. The problem may be resolved by changing the biocide from time to time. Different mechanisms of resistance to biocides can be distinguished, including intrinsic resistance, acquired resistance and adaptation (Payne, 1988). In general, gram-negative bacteria are more resistant to antimicrobial agents than gram-positive species i.e., possessing intrinsic resistance due to basic properties of the individual cells. Microorganisms can acquire resistance to antimicrobial substances through the possibility of genetic changes within the cell. Exposure of bacterial cells to mild doses of a particular biocide may encourage adaption with changes to cell wall composition and structure, enabling resistance to the effects of the biocide. On the other hand, the presence of surfactants can reduce the surface tension at the surface of individual cells so that biocides can more rapidly attack the microorganisms and increase the rate of activity of the biocide.

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The effects on different parts of a cell were listed by Denyer (1990) and are summarised in the following Table 4.1 Table 4.1 Damaging effects of biocides Target Damaging Effect

Wall

Membrane

Abnormal morphology and construction. Initiation of autolysis Inhibition of membrane Increase in permeability of some ions Loss of structural integrity

Consequence

Lysis (cell death).

Restriction of respiratory chain and energy transfer. Interference with normal chemical activity. Leakage of intercellular material. Initiation of lysis.

The detailed effects are extremely difficult to assess or predict, because of the small size of individual cells and their complex internal structure and function. The industrial approach to biocide control of microbial biofilms therefore, is very largely empirical.

4.2.1. Biocides For many years the preferred biocide has been chlorine because it is relatively cheap and readily available. However its use is becoming severely restricted on account of its potential detrimental effects. It has had widespread use in association with the use of cooling water that is often discharged into the local environment such as rivers, lakes or the sea. Under these circumstances, it may detrimentally affect plants and living creatures including humans. Some of the products of chlorination of naturally occurring water use have been found to be carcinogenic. Discharge to a public sewer may be expensive or even prohibited. It would be possible to treat the water before discharge but this would add considerably to the operating costs. It is not surprising therefore, that over the years a different approach to biocides and their application has been apparent. The range of industrial biocides available is extremely large and additions to the range are made from time to time; however it is probably true that the actual number in large-scale operations is relatively small. The biocides discussed in this Chapter are restricted to those in common use. It has to be appreciated however, that the use of biocides is increasingly under the legal control of local governments in respect of the problems associated with

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human safety and the local environment. It is important when making a choice regarding biocide application in a particular situation, that these and other important factors are taken into account including the cost and ease of application under the conditions of effective process operation. Furthermore, because the spectrum of microorganism species present in the industrial system is likely to be large (in cooling water from natural sources for instance) and may change with time, a wide-ranging biocide is likely to be the most effective. In simple terms, the action of a biocide follows a recognisable sequence i.e., adsorption onto the cell surface, penetration into the cell so that chemical reaction with the internal components of the cell can take place and leading inevitably to mortality. Bott, (1992) lists the long-standing desirable properties of biocides that affect the ultimate choice including: 1. Ability to remove established biofilms and reduce regrowth. 2. Activity against a wide range of microorganisms (aerobic and anaerobic). 3. Relatively low toxicity to other life forms (including humans!). 4. Non-corrosive. 5. Water soluble. 6. Effective over a wide range of pH. 7. Does not deactivate other additives e.g., corrosion inhibitors, or scaleforming preventatives that may be included in an overall treatment programme. 8. Effectiveness not affected by a wide range of dissolved inorganic or organic material other than microorganisms. 9. Active in hard or soft water. 10. Not readily deactivated in the presence of a wide range of other materials. A valuable additional property would be biodegradability and particularly, biodegradability to environmentally acceptable products. It is unlikely that every biocide will meet all these criteria so the choice in a particular situation will necessarily be somewhat of a compromise. In addition to the cost of the biocide itself, factors that also affect costs have to be taken into account. In the use of cooling water for instance, this would include: 1. Quality of the water (makeup or once through). 2. The extent and the nature of the microbial contaminants. 3. Exposure to the atmospheric environment from the cooling tower or spray pond operation.

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4. Process leaks that may provide nutrients for the microbial population or indeed act as a biocide! 5. Residence time in the system. Of these considerations the most important are those associated with the quality of the water and the microbial contamination. A principal criterion will be the overall cost. An important aspect concerning the use of biocides is the resistance in one form or another, to the mass transfer of the biocide to and through the biofilm. As discussed in Chapter 2, there will be mass transfer resistance from the slow-moving fluid layers in contact with the biofilm and as mentioned in Chapter 3, the extracellular polymers will clearly offer some protection to the microorganisms making up the biofilm. It would be possible to improve the mass transfer by increasing the driving force, i.e., the concentration of biocide in the bulk flow. Depending on the cost of the biocide this technique could be an expensive exercise. Furthermore, this could have increased undesirable consequences for the environment if circulating water containing the biocide is discharged back to its natural source. Under these circumstances it may be legally necessary to treat the water before discharge, with the additional costs this would entail. An alternative technique to improve the mass transfer of the biocide to the biofilm would be to increase the velocity of the fluid (usually water) thereby increasing its turbulence; again increased costs would accrue from the increased energy usage associated with the higher velocity. Furthermore, there would be implications also for the environment through the higher electrical power required by the pumps and depending on the origin of the electrical power, in addition to the cost of the treatment. Other factors would have to be considered, including the consistency of the microbial content in terms of species and concentration of the microorganisms. There could be changes throughout the year if the water employed was from natural sources. Leakage would have to be considereddwhether from the process side in any cooling water application that may be in effect, a nutrient or an additional biocide or from contamination of the product by water bearing the biocide. The method of dosing will also need careful consideration, based on experience or regular trials. The alternatives include: 1. Continuous dosing, where a fixed biocide injection rate is maintained. 2. Intermittent (pulse) dosing involving dosing at set times on a regular basis. It could be regarded as shock dosing on a more frequent timescale.

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3. Shock dosing involving the injection of a relatively large shot of biocide on a regular basis but less frequent than intermittent dosing. It has the ability to remove rather large quantities of biofilm. The technique can in some circumstances, be very effective in overcoming any resistance that the biofilm may have developed against the biocide by natural selection or the orientation of cells in the biofilm. An alternative to shock dosing that can achieve the same outcome, is to change the biocide from time to time. The permutations of the size of the dose and frequency of application are virtually infinite. In order to maximise the effectiveness of biocide treatment at minimum cost, preliminary investigations in the laboratory or with a side stream should be carried out to give an indication of appropriate strategies. Trials on the full-scale equipment would be greatly assisted with some preliminary data of this kind, thereby avoiding potentially costly operating problems during trials. Grant and Bott (2005) carried out tests on continuous dosing, shock dosing and pulse dosing of biocide. A single species Pseudomonas fluorescens was chosen as a typical slime-forming microorganism for the tests, together with a proprietary biocide containing 2,2–Dibromo-3-nitrilopropionamide. A laboratory pilot plant was used to obtain the data. The tests could have equally been carried out on a side stream from an actual cooling water system for which the data obtained was to be applied. Tests could have been made in a full-scale plant but this could have created operational problems that might have been costly. The measure of the biofilm accumulation was infrared absorbance (described in Chapter 5). Although the actual mass or thickness of the biofouling was not obtained, the absorbance provided a measure of the effects of the different dosing regimes. Because water flow rate is an important factor in mass transfer (See Chapter 2) two different bulk water velocities of 0.5 and 1.3 m/s were used in the tests to cover the likely range of velocity used in water coolers. Continuous dosing of the biocide and concentrations of 100 and 50 mg/l were found to control biofilm formation as demonstrated in Fig. 4.1. A reduction of biocide concentration to 20 mg/1 indicated some biofilm formation detected by the infrared monitor. It was apparent that there was some biocidal effect because the biofouling could not be seen by the naked eye. A reduction of biocide concentration to 15 mg/l allowed the rate of biofilm growth to rise, which was particularly noticeable at the lower velocity of 0.5 m/s. The growth rate at this velocity was close to that obtained when there was no biocide in the circulating water, indicating that

Biofouling Control

1.3 m/s 0.5 m/s Control 1.3 m/s Control 0.5 m/s

0.4 Absorbance

91

20 ppm

0.2

50 ppm

15 ppm

0.0 0

10

5

15

Time. days

Figure 4.1 The effect of continuous dosing of biocide from a starting concentration of 100 mg/l. (Courtesy of Taylor and Francis.)

under these conditions the biocide was having very little effect. At the higher water velocity of 1.3 m/s the biofouling appeared to be under control. Earlier work (Pujo, 1993) with this biocide at a similar concentration, demonstrated control of biofilm growth. The intermittent or pulse dosing tests were designed in the light of information gained from the continuous dosing experiments i.e., to apply the total biocide used in the tests of continuous dosing in four, 15-minute pulses evenly spaced over the course of a day. The calculated peak concentration was 106 mg/l. The results of the test, presented in Fig. 4.2, demonstrated that there was complete control of biofilm growth under these conditions. Control was also achieved if the dosing was made in eight pulses with a peak concentration of 53 mg/1. Earlier work by Taylor (1995) using the same biocide with a peak biocide concentration of 15 mg/1 in four, 30-minute doses provided complete control. The water velocities used were 1.27 and 0.86 m/s. Fig. 4.3 shows the results obtained using eight, 15- minute daily doses with peak dose concentrations of 8.43 mg/l. There was some control for about five days after which biofilm began to develop. It is difficult to appreciate that growth is possible in the presence of a biocide. However, it is suggested by Le Chevallier et al. (1988) that the extracellular polymers in the biofilm can act as an exchange resin thereby preventing the biocide from reaching vital parts of the individual cells. In addition, the rate of mass transfer of the biocide though the laminar layer to the biofouling will

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1.0 1.3 m/s 0.5 m/s Control 1.3 m/s Control 0.5 m/s

Absorbance

0.8

0.6

0.4

0.2

0.0 0

2

4 6 Time. days

8

10

Figure 4.2 The effect of pulse dosing biocide 4  15 minutes/day at a peak concentration of 106 mg/l. (Courtesy of Taylor and Francis.)

0.30 1.3 m/s 0.5 m/s

Absorbance

0.25 0.20 0.15 0.10 0.05 0.00 0

2

4 6 Time. days

8

10

Figure 4.3 The effect of pulse dosing biocide 815 minutes/day at a peak concentration of 8.43 mg/l. (Courtesy of Taylor and Francis.)

depend on the water velocity in the equipment, which will affect the amount of biocide available at the biofilm/water interface. Shock dosing tests based on the results of the continuous dosing data of Grant and Bott (2005) showed that dosing with short bursts of 100 mg/1 biocide over a period of 15 minutes on a daily basis was not effective, as

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0.5 1.3 m/s 0.5 m/s Control 1.3 m/s Control 0.5 m/s

Absorbance

0.4

0.3

0.2

0.1

0.0 0

2

6 4 Time, days

8

10

Figure 4.4 The effect of daily shock dosing 100 mg/l biocide. (Courtesy of Taylor and Francis.)

shown by Fig. 4.4. After two days, biofilm began to develop. In the light of these results tests were carried out using the total biocide in 24 hours of the continuous dosing regime i.e., 28,800 mg as the daily shock dose, administered in 15 minutes. After four days, a biofilm began to appear demonstrating that this technique was not entirely effective. The failure is likely to be due to the relatively short time that the biocide was in contact with the surface that was being colonised. The survivors could then have had a relatively long time in which to develop the biofilm. The inference is that growth under the conditions of the experiment was not arrested, even though the rate of development may have been reduced. These limited tests cannot be universally applied to control biofilm growth but they do illustrate some of the difficulties inherent in the use of biocides to control biofouling. Much will depend on the local conditions and so no hard and fast rules can be made. Probably the best approach is to carry out tests using a side stream or pilot plant, with continuous dosing starting at a relatively high concentration and by carefully reducing it over a period of time till there is an indication that the limit has been reached. Difficult though this is, a shock dosing technique would be much more difficult to establish. It might be prudent to introduce a shock dosing test just before any shut down for regular cleaning. In fact, it is possible that shock dosing might facilitate the cleaning procedure. A major factor in establishing any biocide dosing regime is the associated cost.

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The final choice of technique will, like the choice of biocide itself, be dependent on the local legislation that may be present in respect of discharge in the case of cooling water and the overall cost of the treatment. It is likely that adjustments to any strategy envisaged will be necessary after it has been put into operation and at times when operating changes are made. A change in the origin of cooling water for instance, will certainly need a reappraisal of the biofilm control strategy. Changes in the season may also require changes to the dosing regimen for dealing with water taken from natural resources. Although not strictly biocide control, two other techniques can be related to biocidal technology. They are: 1. High temperature sterilisation or ‘heat shock’ by the circulation of hot water in place of the normal cooling water that kills the residual microorganisms. A problem that can occur from this approach is the potential for release of ‘lumps’ of biofilm that might cause other problems downstream, such as blockage. 2. Osmotic shock, which involves passing salt water through what is normally a fresh water system that will interfere with the microbial metabolism and cause the death of the organisms making up the biofilm. The process is likely to require some time to take effect and there may be associated corrosion and disposal problems. In cooling water systems where the water is taken from natural sources, a wide range of microorganisms is likely to be present and furthermore the distribution of organisms may change with time, for instance, between summer and winter. For this reason, it may be necessary to change the dosing policy to accommodate these changes. It has been usual over many years to discharge the water back to its original source but this practice has come under scrutiny from governmental agencies questioning acceptability, particularly in respect of any biocide that has been employed. The system may be ‘once through’, although in order to reduce costs and meet the demands of regulating bodies recycling to a lesser or greater degree may be employed. Other systems may obviate the use of microbial-laden water altogether (often referred to as ‘zero discharge’) through the installation of some form of closed system. In this technique that uses treated water, its temperature after use may be reduced by air-blown heat exchangers to restore its cooling properties.. Clearly, although this approach avoids problems of discharge, it will incur additional capital and operating costs even though it offsets the need to treat the water before discharge.

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Where the standard practice of discharge back to the environment is employed a number of criteria need to be examined. They include: 1. Quality of the water utilised, particularly in relation to microorganism content. This is generally the most important factor for consideration since it will indicate the potential for biofouling to occur and the most suitable method of control. The chemical composition of the dissolved materials and the pH may also be important since these factors may influence the choice of chemical control technology. 2. The likely extent of microbial contamination retained after a preliminary treatment has been carried out. 3. Contact with the atmospheric environment at the operational site e.g., the potential for the discharge of Legionella bacteria with the associated risk to health for people close by. The problem is particularly acute where evaporative systems, such as spray ponds or cooling towers, are used to cool the circulating water. 4. Process leaks into the cooling water that might act as nutrients or even biocides. 5. Residence time in the equipment that might affect the concentration of the biofouling precursors. In general although not universally, proprietary biocides will include other chemicals to enhance effectiveness and improve stability. The formulation might contain stabilising agents, anti-corrosives, surfactant and chelating agents. For example, peracetic acid is a relatively unstable biocide and is often supplied in equilibrium with water, hydrogen peroxide and acetic acid (Malchesky, 2000). Clearly it is not possible to discuss the details of proprietary biocides but information on the principal ingredient provides a basic understanding of its applicability and effectiveness.

4.2.2. Operation and Biocide Effectiveness An important aspect concerning the use of biocides is the resistance to the mass transfer of the biocide to and from the biofilm. As discussed in Chapter 2, there will be a mass transfer resistance from the slow-moving fluid layers in contact with the biofilm and as mentioned in Chapter 3, the extracellular polymers will clearly offer some protection to the microorganisms making up the biofilm. It would be possible to improve the mass transfer by increasing the driving force i.e., the concentration of biocide in the bulk flow. Depending on the cost of the biocide this technique could be an expensive exercise. Furthermore, this could have increased undesirable consequences

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for the environment if circulating water containing the biocide is discharged back to the natural source from where it was obtained. Under these circumstances it may be legally necessary to treat the water before discharge, with all the additional cost this would entail. An alternative technique would be to increase the velocity of the liquid, usually water, thereby increasing turbulence and reducing the resistance to the mass transfer of the biocide. Increased energy costs would be the penalty of this action. In addition, there would be implications for the environment through the higher energy usage required by the water pumps that in turn, depend on the origin of the electric power. It might be accepted without question, that a particular biocide is the ultimate answer to all biofouling problems. The final choice however, must depend on the local conditions where the biocide is to be used, including any safeguards required in respect of personnel safety and damage to the local environment in addition to the microorganisms involved. Patching and Fleming (2003) make some very important points in respect of choice of biocide. The following are some aspects of biocide application that should be considered: 1. Mode of operation and spectrum of activity. 2. Environmental aspects of activity. 3. Development of resistance. 4. Effects on process plant. 5. Effects on product. 6. Effects on the environment. 7. Ease of handling including potential health risks. 8. Cost. Patching and Fleming (2003) emphasise the point that oxidising biocides tend to react with organic material that in addition to microorganisms, may be present in the stream to be treated, which reduces effectiveness. However, where relatively large quantities of organic material are present in the system, it may help to ameliorate any adverse environmental effects on discharge. It is not surprising that as living entities microbial populations can display a measure of resistance to the effects of chemical biocides. A valuable outline of the potential resistance of microorganisms to biocides has been made by Russell (2003) covering different aspects of the problem. Bacterial resistance mechanisms fall into two broad groups: 1. Intrinsic resistanceda natural property of an organism. 2. Acquired resistancedresulting from genetic changes in an organism.

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By way of introduction, the wide range of differences with different organisms is presented in Table 4.2. Included as a matter of general interest, are some microorganisms that are not associated with biofouling. In general terms, spores and some protozoa notably Cryptosporidium are resistant to biocides. Table 4.2 Susceptibility of microorganisms to biocides Microorganism/ entity Comment

Prions Bacterial spores, coccidian Mycobacteria Non-enveloped viruses Yeasts and moulds Gram-negative bacteria Gram-positive cocci

Most resistant Highly resistant to many biocides. Intermediate resistance. Small non-lipid viruses e.g. poliovirus, resistant to many biocides. Important spoilage organisms. Pseudomonas aeruginosa, Proteus spp., Providencia spp. have above average resistance. Enterococci less susceptible than staphylococci.

Russell (2003) pointed out that microorganisms can differ greatly in their responses to biocides with a wide variation between different species of bacteria. It is also apparent that it is not only the type of microorganism within a life-cycle that can influence the efficacy of the biocidal agent. In addition to concentration of biocides, other factors that influence the activity of biocides include: 1. Temperature increase, which enhances activity. 2. Prevailing pH, which may facilitate or reduce activity dependent on the biocide. 3. The presence of organic matter, which interferes with activity. 4. Density of the biofilmda dense biofilm is probably more biocide resistant than one with a more open structure. 5. Flow velocity, which will influence the mass transfer of biocide to the biofilm as described in Chapter 2. High velocities will improve the rate of mass transfer. Factors that can influence the activity of biocides are principally the biocide concentration and the operating parameters of the industrial operation concerned i.e., pH, temperature and flow velocity. These parameters can affect the morphology of the biofilm and the resistance to biocides. For

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instance Chen, Zhang, and Bott (2001) studied the effectiveness of the biocide hydrogen peroxide on biofilms of Pseudomonas fluorescens and found that the efficacy of the biocide showed a marked dependence on the biofilm structure and the hydrodynamics of the system defined by the magnitude of the Reynolds number. The biofilms formed at the higher water velocities demonstrated higher resistance to the applied biocide. In general, biofilms in a full-scale cooling water system for instance, will be of variable morphology dependent on the conditions prevailing in different parts of the particular system. Furthermore, they are likely to be composed of a variety of microorganisms of different species and developed under variable growth conditions, such as low carbon source, not all of which is biodegradable. The structure of these biofilms is also likely to vary according to seasonal changes as is the effect of these changes on the predominant microbial species present. The presence of extraneous organic matter can also reduce effectiveness. The biofilm itself may also act as a barrier to the penetration of the biocide. Empirical collective data on a particular system that does not differentiate between the different aspects of resistance to biocide penetration can be obtained by effective monitoring (see Chapter 5). Russell (2003) cites mitigation and adaption as means of resistance to biocides through repeated exposure to the antibiotic. The resistance may be ‘high-level’ or ‘low-level’. The resistance may be stable after removal of the biocide following sufficient exposure. Alternatively, it may revert to susceptibility after withdrawal of the biocide dosing. Youngblood and Stratton (2009) conclude that in the medical world there is a need to develop new microbiocides to combat increasingly resistant bacteria. Major epidemics like anthrax attacks, outbreaks of avian and swine flu and the increase in methicillin-resistant Staphylococcus aureus infections, have led to an increasing interest in alternative and stronger biocides. These authors suggest that polymer-based biocides are becoming a viable alternative to the more traditional biocides, such as chlorine dioxide. Although cost is an important factor in medical applications, the cost of these alternatives may be a severe restriction to their potential use in industrial plants. Nevertheless, it is important that development of new antibacterial substances is undertaken against the possibility that the microorganisms that are responsible for biofouling become increasingly resistant to the biocides currently employed. As pointed out in Chapter 3 the attachment of bacteria to surfaces is of vital importance to survival. It can be readily appreciated that sessile cells are less susceptible to online control than their planktonic counterparts.

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The biofilm offers resistance to the penetration of the biocide. Furthermore, the presence of extracellular polymers will offer physical protection to the embedded cells. Cells at the base of the biofi1m are likely to be starved of nutrients. It has long been known that cells limited in nutrient availability are more resistant to biocides than freely developed cells (Gilbert, Collier, and Brown 1990). This means that the cells in a biofilm attached or close to the solid surface, will be more resistant to the biocide than those in contact with the fluid stream. Where water from natural sources is utilised it is very possible that the season will affect the distribution of different microorganisms so that the apparent effectiveness of a particular biocide may appear to change. Dosing may therefore have to be modified to maintain effectiveness of control. Russell (2003) stated that microbial and particularly bacterial, response to biocides was being extensively studied. He cited the reasons for this interest as: 1. The possibility that bacterial resistance to biocides is increasing. 2. Concern that biocides might be selecting for antibiotic-resistant bacteria in hospital, industrial and domiciliary environments. 3. The role played by biofilms in nature and disease in reducing the efficiency of biocides and where relevant, of antibiotics. 4. Emerging infections, including bacterial diseases and the organisms responsible for them, may be refractory to biocides and antibiotics. As a result of these factors it might be necessary to change the biocide from time to time to maintain maximum effectiveness against biofouling.

4.2.3. Classification of Biocides Classification of biocides by their chemical activity is possible and may give some guidance in the choice of biocide for a particular set of circumstances. An obvious choice for distinguishing the chemical activity of a biocide is to ascertain whether it is oxidising or non-oxidising. 4.2.3.1. Oxidising Biocides 1. Chlorine along with a wide range of alternative methods of application including liquid (compressed gas), sodium hypochlorite in solution or powdered, organic chlorine donors and generated in situ by electrolysis of brine. 2. Chlorine dioxide an unstable water-soluble gas, generated on site by various chemical and electrochemical processes, for example, reaction between sodium chlorite and chlorine.

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3. Bromine, provided as a liquid (compressed gas) by hydrolysis of suitable compounds and displacement by chlorine from dissolved halogen compounds. 4. Iodine used in solution. 5. Hydrogen peroxide used in solution. 6. Ozone, because of its short life is necessarily generated on site by electrical discharge in dry air followed by absorption in water. Of the many applications of peroxygens used for biocidal purposes those that are suitable for some specialised industrial operations are given in Table 4.3 (McDonnell 2007). Table 4.3 Some specialised applications of peroxygens. Application Description

Water disinfection

Surface disinfection

Antimicrobial reduction of viable microorganisms to a safe and defined level in drinking water. Antimicrobial reduction of viable microorganisms.

Examples

Chlorine dioxide. Ozone Electrolysed water. Liquid or gaseous hydrogen peroxide.

The action of common peroxygens is to react with the essential macromolecules that make up microbial life, including the oxidation of various proteins, carbohydrates, lipids and nucleic acids (McDonnell and Russell, 1999). These reactions will lead to the loss of cell structure and the loss of the essential functions of these compounds, including unfolding, fragmentation and cross reaction with oxidised groups. Proteins, carbohydrates and liquids on the surface of microorganisms are the initial accessible targets. The various structures of these materials are essential to the survival, pathogenicity and basic structure of microorganisms. The loss of coherent structure and function is sufficient to produce loss of viability (McDonnell, 2007). 4.2.3.2. Non-oxidising Biocides The range of non-oxidising biocides is extremely large including both organic and inorganic compounds. They may be added as solutions or emulsions in water or in an organic liquid. Proprietary biocides may involve blends of a range of different biocides and other chemicals such as biodispersants and surface active agents to widen the effectiveness across a range of microbial systems. These blends of active components

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are usually under some sort of trade name. Patching and Fleming (2003) list some non-oxidising biocides are: 1. Aldehydes. 2. Halogenated bisphenols 3. Isothianzolones 4. Organosulphurs 5. Quaternary ammonium compounds Toxic organic and inorganic metal compounds may also be classified as non-oxidising biocides.

4.2.4. Common Biocides in More Detail It is not possible to discuss each of the large number of chemicals employed for biofouling control but a selection of common biocides currently in use is made in order to illustrate the possibilities of chemical control. Chlorine Although the use of chlorine has diminished in recent times, the fact that it has been used as an effective biocide for many years means that there is more published on the effects operating parameters than other more recently introduced biocides. It is useful therefore, to examine the way some operating parameters affect efficiency of its use, since this may provide pointers to their effects in association with other biocides. The usual application has been in connection with cooling water particularly in electric power production. When chlorine comes into contact with water, it hydrates to form hypochlorous and hydrochloric acids, according to the following equations: CI2 þ H2 O ¼ HOCI þ HCI

(4.1)

The acids will exist as ions HOCI/Hþ þ OCI

(4.2)

HCI/Hþ þ CI

(4.3)

Hypochlorous acid is a powerful oxidising agent; it diffuses through the cell wall of microorganisms to react with the cytoplasm to produce chemically stable nitrogen-chlorine bonds with cell proteins. Hypochlorite ion is much less effective.

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The pH of the water determines the extent of the ionisation of the hypochlorous acid and it is the amount of hypochlorous acid that determines the effectiveness of the control. Fig. 4.5 shows the effect of pH on the ionisation of hypochlorous acid. At pH 5 there will be little ionisation and at pH 7.5 it is about 50%. As the pH is raised as a result say, of the accumulation of alkaline salts and the frequency of ‘blowdown’ is reduced, there is a rapid fall in the extent of OCl ions. A pH in the range 6.5–7.0 can be considered suitable for effective biocidal activity. Lower pH is likely to stimulate corrosion. At a pH of 9.5 or higher, the effectiveness as a biocide, is considerably reduced.

100

Available Chlorine Present As HOCl %

90 Cl2 80 OCL 70 60 HOCl 50 40 30 20 10 0

1

2

3

4

5 6 pH Value

7

8

9

10

Figure 4.5 The effect of pH value on form of free available chlorine in water. (Courtesy of Drew Principles of Industrial Water Treatment.)

Fig. 4.6 illustrates the striking effectiveness of chlorine as a biocide (Cowbourne et al., 2001), indicating why it has been used extensively for biofouling control. Biofilms of Pseudomonas fluorescens were produced on the inside of glass tubing and on glass slides in a Robbins device at different water velocities. A biofilm was allowed to develop over a period of about 340 hours before a continuous dose of 5 mg/l chlorine (derived from

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2.5 2 m/s glass 0.5 m/s glass 1 m/s glass 1 m/s glass-Robbins

Absorbance

2

1.5

1 Biocide addition

0.5

0 0

50

100 150 200

250 300 350 400

450

Time (hours)

Figure 4.6 The effect of 5 mg/l chlorine. (Courtesy of Publico publications, Essen.)

sodium hypochlorite) was introduced. It is evident from Fig. 4.6 that biofilm removal was very efficient, with the majority of the biofilm being removed in about 96 hours. The chlorine however, did not remove all the biofilm. When chlorine is introduced into cooling water, it will react with the many different materials that may be in the water, including organic materials, inorganic compounds such as ammonia, ferrous ions or sulphites. As a result, some of the chlorine will be fixed and will therefore cease to have biocidal properties. In order to overcome this problem sufficient chlorine must be added to satisfy this chlorine demand in addition to the chlorine necessary to perform the biocidal function. If the chlorine demand is high, as a result of the use of poor quality water, the operational costs may be excessive. In addition to this chlorine demand, if

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the water is contained in a recirculating system, which in cooling operations may be reduced in temperature by the use of a cooling tower or spray pond, chlorine will be lost to the atmosphere. Dosing will have to be sufficient to replace this loss in addition to the loss due to chemical reactions to maintain the biocidal activity, thereby increasing the cost of operation. The level of free chlorine residual required will usually be a matter of trial and error in relation to the conditions prevailing at the particular site. Nevertheless it is important that such potential problems are recognised and allowed for as far as possible, at the design stage of the plant. The use of chlorine (liquid or gas) as a biocide in water produces acid conditions so that if excessive use of chlorine is employed, say in response to high levels of biofouling, corrosion of metal surfaces may occur. This is not the situation when hypochlorite is employed. The transport and storage of large quantities of chlorine for use as a biocide in the cooling water of a large power station represents potential hazardous and environmental problems. As a result it may be necessary to consider on site generation or revert to the use of hypochlorite solutions. Reaction of chlorine with organic materials that are present in water from a river, a lake or the sea produces compounds that are toxic or carcinogenic. It is generally acknowledged that even small concentrations of chlorine constitute a hazard in discharge water. In continuous discharge with the residual concentration of chlorine as low as 0.01 mg/l, it would not protect salmon and trout and some important fish-food organisms. This implies that the concentration would have to be very much lower to protect most aquatic creatures. For intermittent discharge for a period say of two hours per day, a slightly higher concentration should protect most species of fish. In order to meet legal requirements on the concentration of chlorine permitted in water discharge to the environment, it may be necessary to modify the use and handling of chlorine; for instance, the use of targeted chlorination. The technique is particularly suitable for application to the control of biofouling in heat exchangers such as the steam condensers on power plants. It involves the application of bursts of chlorine for short periods of time lasting only a few minutes with relatively high doses of chlorine, depending on the severity of the particular biofouling problem. Higher doses will of course, provide more effective control. The local high concentrations will be reduced to acceptable lower concentration through mixing with untreated water. It is directed to selected fractional areas of the

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cooling water inlet to a tubular heat exchanger. Each fractional area is treated in sequence till the whole exchanger has been treated. The principal objective of the technique is to reduce chlorine usage, and hence reduce operating costs but perhaps more importantly to meet regulations that might be in place in respect of chlorine discharge. The detail of a particular application will depend on the plant design, the location and the origin and distribution of the water. One method of coping with discharge restrictions is to dechlorinate the water before discharge. As long ago as 1984, the Electric Power Institute in the U.S.A. issued a wide-ranging manual on dechlorination. Its stated objective was to provide information to allow utilities to design and operate systems to keep effluent chlorine within the prescribed limits, while at the same time preventing biofouling of equipment in their cooling water systems circuits. The different opportunities available for dechlorination discussed included chemical dechlorination using sulphur dioxide, sodium sulphate and sodium bisulphate, adsorption with granular activated carbon and physical treatment such as air stripping. It is clear that there could be considerable additional investment in equipment costs, together with the need to develop operational skills. The use of chlorine as a biocide for many years has created a wide range of experience in its use for many different applications. However, increasing legislation in respect of chlorine use and indeed public opinion against its continued application where chlorinated water is discharged to the environment, is stimulating interest in alternative biocides that do not present environmental hazards. Many such biocides have been developed and marketed over the last few years. There is also the possibility of using physical methods of control (See Section 4.3). However the cost of these alternative chemicals and the equipment depreciation costs of some of the physical methods are likely to be substantially greater than the cost of chlorine that would provide the same protection. Chlorine Dioxide Chlorine dioxide is known to be an effective biocide particularly in cooling water systems (Bartole and Bressen 1993). It is an unstable, water soluble gas. As a result of its intrinsic instability, it is usually generated at the site of application from sodium chlorite. The method of generation will depend largely on the prevailing site conditions and the relative local cost. Various chemical and electrochemical methods may be employed. The basic chemical methods available are based on the following reactions:

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2NaClO2 þ Cl2 ¼ 2NaCl þ 2ClO2

(4.4)

5NaClO2 þ 4HCl ¼ 5NaCl þ 4ClO2 þ 2H2 O

(4.5)

It has to be appreciated that ClO2 is not a chlorinating agent that in many ways is a valuable asset since it does not produce chlorinated compounds that might have environmental implications. It is active over a pH range of 5–9 and rapidly breaks down into innocuous compounds. Petrucci and Rosellini (2003) outline chlorine dioxide generation. Employing chlorine (Equation 4.4), sodium chlorate is injected into chlorinated water with a concentration of around 0.5% chlorine. For the use of HCl (Equation 4.5) vacuum generators may be employed. These authors suggested that the main features of chlorine dioxide as a biocide include: 1. Does not react with bromides to form hypobromites, which could contribute to the formation of organobromine compounds. 2. Does not react with ammonia. 3. High reactivity with regard to a number of structures, including secondary and tertiary amines, organosulphur compounds and activated aromatic rings. 4. Is not a chlorinating agent and does not lead to any significant formation of halogenated organic compounds. 5. Is efficient over a wide range of pH (5.0–9.5). Chlorine dioxide is directly used in liquids (including water), in formulations that use two components, which are mixed to generate the biocide in addition to other materials that together form the disinfecting biocide (McDonnell, 2007). It can be used in gaseous form, for instance, for disinfection of some items but not for the control of industrial biofouling. Bott and Mott (2003) made a comparison of the operating characteristics of two cooling water systems; one with chlorine, and the other with chlorine dioxide as biocides. The conclusions of these tests were as follows: 1. A tighter control of biocide concentration could be obtained by a chlorine based programme of 0.5–4.5 mg/l, compared with the use of chlorine dioxide in the concentration range up to 0.57 mg/l. 2. Higher corrosion rates of 1.5 mg/y were obtained for the chlorine dosed system compared with 1.0 mg/y for the chlorine dioxide treatment. A change in chlorine concentration from 0.5–4.5 mg/l resulted in an

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increase in corrosion rate of 0.5 mpy. There was no apparent increase in corrosion rate when the chlorine concentration was raised from 0.1 to 0.57 mg/1. 3. An increase in temperature from 38 to 43  C did not appear to influence the corrosion rate for the chlorine dosed system over the concentration range of 0.5 to 4.5 mg/l. for the identical temperature range a 0.5 mpy increase in corrosion rate was observed for the chlorine dioxide-dosed system in the same concentration range of 0.1 to 0.57 mg/l. Ozone Ozone is a reactive, short-lived oxidising agent. It rapidly reverts to oxygen. The reactivity of the ozone molecule makes it an effective antimicrobial agent in liquid and gaseous applications. In addition, during generation and degradation of ozone, several other oxygenated species are produced that contribute to the overall biocidal activity attributed to ozone alone (McDonnell, 2007). Background to the use of ozone as a biocide was presented by Bott (1991). It has been used to improve the quality of drinking water in respect of microbial content, particularly in France, for many years. It acts by rupturing the microbial cell membrane. There was considerable interest in ozone as a biocide in the 1980s, in the light of environmental concerns about the continued widespread use of chlorine and its derivatives with the likely impact of regulatory control of their use. Despite the fact that ozone treatment had been used earlier, a US. Patent was issued in 1979 (US. Patent 4, 172, 786) titled ‘Ozonation of Cooling Tower Water’. The following is an extract from the summary of the invention: ‘It has now been discovered that the maintenance of high levels of ozone in cooling water in absence of any other chemical treating agents or electrochemical or electro-mechanics approach prevents further scale formation, removes existing scale, passivates the metal surfaces to inhibit further corrosion and at the same time is an active biocidal agent, which fully disinfects the water to prevent algae growth.’ Cooling towers usually contain packing of some sort that provides a large surface area to facilitate evaporation that in turn, removes heat as latent heat, thereby reducing the temperature of the water for reuse. In the past the tower packing often consisted of wooden slats. Unfortunately some forms of fungi attack wood so that a major problem was the maintenance of the integrity of the interior wooden slat structure. It seems rather strange that there was no direct reference in the patent to the problems apart from algae

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associated with cooling towers or the formation of biofilms on water-cooled heat exchangers. The use of alternative materials of construction for contact devices such as the fabrication of air/water contact devices with polymeric materials has in effect, redirected the use of ozone as a biocide to other areas of cooling water systems. A few years after the granting of that patent on ozone use for cooling water treatment, an extensive and very readable paper was presented by Edwards (1983). The paper was, at least in part, in response to the restriction promulgated by the U.S. governmental authorities that seriously restricted many of the practices that had been considered ‘standard practices’ in the business of treating cooling tower water. It was evident that alternative treatments would be required. Ozone was put forward as a possible replacement. In the discussion of the use of ozone as a treatment for cooling water, it is informative to note the observations made by Edwards (1983). These advantages are, in many respects, still relevant today and include: 1. Bacterial disinfection. 2. Viral inactivation. 3. Oxidation of soluble iron and manganese. 4. Decomplexing of organically bound manganese. 5. Colour removal. 6. Taste and odour removal. 7. Algae removal. 8. Oxidation of organics e.g., phenol, detergents and pesticides. 9. Micro-flocculation of dissolved organics. 10. Oxidation of inorganics e.g., cyanides, sulphides and nitrites. 11. Turbidity or suspended solids removal. 12. Pre-treatment for biological processes involving sand or other surfaces. Edwards (1983) elaborates on this list with some examples: 1. Disinfect bacteria e.g., E. coli, Streptococcus faecalis, Mycobacterium tuberculosis, Bacillus megaterium spores, 2. Destroy viruses e.g., Poliovirus, Porcine Picornavirus, Coxsasckie virus. 3. Improve coagulation and increase settleability. 4. Remove by oxidation reaction: a. Colour e.g., humic, fulvic and tannic acids. b. Taste e.g., natural and synthetic materials. c. Odour e.g., ortho-chlorophenol. 5. Remove algae.

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Although the wide-ranging advantages are directly related to the enhancement of the quality of drinking water, it has to be appreciated that these advantages will be applicable to a lesser or greater degree to the specialised control of biofouling in industrial equipment, particularly cooling water treatment. Rice and Wilkes (1991) however, raise questions regarding its universal use without critical thought to the underlying chemistry of particular systems. They suggest that these queries are of more significance in industrial cooling water circuits, than for instance, in air conditioning systems. Of particular respect is the inherent pH of the water. At pH 8 and higher pH values ozone decomposes to form hydroxyl radicals, which are stronger oxidising agents than molecular ozone itself. Puckorious (1991) points out the problems of using ozone in large scale industrial cooling water systems, particularly those that use raw or partially treated water, contaminated with by-products of a particular industrial process. Oil refining, chemical manufacture and power generation from fossil fuels are cited as examples where the use of ozone could be difficult and costly. Of particular influence is the basic design of the cooling water system and the heat exchangers it serves. An example of what could happen in an industrial system is the reaction of ozone with bromide ions that might be present in cooling water through leakage or for some other reason. Ozone readily reacts with bromide ions where they are present, to produce hypobromite ions (OBr). Hypobromous acid is a good biocide and does not decompose as ozone itself. There are industrial organisations that specialise in this kind of technology. The principal advantage of ozone is that it is a more powerful oxidising agent than chlorine and therefore it is thought to be the more effective as a biocide. Furthermore, it is considered not to produce compounds during its application that have anything like the toxic qualities of the products of chlorination. A potential problem in respect of the use of ozone as a biocide, is the fact that it reverts rapidly back to oxygen. Its half-life is of the order of 20 minutes. As a result it cannot be stored and therefore ozone has to be generated in situ, in sufficient quantities to meet the biocidal demand. In a different respect, this could be considered to be an advantage since it means that it will not persist when for instance, cooling water is discharged back to the environment. The usual method of in situ generation is by passing dry air through a field of silent electrical discharge produced by a high voltage alternating power supply. The use of oxygen for generation rather than air, would add

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considerably to operating costs, in addition to the investment in the generation equipment; there will also be the cost of electrical energy associated with the production of the ozone. The use of air rather than oxygen means that the ozonised air produced is in effect, diluted with nitrogen. The strong oxidising qualities of ozone mean that it has corrosive properties in respect of some materials of construction, such as copper, rubber and some plastic materials. It may be necessary therefore, where ozone is employed, to consider the use of materials of construction that are more expensive than those usually specified for plant construction. The intrinsic characteristics of ozone in terms of its stability, imposes some restrictions on its absorption into water. For this reason careful attention must be paid to efficient mass transfer of the ozone into the liquid water phase. The rather obvious method of achieving this is to maximise the contact surface area between the ozone-laden air and the water phase, by the generation of small-diameter bubbles. A further operating problem that has to be recognised is the possible extent of ozone decomposition within the water environment. The pH of the ozone solution is likely to affect the rate of decomposition. Rice and Wilkes (1991) listed and discussed some of the important aspects regarding the chemistry of ozone in water. The following notes are taken from their paper: 1. Ozone decomposes rapidly when added to water as compared with other biocidal oxidants used in water treatment. In very pure waters, this decomposition rate is slow and the half-life of ozone is of the order of hours. However, in real world recycling cooling tower waters that contain relatively large quantities of ozone-demanding impurities, the half-life of ozone is measured in terms of minutes, sometimes in the 1 to 10 minute range, after the immediate ozone demand of the water has been satisfied. With very dirty water the half-life of ozone can be even shorter. 2. The rate of ozone decomposition in water increases as the pH increases. At low pH (<7) and in the absence of UV radiation or hydrogen peroxide, the predominant form, is molecular ozone O3. However as the pH increases, the rate of decomposition of ozone to produce the very short-lived (in microseconds), hydroxyl free radical (HO) increases. Thus above pH 9, and particularly as the pH approaches 10, very little molecular ozone can be detected in solution compared to the amount dosed.

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3. Bicarbonate and carbonate anions are excellent scavengers (destroyers) of hydroxyl free radicals. In fact, carbonate ion is approximately 27 times more effective in scavenging hydroxyl free radicals than bicarbonate ion. Consequently, when the alkalinity of recycling cooling water is high (>200 mg/l) and the pH is also high (>9), the addition of ozone results in very little benefit. Ozone decomposes rapidly to hydroxyl-free radicals (at elevated pH), which are lost even faster from the system by reaction with bicarbonate and carbonate anions. 4. When bromide ions are present in water being treated with ozone, it is quickly oxidised to hypobromite ion (OBr-), which hydrolizes to hypobromous acid (HOBr). This reaction can take place if bromide ion happens to be present. Hypobromous acid is a good biocide and does not decompose as ozone does. Thus any free bromine can (and probably does) provide much of the biofouling control usually attributed to ozone alone. If free ammonia is present, just as bromide ion is oxidised by ozone bromamine will be produced, which is a biocide of somewhat less efficiency than hypobromous acid (Mitchell, 1985). 5. Some inorganic materials react very rapidly with ozone, in a matter of seconds, particularly sulphide, nitrite, bromide, iodide and cyanide ions. However, most inorganic materials cannot react with ozone (calcium, magnesium, silica, carbonate, bicarbonate and sulphate). Since some amounts of these materials are removed from solution when cooling waters are treated with ozone, their removal must be a result of solubility limitations or secondary reactions. Ozone produces some products of water constituents that react further with these inorganic materials to produce something insoluble, which can then be removed by filtration. 6. Even though some organic materials react rapidly with ozone (nonchlorinated alkenes and phenols), organic materials are rarely converted completely to carbon dioxide and water. Normal cooling tower ozonation conditions are insufficient to convert most of the organics present completely to CO2 and water. Most organics react only slowly with ozone (many aromatics, chlorinated organics, low molecular weight acids, aldehydes, mixed aldehyde-acids etc.). Conditions which favour the production of hydroxyl free radicals (pH 8–9, low alkalinity) also favour the more complete oxidation of most organic materials. 7. Since organic compounds form carbon dioxide only rarely, other (organic) end products of oxidation must be produced, particularly under cooling water ozonation conditions. These ozone end products

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generally are low molecular weight aldehydes, carboxylic acid, ketones and mixed aldehyde acids. The most stable organic end product of ozone oxidation of organic materials is oxalic acid. This compound is unaffected by molecular ozone but can be converted to CO2 and water by hydroxyl-free radicals. 8. Hypochlorite ion is oxidised by ozone to produce chloride ion (77%) and chlorate ion (23%). Hypochlorous acid is unaffected by ozone. Therefore, ‘free chlorine’ at pH well above 7.0 (at which it is primarily in the hypochlorite ion form) will be destroyed during ozonation. 9. Monochloramine also is destroyed by ozone but more slowly, forming chloride and nitrate ions. 10. Primary oxidation products formed by ozonation may result in the formation of secondary oxidation products (e.g., the formation of OBr/HOBr when ozone oxidises bromide ions; the precipitation of calcium ions as CaCO3 or as an insoluble organic. Any or all of these reactions may occur simultaneously. In practice however, one or the other reactions may predominate, depending on the reaction conditions and the chemical make-up of the water being treated with ozone. It is important in the light of these wide-ranging properties of ozone in water that careful consideration should be made, of the potential problems as well as the benefits that accrue from the use of ozone as a biocide. An additional factor that could influence decisions regarding the use of ozone concerns potential corrosion problems. Videla, Viera, Guiamet, and Staibano-Alais (1995) make the following point in respect of ozone in contact with two materials of construction used in process plants. In their laboratory tests they discovered that the corrosion potential vs time evolution for carbon steel is not markedly modified by ozone or ozone/ bromide mixtures. They found however, that a potential shift in the noble direction was observed for samples of stainless steel. Some work with ozone as a biocide (Kaur, Curan, and Bott 1991) confirmed that ozone first acts on the cell wall of microorganisms with which it comes into contact with cytoplasmic degradation (see Section 3.2), confirming much earlier work on E. coli, reported by McNair-Scott and Lesher (1963). Concentrations of less than 0.l mg/1 ozone are likely to affect cell metabolism but for a complete kill, concentrations of the order of 4 mg/1 are required. These data are in agreement with the standard concentration considered suitable for the disinfection of drinking water. In these tests the presence of the ozone did not seem to affect the carbon content.

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Tests on the effectiveness of ozone as a biocide were carried out on an experimental laboratory simulated cooling water system (Kaur, Bott, & Leadbeater, 1992). A single bacterium Pseudomonas fluorscens was used as the test bacterium as in other studies, on account of its slime-forming properties and association with industrial biofouling. From the single applications of ozone to an established biofilm, the importance of biofilm structural quality (or morphology) was identified. These qualities included the age of the biofilm, the biofilm mass and the conditioning of the biofilm by the presence of chlorine. The water velocity was 1 m/s. Some of the data from the tests are presented in Table 4.4. Table 4.4 Intermittent dosing with ozone Dose no.

1 2 3 4

Ozone Residual (mg/l)

Duration of Ozonation (hr)

Initial removal rate (%/min)

Biomass regrowth after 40 hours (mg/cm2)

0.06 0.04 0.03 0.02

3 2 3 3

1.56 1.35 0.93 1.30

2.24 13.98 4.68 5.09

The results suggest that there is virtually complete removal over a period of about three hours with relatively low ozone concentrations. Rapid regrowth occurs however, following the dosing period. The following points summarise the results of the tests as a whole: 1. As would be anticipated, the age of the biofilm influences the effectiveness of the ozone dose applied; as the age of the biofilm increases the resistance to removal increases. This is likely to be due to the consolidation of the biofilm with time and the production of EPS. 2. The greater the initial biomass prior to an application of ozone, the lower the rate of its removal under otherwise identical conditions. Again, this is almost certainly due to the consolidation effect with the greater mass of biofilm. 3. Biofilms that were conditioned by high total chlorine in the circulating water required a higher ozone residual (concentration), compared to biofilms grown in the presence of lower chlorine concentrations. The effect of the higher shear force as the water velocity is increased, on the removal of biofilm is illustrated on Fig. 4.7. The effects of some operating parameters on the efficiency of ozone as a biocide were studied by Sinha (2000). Although the work was directed

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100 0.6 m/s 1.0 m/s 1.4 m/s

% biofilm removed

80

Ozone residual 0.19 mg/l

60

40

20

0 0

1

2 Time (hours)

3

4

Figure 4.7 The effect of velocity on bioflim removal. (Courtesy of Ozone Sci. and Eng. 14, 6, 1992.)

towards the treatment of poultry waste, it does have a general relevance to the use of ozone as a biocide: 1. Ozone does not seem to distinguish between negative and positive bacteria. 2. Temperature in the range of 5–20  C does not appear to have a significant impact on the biocidal efficacy. 3. Increasing the pH from 6 to 9 enhances the biocidal efficacy. 4. Increasing the total organic carbon (TOC) from 2.00–170.00 mg/l) reduces biocidal efficiency. 5. Calcium carbonate in solution at a concentration of 3 mg/1 does not affect biocidal efficiency. The opportunities for the use of ozone in cooling water systems are very evident. On the other hand, despite its many assets as Puckorius (1991) states: ‘Ozone is not a "panacea”, nor is it a “stand-alone” treatment in most potential applications, It’s applicability depends upon specific criteria that must be evaluated prior to consideration or use. If it is to be “tried”, then it is extremely critical to have adequate and rapid monitoring tools in place to evaluate its performance.’ Glutaraldehyde Glutaraldehyde had, even in 1980, been in use for many years to control the growth of microorganisms (Gorman, Scott, and Russel 1980). It was

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initially used to disinfect medical equipment but subsequently it has found application in many industrial operations including oil fields, cooling towers, farm animal housing and metal working fluids (Grab, Emerich, Baron, & Smolik, 1990). It is potentially effective against a wide range of microorganisms, including gram-positive and gram-negative bacteria, bacterial spores, fungi, algae and viruses. For instance, the cell walls of bacteria contain free amino acids which can react with glutaraldehyde. As a consequence of its versatility and efficacy, it is to be found as the basis of many proprietary biocide formulations. Glutaraldehyde is a three carbon aliphatic chain with aldehyde groups at either end. OHC  CH2  CH2  CH2  CHO The function of glutaraldehyde is associated with dialdehyde chemistry but strongly influenced by the aromatic ring to which the aldehyde groups are attached, i.e., reacting with amine groups on proteins, nucleic acids and other microbial structures, such as cell walls (Ascenzi, Chen-Myers, and Gordon 2007). Stability is assured by cross links due to their complex nature (Cheung & Nimni, 1982). Grab et al. (1990) concluded that as the external surfaces of microorganisms all contain proteins that are comprised of amino acids, which may contain free amino groups, they are cross linked by glutaraldehyde. This reaction makes the cell membrane impermeable (Munton and Russell, 1973), which prevents the uptake of food and the discharge of waste products that result in cell death. System variables that affect the efficiency of glutaraldehyde as a biocide include its concentration, temperature, and flow velocity but the most important variable is pH. Grab et al. (1990) report the effect of pH on the effectiveness of glutaraldehyde as a biocide against E. coli, a gram-negative bacterium. The tests were made using 88 ppm of the biocide over a range of pH. The results of the tests are presented on Fig. 4.8. The figure demonstrates that the rate of kill was approximately twenty times faster at pH 8.5 than at pH 5.0. Even under the conditions at the lowest pH however, the biocide reduced the population of Escherichia coli to zero in approximately 22 hours. At a pH of 8.5 the time for a total kill was under an hour. Henry (1993) presents data on temperature and the effectiveness of glutaraldehyde demonstrating that the higher the temperature the shorter the time to inactivation of Bacillus subtilis spores. The effectiveness of gluteraldehyde has been studied by Taylor (1995). Results on a proprietary biocide containing 50% gluteraldehyde were

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108

Microbial populations, cfu/mL

107 106 105

pH 5.0

104 pH 8.5

103

pH 8.0

102

pH 7.5 pH 6.5

101

0

2

4

6

8

10 12 14 16 18 20 22 24 TIME (HOURS)

Figure 4.8 Glutaraldehyde rate of kill vs. pH. (Courtesy of Tappi Press, Atlanta, 1990.)

reported. The biocide solution was pulse fed into a laboratory pilot plant at the rate to yield a peak concentration of 100 mg/l. The pH was maintained at 7.0. Once biofilm development was observed in the biocide-free control equipment, the concentration of the biocide supplied to the test pilot plant was successively reduced till the initiation of biofilm development was observed. The results are shown in Fig. 4.9, (absorbance is a measure of the biofilm accumulation). It will be seen that the initiation period before the Pseudomonas fluorescens biofilm began to develop in the test facility without biocide was quite short, about two days. In comparison, no attached microbial growth was found to occur in the pilot plant while dosing with gluteraldehyde at a concentration of 100 mg/l. On the fourth day of the test, the concentration of the biocide was reduced to 50 mg/l but only an extremely small amount of biofilm was apparent as a result. The dose was reduced to 20 mg/l after six more days, producing a further small increase in biofilm accumulation. After fifteen days the application of glutaraldehyde was terminated, which resulted in a rapid increase in biofilm at the water velocity of 1.27 m/s. The indications were that there would have been an increase in biofilm accumulation at the lower velocity if the test had continued.

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2.5 Control : Velocity = 0.86 m/s Control : Velocity = 1.27 m/s

2.0

Infra-red limit

Absorbance

Test : Velocity = 0.86 m/s Test : Velocity = 1.27 m/s

1.5

1.0

0.5 100 mg/l

20 mg/l

No biocide

50 mg/l

0.0 0

2

4

6

8 10 Time (days)

12

14

16

18

Figure 4.9 Inhibition of bioflim development in the test rig with 50% glutaraldehyde biocide. (Courtesy of R. J. Taylor Ph. D. Thesis, University of Birmingham, 1996.)

Sim oes, Pereira, and Vieira (2003) have also investigated the effect of flow conditions on the effectiveness of glutaraldehyde. The results indicate that biofilms formed under laminar conditions are more easily inactivated than those formed under turbulent conditions. Biofilms formed under turbulent conditions are denser than their laminar flow counterparts with a substantial matrix of extra-polysaccharides (EPS). On the other hand, biofilms formed under laminar flow conditions are scattered on the surface to which they are attached and they have a rather uneven surface. The biocide therefore is relatively more able to penetrate this biofilm than those formed under turbulent conditions. Consequently, the glutaraldehyde demand is more substantial from a biofilm formed under turbulent conditions of flow than under laminar flow. The effectiveness therefore, of a given dose of the biocide could be less for biofilms formed under turbulent conditions but the difference could be reduced by the increased mass transfer under the turbulent flow regime. Other aldehydes that may be used as biocides include those that release formaldehyde or formaldehyde itself. A range of biocides that release formaldehyde has been studied by Rossmore and Sandorsi (1991). Some bacteria are resistant to formaldehyde.

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Hydrogen Peroxide Hydrogen peroxide is a powerful oxidising agent with biocidal properties. It is an environmentally friendly biocide in that it breaks down to water and oxygen. Although widely used in the medical world as an effective antiseptic wound cleaner and surface disinfectant, it does not appear to be used extensively in processing industries. The reason for this is not entirely clear but it is probably associated with the fact that it is available in solution, which would incur relatively high transportation costs between point of manufacture and point of application. It has been used in gaseous form for fumigation purposes (McDonnell, 2007). Its potential as a biocide was demonstrated by Taylor (1995) in a circulating test facility having an established biofilm of approximately 80mm thickness and a hydrogen peroxide concentration maintained at 0.22 mg/l. The result is demonstrated in Fig. 4.10. Over the space of two hours, the reduction of biofilm was considerable i.e., of the order of 60% at the two water velocities employed (0.8 and 1.27 m/s). 0.30

Absorbance

0.25 0.20 0.15 0.10 0.05

Velocity = 0.86 m/s Velocity = 1.27 m/s

0.00 0

20

40

60 80 Time (minutes)

100

120

140

Figure 4.10 Bioflim removal using 0.22 md/l of hydrogen peroxide. (Courtesy of R. J. Taylor Ph. D. Thesis, University of Birmingham, 1996.)

2,2–Dibromo-3-nitrilopropionamide (DBNPA) This biocide is considered to be environmentally friendly but for universal acceptance its effectiveness must be seen to at least, approach that of more traditional biocides, such as chlorine. Some tests to establish effectiveness in comparison to chlorine were carried out by Cowbourne, Lester, and Bott (2001) using a laboratory pilot plant with planktonic and sessile Pseudomonas fluorescens as the biofouling organism and a water velocity of 1 m/s across the

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test surfaces of glass and stainless steel. The velocity was chosen on the grounds that it was typical of velocities often used in industrial cooling water applications. Stainless steel is often the material of construction in industrial equipment, whereas glass is only employed in special circumstances. Nevertheless, a comparison between these two surfaces is a useful demonstration of the opportunities for the use of DBNPA. The results are presented in Tables 4.5a and 4.5b. From the results it would appear that cells residing on the stainless steel took longer to eradicate compared to those on the glass surfaces. In addition, the results indicate that for the same conditions, more cells are able to adhere to the stainless steel. The greater roughness of the stainless steel is likely to influence this result (see Chapter 3). It is not surprising therefore, that it took longer generally, to obtain a total kill of the cells resident on the stainless steel. All the cells were destroyed on both surfaces in up to 120 hours using a biocide concentration of 25 mg/1. The dosing regimen required for an industrial application would necessarily be related to the severity of the particular biofouling problem being tackled. The optimum cost would depend on the dose levels coupled to the dosing strategy, whether continuous or intermittent.

Table 4.5a Viable cell counts before biocide dosing Cell count (cells/ml) before dosing Dose of DBNPA mg/l Planktonic Sessile on glass Sessile on stainless steel

2.1  107 2.1  107 2.2  107

100 50 25

2.6  104 2.1  104 2.0  104

1.9  105 2.3  105 2.1  105

Table 4.5b Cell death after dosing Cell count (cells/ml % of original) after dosing Planktonic after DBNPA dose mg/l 24 hours Sessile on glass Sessile on stainless steel

100

100

50

100

25

100

95 after 24 hours. 100 after 72 hours. 95 after 24 hours. 100 after 72 hours. 98 after 96 hours. 100 after 120 hours.

55 after 24 hours. 100 after 72 hours. 75 after 24 hours. 100 after 95 hours. 98 after 96 hours. 100 after 120 hours.

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Amines There is a wide range of compounds that may be grouped under this heading, including straight chain amines to quaternary compounds. These compounds are surface active and therefore can disperse slime masses. If they are employed in conjunction with biocides such as chlorine, they facilitate access thereby improving control. The general structure of a straight chain is: R  CHO and that of a quaternary compound is +

R1 R4

N

R2

Halide -

R3

R represents alkyl, aryl or heterocyclic radical substitute containing 8 to 25 carbon atoms bound to the nitrogen atom. Quaternary compounds are generally most effective against algae and bacteria in the alkaline pH range. Their biostatic action is attributed to the cationic charge, which forms an electrostatic bond with any negatively charged sites on the cell wall of the microorganisms. As a result, stresses are created in the cell wall that cause cell lysis. Cell death is also brought about by protein denaturation, which affects the flow of nutrients into the cell. The dispersant quality of these compounds can lead to complications with other additives that may be in use such as corrosion inhibitors and scale preventatives thereby affecting their effectiveness. Leaks of organic process liquids and other contaminating substances derived from the origins of the water in use as a coolant may lead to operating problems such as foaming in cooling towers and reduced biocidal effectiveness. Organo-sulphur Compounds Organo-sulphur compounds are generally applicable to cooling water systems. They function either by competitively or non-competitively inhibiting cell growth. The pH of the system affects their function. In the respiration of microorganisms, a low energy ferric (Fe3þ) cytochrome accepts an electron and is transformed to a high energy ferrous ion (Fe2þ) cytochrome state. These reactions provide energy that is vital to the life of the microorganism. Through competition, organo-sulphur compounds remove the ferric ion by incorporating it in an iron salt, thereby preventing

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the transfer of energy which causes immediate cell death. Inhibition that is not based on competing reactions is also possible with organo-sulphur compounds. In simple terms, the compound induces the microorganism to accept a chemical substance that will ultimately lead to cell death. Methylene bis-thiocyanate is an organo-sulphur compound that is effective against algae, fungi and bacteria, particularly species employing sulphate reducing reactions through competitive action. Although it is an effective biocide in cooling water systems, unfortunately methylene bisthiocyanate is not very soluble in water, so proprietary formulations include dispersants. The presence of a dispersant enhances effectiveness such that the biocide can penetrate algal and bacterial slime layers. It is pH sensitive so that it is not recommended for water systems with a pH value of 8.0 or higher. Non-competitive organo-sulphur compounds decompose in water to form solutions similar in character to microbial metabolites and through their substitution cause cell death. The pH of the water is an important variable. Sulphones are most effective in water with a pH in the range 6.5– 7.5, whereas for thiones the optimum pH is in the range 7–8. Sodium dimethyl-dithiocarbamate and disodium ethylene bis-dithiocarbamate are organo-sulphur compounds that are very effective biocides. They are readily soluble in water and most effective in water with a pH of 7 and above.

4.2.5. Immobilised Biocides Hu¨ttinger (1987) made the suggestion of binding biocides to the surface that was likely to be the host for a biofilm. It is suggested that binding could be accomplished without a spacer or coupling agent. The biocide must have a reactive group which cannot be the group that performs the biocidal function. Special biocides have been developed, which are able to be bound onto the surface of different materials of construction made possible by applying silane coupling agents (Hu¨ttinger, 1987). According to this author, application of the products to the required surface is not difficult. Despite the possibilities of the technology it would appear that it has not been widely adopted. This may well be due to the difficulties of maintaining the integrity of the biocidal film, particularly during off-line cleaning operations that might become necessary and the potential difficulties associated with renewing its activity. However, it would appear to have a potential for the control of biofilm formation in certain operations.

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4.2.6. Microparticle Transport of Biocides In medical technology, the transport of drugs to the point of need can be facilitated by using microparticles as drug carriers. A report by Ferreira et al. (2009) examines the concept for the control of biofouling in industrial equipment. The efficiency of microparticles (average size 4 mm) carrying the quaternary compound benzyldimethyldodecylammonium chloride (BDMDAC) was assessed in relation to Pseudomonas fluorescens in planktonic and biofilm states. The preparation of the impregnated polystyrene particles was done by a layer by layer technique. Oppositely charged molecules of polyethyleimine, sodium polystyrene and the BDMDAC were assembled onto core particles of polystyrene. The tests were very revealing. The survival of planktonic bacteria in contact with the biocide particles was zero. The effects of the application of the biocide-coated particles at 0.87 mg/l for 30 and 60 minutes against biofilms were again assessed in terms of bacterial survival ratio. Figure 4.11 provides the results. Exposure of the biofilm to the biocide-carrying particles resulted in a viability decrease of 60.5% and 66.5 % of the total biofilm population for 30 and 60 minute exposure times respectively. Although the concentration of biocide in these tests did not completely eliminate the bacterial activity, Ferreira et al. (2009) suggested that raising the biocide concentration would increase the level of deactivation. The technology shows promise but there are three important questions that need reliable answers: 1. The cost of preparing the biocide impregnated particles. 2. The behaviour of the particles in a circulating cooling water system. Could they accumulate for instance, in cooling towers? 1.4

Survival ratio

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t = 30 min

t = 60 min

1 0.8 0.6 0.4 0.2 0 Saline solution

PS-PEI/PSS/BDMDAC

PS

Figure 4.11 Survival ratio of Pseudomonas fluorescens bioflims exposed to saline (control), 0.87 mg/l of BDMDAC in coated particles and PS for two different exposure periods. PS (polystyrene cores), PSS (sodium polystyrene sulphonate), PEI (polyethyleneimine), BDMDAC (benzyldimethyldodecylammonium chloride).

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3. Since the particles are quite small, any necessity for the removal of the particles before disposal of the water could be a serious difficulty and hence expensive. It will be of considerable interest to see whether or not these questions receive encouraging answers.

4.2.7. Removal of Biofouling with Biocides In general, the philosophy governing the use of a biocide is to prevent biofouling from occurring. It may be helpful however, to consider the removal of biofilms by chemical means as an alternative to off-line cleaning (discussed in Chapter 5) so that production could be maintained. A serious difficulty that could arise might be the disposal of the potentially concentrated wash-liquor after the cleaning operation. Research by Bott and Taylor (1997) illustrated the possibilities of chemical cleaning. The laboratory pilot plant study involved passing simulated cooling water through tubes at two different predetermined bulk velocities of 0.86 and 1.27 m/s. These velocities were chosen on the basis that a rule of thumb in cooling water applications is to operate at a velocity of around 1 m/s. The dimensions of the tubes were chosen so that at these two velocities, the Reynolds number was 14,000. Previous research (Pujo and Bott, 1991) had demonstrated that maximum thickness of the slimeforming bacterium Pseudomonas fluorescens occurred at Reynolds numbers of 10,000–15,000 in tubes of similar diameter to those commonly found in shell and tube heat exchangers used in power plant steam condensers. Infrared absorbance was used to assess the quantity of biofilm on the test surfaces. Removal of established biofilms consisting of Pseudomonas fluorescens was investigated using four biocides: chlorine derived from sodium hypochlorite, hydrogen peroxide, peracetic acid and potassium permanganate. The data were obtained over a test period of 24 hours. The operating conditions used in the experiments were: Temperature pH Variable cell count Water velocities Reynolds number

30  C 7 1e2  107 cfu/ml 0.86, 1.27 m/s 14,000

The data recorded in relation to the four biocides are presented in Figs. 4.12 to 4.17.

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Chlorine From the data presented in Figs. 4.12 to 4.14 .it will be seen that total biofilm removal was only achieved under conditions of the experiments at the highest chlorine concentration of 13.6 mg/l of active chlorine. The effect of increasing the velocity from 0.86 to 1.27 m/s is clear from the data. At the lowest chlorine concentration and lowest velocity, only 30% of the 100 1.27 m/s 0.86 m/s

% Biofilm remaining

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Figure 4.12 Removal of established bioflim using 4.7 mg/l of active chlorine. (Courtesy A. I. Ch. E.) 100 1.27 m/s 0.86 m/s

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Figure 4.13 Removal of established bioflim using 6.3 mg/l active chlorine. (Courtesy A. I. Ch. E.)

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Figure 4.14 Removal of established bioflim using 13.6 mg/l active chlorine. (Courtesy A. I. Ch. E.)

biofilm was removed; whereas if the velocity was increased to 1.2 m/s, removal was raised to 50% of the original biofilm. Hydrogen Peroxide The data obtained from the use of hydrogen peroxide is presented in Fig. 4.15 (using data identical to that in Fig. 4.10 but differently presented). 100 1.27 m/s 0.86 m/s

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Figure 4.15 Removal of established bioflim using 0.22 mg/l hydrogen peroxide. (Courtesy A. I. Ch. E.)

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Under the conditions of the tests it would appear that raising the velocity to 1.27 m/s from 0.86 m/s makes very little difference to the amount of biofilm removed. A possible reason suggested for this similarity is that the hydrogen peroxide concentration at the water/biofilm interface was sufficiently high to sustain a suitable rate of reaction. Peracetic Acid The results from the application of 0.4 mg/l of peracetic acid are presented in Fig. 4.16. The removal of the established biofilm amounted to about 35% and 53% for velocities of 0.86 and 1.27 m/s respectively. The period of exposure was less than in the previous experiments but it was evident that the biocide is less effective at the lower velocity. It is not clear why this should be so. 100 1.27 m/s 0.86 m/s

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Figure 4.16 Removal of established bioflim using 0.4 mg/l peracetic acid. (Courtesy A. I. Ch. E.)

Potassium Permanganate In the experiments with potassium permanganate (Fig. 4.17) there appears to be an increase in the biofilm accumulation in the period of the test. A possible explanation for this is the possibility that manganese dioxide, a by-product of the reduction of the potassium permanganate being deposited on the biofilm, could have affected the effectiveness of the biocide or made the deposit less susceptible to the removal forces.

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Figure 4.17 Effect on established bioflim using 10 mg/l potassium permanganate. (Courtesy A. I. Ch. E.)

4.2.8. Biodispersants Biodispersants, including synthetic organic compounds, polymers and surface active agents are generally applied to penetrate and disperse the microorganisms in a biomass. Some dispersants also act as biocides. Often a biodispersant is used in conjunction with a biocide since the effectiveness of the biocide is enhanced. The scattering of clumps of biomass or individual microorganisms, makes them more vulnerable to the biocide by the associated increase in exposed surface area. The selection of the proper dispersant for any operating system is based on actual analysis of the deposit associated with the particular carrier e.g. water. The effectiveness of a biodispersant lauryl dimethylamine oxide was tested in a laboratory pilot plant by Cowbourne, Lester and Bott (2001) for effectiveness against Pseudomonas fluorescens biofilms. A concentration of 10mg/l was applied from the beginning of the test, which lasted for almost one thousand hours. Four water velocities were used in the test; 0.3, 0.5, 1.0 and 2.0 m/s. There was little growth on the glass surfaces in the first 200– 400 hours with a very small amount of biofilm recorded at each velocity. After 528 hours, the addition of the biodispersant was stopped but the inhibition of growth continued for a further 200–300 hours. It may be assumed that that this effect was due to the retention of the biodispersant on the test surface till it was washed away by the flowing water. It is apparent

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from the data obtained that the extent of the biodispersant retention when the velocity was 2 m/s was much less than at the lower velocities. At the lowest velocity of 0.3 m/s there was a rapid increase in biofilm accumulation after 900 hours. It is possible that this effect was due to the low shear forces acting at 0.3 m/s enabling the microorganisms to attach where the biodispersant had become depleted. It is likely that these results are dependent on the quality of the glass surface and if the test surface had been a different material other than glass, the result could have been different. Although the data obtained in this test may be limited it does demonstrate the potential of biodispersants for biofouling control.

4.2.9. Concluding Remarks on Chemical Control When planning for biocide treatment to control biofouling, Grab and Rossmore (1992) state that it is essential to consider the possibility that a particular biocide may be species specific. These authors also point out that the method of assessing the species present in a system might be misleading. The results of their study indicate that biocide levels normally considered to be effective may actually be unable to control certain microorganisms. It is clear that the use of an appropriate biocide for a particular defined problem in biofouling can be effective in control. It has to be borne in mind, however, that process leaks can reduce biocide effectiveness. The pH of the water may be an important factor in the choice of biocides. It is also necessary when choosing a suitable biocide that its ultimate destination is recognised. Discharge to the environment may be covered by regulations, for instance, the European Union Biocidal Product Directive 98/8/CE, so that it may be necessary to remove or destroy the biocide before discharge or employ a different and legally acceptable biocide. The supplier of a biocide will be keen to offer advice in this respect. The technique of destruction will, to a larger or lesser degree, increase the plant operating costs. An alternative possibility would be to dilute the concentration of the biocide to acceptable limits. The employment of this method of disposal may not be feasible due to limited water availability. By way of illustration of the choice of alternatives, Casanueva-Robles et al. (2005) made some practical comments concerning the use of peracetic acid to replace sodium hypochlorite, based on direct in situ tests, with sea water used for cooling in a large power station. They concluded that peracetic acid is entirely satisfactory as a substitute, particularly in the light of the current and future legislation that covers the discharge of organochlorines. A major problem however, is that peracetic acid is ten

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times the cost of sodium hypochlorite! Attention to the dosing strategy (discussed earlier in this chapter) coupled with the potentially lower discharge cost could make the alternative an attractive proposition. In order to facilitate the use of biocides for control of biofouling, it is worth considering the use of a biodispersant to reduce the required concentration of biocide for control. There may be advantages in this policy in respect of cooling water discharge back to the environment. Many proprietary slimicides contain dispersants to enhance effectiveness. An important consideration would of course be the cost.

4.3. PHYSICAL METHODS OF CONTROL 4.3.1. Introduction An alternative to the use of chemicals for the control of biofouling (thus avoiding potential problems associated with their use, such as cost and environmental hazards) are methods based as the name implies, on physical technologies. An additional incentive to investigate the opportunities of physical control is the possibility of increased and stricter legislation concerning the development and use of biocides. Probably the earliest technique of any kind to combat the accumulation of microorganisms on the surfaces of industrial process equipment was reversal of flow. In Chapter 3 it was pointed out that it is often the case that microorganisms forming biofilms on the surfaces or ducts through which they are being carried by flowing water, can become oriented to minimise the effects of the imposed removal forces created by the flowing water. Under the reverse flow conditions the microorganisms are therefore more vulnerable to removal. It can be an automatic procedure that eliminates the need for action by an operator. The technique however, is likely to be not all that effective, which over the years, has meant that alternative techniques have been employed. Pulsed flow is another relatively simple method used to control biofouling. The associated rapid changes in velocity impose removal forces on the biofilm. A difficulty however, is to ensure that the changes in velocity are rapid enough to be effective. Over relatively long distances therefore, the effectiveness is likely to be reduced.

4.3.2. Ultraviolet Light and Gamma Rays Ultraviolet light and gamma rays have long been used to sterilise liquids, particularly in medical applications. Although attractive in that no

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chemicals are involved, a serious drawback to use in industrial operations is that microorganisms have to be ‘seen’ by the radiation. Such a requirement is extremely difficult or often impossible to achieve, in industrial equipment largely due to the scale of industrial operations and the fact that the techniques are essentially applicable to the control of planktonic microorganisms. Penetration into equipment, such as the inside of tubes in a shell and tube heat exchanger, to tackle biofouling on the inside of the tubes would be virtually impossible. The technique could have some limited application in say, the treatment of cooling water to reduce biofouling, if the incoming bulk water was subjected to ultraviolet light or gamma rays so that the majority of the planktonic microorganisms would be killed. The survivors however, would still have the ability to colonise and cause biofouling of the heat transfer surfaces that would ultimately require treatment to maintain operational efficiency. Nevertheless, the technique may be useful under certain circumstances and it should be considered when alternative solutions to a potential biofouling problem are being reviewed.

4.3.3. Sponge Rubber Ball Circulation A physical technique that has been used for many years in connection with the control of biofilm formation on the cooling water side of power station condensers is the circulation of sponge rubber balls (the so-called Taprogge System). The technique involves the use of sponge rubber balls that have a slightly larger diameter than the internal diameter of the condenser tubes through which the cooling water passes. A schematic diagram of the system is shown on Fig. 4.18 demonstrating the arrangement that could be used on a power station condenser system. The oversize balls are squeezed as they pass randomly through the tubes, to ensure that the surfaces of the balls are in contact with the tube surface. The concept is that as the balls are pushed through the tubes, they wipe away any biofilm that has accumulated on the inside surface of the tubes since the previous passage of a ball through the tube. Before the water is discharged or passed to a cooling tower for recycling, the balls are filtered off and returned to the inlet of the condenser. The quality of the balls is such that the amount of wear of the tube surfaces is a minimum. It has to be recognised however, that the circulating balls themselves wear and so they gradually become less and less effective. In order to overcome this problem, balls that become worn are filtered off to be replaced by new balls. Balls with an abrasive

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Once through tubular heat exchanger

Ball collector Cooling water in

Ball pump

Strainer (Ball recovery)

Cooling water out

Figure 4.18 The Taprogge system for the circulation of sponge rubber balls. (Courtesy Elsevier.)

surface can be used for tenacious deposits or for periodically cleaning the tube inner surfaces, for instance, to restore effective operation after shutdown for periodic maintenance. The effectiveness of the system is clearly shown in Fig. 4.19. The circulating balls maintained heat transfer in a steam condenser in a nuclear power station, in comparison with the situation when no balls were present in the system. The application of balls to the individual tubes is a random process, but a sufficient quantity of balls is maintained in the system to ensure, even though it is a random process, that each tube ‘sees’ a ball on an average regular basis. Elmir (1985) suggested that an optimum circulation rate ensured that each tube in the system received a ball 12 times per hour. Problems can occur if for some reason a tube becomes blocked by a ball that has the effect of reducing the heat transfer area available or tube plate fouling redirects the flow distribution pattern in some way. The application of the balls need not necessarily be continuous. Where the problem is not likely to be too severe, the injection of balls could be suitably planned on an intermittent basis. Elmir (1985) pointed out that a system of one hour dosing daily, with the standard frequency of 12 balls per tube per hour, could be replaced with six balls per tube per hour for a period of two hours. Changes of this kind are probably best undertaken through experience with the

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Figure 4.19 The effectiveness of the Taprogge system. (Courtesy Taprogge Gesellshaft, Wetter.)

particular system in mind. Furthermore, it must be appreciated that in this connection, wide deviations from the standard of 12 balls per tube per hour could lead to accumulation problems. In making judgements regarding the frequency of ball injection it would be necessary to take account of a number of other factors including primarily water quality, particularly in respect of deposit accumulation. The unwanted deposit may contain particles of one sort or another depending on the source of the water, in addition to microorganisms. Another factor that requires careful attention is the material of construction of the condenser tubes and any associated corrosion protection that might be affected by the passage of the balls. Corrosion prevention is often dependent on the establishment of a protective layer on the tube surface. It is imperative that any sort of online cleaning technique, such as the passage of balls, whether with abrasive surfaces or not, does not remove the protective layer along with the biofouling accumulation. If this should be likely to occur, then continuous or regular intermittent application of corrosion inhibiters would be required. The programme would most certainly have to be devised largely

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on experience of the particular plant involved, as it is impossible to provide hard and fast rules. Each system will be unique. The steam condensers represent only part of the investment in the cooling system on a power station. Other parts of the cooling water system are not amenable to the circulation of sponge rubber balls to prevent potential biofouling, such as water pipelines, cooling towers and their basins. Other supplementary techniques, such as biocide dosing would need to be considered.. It is probably true that the technique of circulating sponge rubber balls is only suitable for ‘once through’ condenser tubes, although it may be possible in ‘u-tube’ coolers or possibly two passes on a normal design of shell and tube condenser. It will be obvious that the system cannot be employed for the shell-side of shell and tube heat exchangers or plate heat exchangers.

4.3.4. Brush and Cage System The principle of the brush and cage system is similar in concept to the circulation of sponge rubber balls, in that it uses a projectile to remove deposits from the inside of straight tubes. In simple terms it involves passing through each tube, a brush usually made from metal wires or plastic filaments under the action of water flow. At either end of the exchanger there is a cage which “collects” the brush projectile at the outlet from each tube. After a period of time based on operating experience, the flow is reversed and the brush projectile is carried through the tube back to the header box at the end of the tube. The frequency of reversal is based on experience of the biofouling potential. The shuttling of the brush back and forth, maintains a clean inner surface of the tubes. It is claimed that the maintenance of the brush and cage system is less than the circulation of sponge rubber balls. One disadvantage can be envisaged but which might not be significant, is that there will be momentary interruptions in the water flow at the time of the flow reversal. Because of the complex pumping arrangement that will be necessary and the automatic control equipment required, it is unlikely that this technology could be used for power station condensers. The application is probably best suited to heat exchangers, such as product condensers or coolers.

4.3.5. Air or Gas Injection A technique similar in principle to the use of projectiles, is the injection of ‘slugs’ of air or a suitable gas. The method is often referred to as ‘rumbling’.

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The principle is that extensive turbulence is created by the passage of the slugs of gas at the tube wall that knocks off any accumulation of biofilm. The technique is usually applied where accessibility is limited, as for instance, in the shell-side of a shell and tube heat exchanger. It has to be recognised however, that the passage of water through a shell that is likely to form a biofilm cannot be considered as good practice because of the difficulties of maintaining efficient heat transfer.

4.3.6. Use of Inserts An insert is primarily designed to disrupt the laminar sub-layer on the inner walls of tubes in a tubular heat exchanger and to improve heat transfer efficiency (see Chapter 2). Inserts have been used extensively in the petroleum industry with success. The general advantages of the use of inserts in heat exchangers have been listed as energy savings, extended run lengths (i.e., between ‘cleans’), reduction in maintenance costs, production enhancement and debottle-necking, with reduction of emissions as a side effect (Krueger and Pouponnot, 2009) At the same time, it has been established that inserts have the capacity to limit deposits through the accompanying increase in the drag forces applied to any deposit on the tube wall resulting from flow conditions at the wall. The penalty of this technology however, is that the back-pressure is increased due to the flow resistance of the insert, thereby increasing the pumping costs. Nevertheless drawbacks that may be associated with the use of chemicals for control, such as high cost and environmental problems, have prompted examination of possible alternative technologies in water systems, including the use of inserts (Bott, 2001, 2009). There would appear to be no reason why inserts should not be used more extensively to combat the formation of biofouling in water systems, except perhaps the investment cost associated with the installation and the cleaning costs that might be involved. The additional power consumption resulting from the increased back-pressure could easily be accommodated by reducing the inefficiencies in the heat transfer caused by the biofouling. 1. Static Inserts An example of a static insert (Hitran, fabricated by Cal Gavin) consists of a wire-wound device (see Fig. 4.20). The original concept was described by Gough and Rogers (1987). The loops of the insert are arranged to press against the inner tube wall through which say, cooling water is flowing, so that the insert is held firmly in place. The number of loops per unit length and

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Figure 4.20 An example of a wire wound matrix insert. (Courtesy of Cal Gavin Ltd., Alcester.)

the material, from which the device is made, will be influenced by the particular application and the allowable pressure drop through the tube. There will be an increase in the pressure drop due to the flow resistance of the insert. Its magnitude will depend on the number of loops there are per unit length of tube. There are other examples of static inserts that are available. The possibilities of static inserts for biofouling control have been demonstrated. Work by Wills, Bott, and Gibbard (2000) has illustrated the potential of the Hitran device to combat biofilm formation. Using a laboratory simulation of a cooling water circuit, the effectiveness of the insert was established. Figure 4.21 (Bott, 2001) demonstrates the reduction in biofilm formation at two different velocities (0.86 and 1.27 m/s) by the inclusion of the insert in the tube. The water flow velocities are based on the volumetric flow rate and empty tube flow area. The vertical axis on the figures, labelled ‘Absorbance’, refers to infrared absorbance and is a measure of the biofilm accumulation on the tubeda technique developed at the University of Birmingham (Bott, 2003) to assess biofilm growth at specified points on the inside surfaces of tubes in laboratory simulated cooling water systems. The figures show the usual sigmoidal growth curve discussed in Chapter 3. Figure 4.21 also demonstrates the reduction in biofilm formation at the higher velocity, due to the increased removal forces at the velocity of 1.27 m/s as compared to the lower velocity of 0.86 m/s. The lengthening of the induction phase at the higher velocity can be attributed to the larger removal forces at the tube surface, as a result of the effect of the insert on the ability of fresh cells to colonise the exposed surface. The shorter exponential phase could be due to the increased nutrients available to bacteria already

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2.5

0.86 m/s flow velocity - no insert 1.27 m/s flow velocity - no insert 0.86 m/s flow velocity - with insert 1.27 m/s flow velocity - with insert

ABSORBANCE

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Figure 4.21 The effect of inserts on microbial accumulation. (Courtesy I. Chem. E.)

attached to the surface, resulting from the increased mass transfer due to the greater turbulence brought about by the presence of the insert. After a period of time, visual examination of the insert revealed as might be anticipated, that bacteria had colonised the core of the insert and there were also ‘streamers’, presumably consisting of cells and extracellular material. Although these appendages are not likely to affect heat transfer they could well increase the pressure drop through the exchanger. In addition there were ‘clean’ areas on the associated tube surface demonstrating that the disruption to the flow regime that the insert caused was sufficient to eliminate the possibility of biofilm growth. There are many different arrangements of wires and loops that may be made on the Hitran insert. Since the insert tested was picked at random, there could be better arrangements for improving the reduction in the accumulation of biofilms under the test conditions. The final choice of structure would depend on a compromise between reduced biofilm formation and increased pressure drop and the associated pumping costs. 2. Oscillating Inserts The technology is based on the oscillating action of a metal device which is inserted into the metal tubes of a tubular heat exchanger, such as a steam condenser in a power station or a product cooler in a chemical plant. Due to

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the increased turbulence in the flowing water resulting from the movement of the oscillating device and its scraping action on the tube walls any deposits are removed. An example is the patented ‘Spirelf ’ system described by Baidelet and Krueger (2000). The device consists of a flexible metal spiral device stretched inside the heat exchanger tubes and held in place by straight fixing wires. Attachment of the device at both ends of a tube permits controlled vibrations and the avoidance of mechanical tube erosion. The vibrations are generated both radially and axially, so that removal of deposits may be made by a scraping action in different directions, thereby ensuring that there are no areas that remain unchallenged by the device. There are other devices available that create scraping or other forces at the surface through some sort of movement originally devised to improve heat transfer that could be used to remove deposits. The penalty in the application of these oscillating devices are the additional capital cost, the additional pumping energy requirement and any associated maintenance costs. Nevertheless in many examples, these costs will be justified by the improved process plant performance.

4.3.7. Flexible Tubes A simple physical technique involving the use of flexible plastic tubes in shell and tube heat exchangers has been used to counteract biofouling. The tubes oscillate in response to the flow conditions and the movement is intended to shake off any deposit that has formed and prevent attachment. Due to the rather fragile nature of the tubes it probably has limited application. It could possibly be best used in conjunction with a suitable biocide.

4.3.8. Fluidised Beds The use of fluidised bed technology for heat transfer introduces the concept of self-cleaning heat transfer. The operation is simply to apply a fluidised bed of particles to the tubes of a vertical shell and tube heat exchanger. Although the technology has been available since the 1970s (Klaren, 2003) and improved over the years, it does not appear to have been widely used for cooling water systems. Nevertheless it may prove useful in some applications, although where considerable volumes of water are involved, as in a large power station, it might prove to be difficult to maintain the particles in suspension. A potential higher capital cost may also be a deterrent.

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Despite these reservations however it is worthy of careful consideration for the control of biofouling. Klaren (2003) outlines the principle of operation. The fouling liquid– cooling water for instance, is fed upward through the tubes of a vertical shell and tube heat exchanger that has specially designed inlet and outlet channels. Solid particles are also fed into the inlet where an internal flow system provides uniform distribution of the liquid and suspended particles across the internal surfaces of the tube bundle. The particles are carried through the tubes by the upward flow of the liquid and they impart a scraping effect on the walls of the tubes, thereby removing any deposit at an early stage in the formation of a fouling layer. The particles can be cut metal wire, glass or ceramic balls, with diameters in the range of 1–4 mm. In a separator at the top of the vertical exchanger, the particles disengage from the liquid and are returned to the inlet channel and so the cycle of operation is continuous. Amongst the examples of fluidised bed heat exchangers, Klaren and de Boer (2007) cite a unit using untreated sea water with glass particles to provide the cleaning action. It is suggested that the technology would be suitable for off shore applications.

4.3.9. Coating of Surfaces As indicated in Chapter 3 the adhesion of microorganisms to surfaces is affected by the quality of the surface both in terms of its physicochemical properties and the roughness of the surface. Both these characteristics can be modified by the application of a suitable coating but surface modification by coating is a relatively modern technology. It is particularly relevant to the control of biofouling. The use of polymer based coatings e.g., Polytetrafluoroethylene (PTFE), could facilitate application of the technology. Although the surface free energy (See Chapter 3) of polymers is relatively low compared with other materials providing counter adhesive properties, their abrasive properties are rather poor. As a result, newer polymeric coating materials that combine reduced adhesion and enhanced abrasion power have been developed. It has to be appreciated however, that a surface treatment may be vulnerable to mechanical off-line cleaning procedures. Paints, some containing biocides, are commonly used to reduce the adhesion of biofilms to ships’ hulls (Railkin, 2004). The use of paint as such, is not likely to be appropriate in industrial operations due to the difficulties associated with maintaining the integrity of the paint film in processing equipment such as tubes and enclosed vessels subject from time to time, to off-line cleaning procedures (See Chapter 5). Furthermore where heat

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transfer is involved, the paint layer would act as a resistance to the transfer of heat thereby reducing the efficiency of the heat exchanger. A fouling layer in its own right! A technique that is used in medical applications is to coat the surface either to reduce biofouling (sterilisation) or in diagnosis, to increase the quantity of a microorganism retained on a surface. Santos (1993) studied the application of Glassclad HP. and Glassclad IM (manufactured by Fluorochem Ltd. principally for use in medical applications) as a possible means of controlling industrial biofouling. The former is a heparin-modified siloxane used to reduce adhesion on glass exposed to human fluids. Glassclad IM. is a polyethylenimine-modified polymer used to coat glass surfaces to improve attachment of microorganisms for medical investigations. It was thought that the technology might be applied to surfaces to reduce unwanted industrial biofouling or to enhance biofilm retention where the biofilm plays an important role in processing, such as with trickle filters. Tests with these coatings on the inside of glass tubes through which distilled water contaminated with Pseudomonas fluorescens flowed at two extremes of velocity (0.5 and 2.5 m/s) were carried out. The biofilm accumulation was determined using infrared absorbance. The tests lasted for over 1000 hours. The results are presented in Figs. 4.22 and 4.23. The results indicate that at a velocity of 2.5 m/s there was virtually no biofilm on any of the surfaces tested caused by the high shear effects at this 0.20 GC HP 0.5 m/s GC HP 2.5 m/s

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Figure 4.22 Bioflim development on glass coated with Glass Clad HP at flow velocities of 0.5 and 2.5 m/s. (Courtesy of Santos, Ph.D. Thesis, University of Birmingham, 1993.)

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Time (hours)

Figure 4.23 Bioflim development on glass coated with Glass Clad IM at flow velocities of 0.5% and 2.5 m/s. (Courtesy of Santos, Ph.D. Thesis, University of Birmingham, 1993.)

relatively high velocity. Differences however, were obtained at the lower velocity confirming that the adhesion of Pseudomonas fluorescens was less on the surface treated with Glassclad HP. resin (Fig. 4.22). Fig. 4.23 shows that the accumulation on the glass surface treated with Glassclad IM. was very much greater. The results are in agreement with the expected performance of these applied resins at least at the lower velocity of 0.5 m/s. Although glass is not a common material of construction in industrial equipment, coated surfaces of glass demonstrated the action that a suitable coating can have on the accumulation of biofouling, at least at specified fluid velocities. The anti-biofouling effectiveness of a PTFE coating containing nickel, copper, and phosphorous, was investigated by Zhao, Su, Wang, and Mu¨llerSteinhagen (2005). It was discovered that changing the PTFE content of the coating had a significant influence on bacterial adhesion through the associated change in the surface free energy. Coatings with a free energy in the range 21–25.3 mJ/m2, reduced the adhesion of E. coli Rosetta by over 96–98%, compared to stainless steel or titanium surfaces. In a related paper Zhao et al. (2009) commented that silicone coatings based on polydimethylsiloxane that are being investigated as fouling release coatings for various applications including ships’ hulls are not suitable for use in heat exchangers. The reasons given include poor thermal

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conductivity, abrasion resistance and adhesion to metal substrates. These authors, quoting Grill (1993), point out that diamond-like carbon coatings are attracting attention due to their excellent thermal conductivity (similar to metal), low flow resistance, smooth surface, hardness and wear and corrosion resistance. From their tests, Zhao et al. concluded that diamondlike carbon coatings have the potential to reduce biofouling in heat exchangers. The cost which the plasma coating process involves could restrict the process to smaller units.

4.4. POTENTIAL PHYSICAL TECHNIQUES FOR BIOFOULING CONTROL Because of the vital need to control biofouling in industrial equipment to improve operation and reduce operating cost, the problem has attracted the attention of researchers into physical techniques. A number of opportunities have been or are being studied:

4.4.1. Circulation of Polymer Fibres Some preliminary studies of the effects of circulating polymer fibres through tubes together with microbial laden water to control biofilm formation demonstrated that the technology could be very effective (Bott, 2001). Polymer fibres at concentrations of 100 and 200 ppm in circulating water at three different velocities of 0.6, 1.0 and 1.6 m/s, demonstrated the possibilities. As might be expected, the best results in terms of control, were obtained at the highest velocity (1.6 m/s). The data are presented in Figs. 4.24 and 4.25. The results at 0.6 and 1.0 m/s, although demonstrating the possibilities of the technique, contained considerable scatter. The suggested explanation for the scatter is possibly that it is due to sloughing of the biofilm in response to the action of the fibres; the effect was more noticeable in the presence of the lower fibre concentration. In the tests at the highest velocity the control was more uniform. In the tests it was evident that when the fibre injection was stopped there was an almost instant reaction with a relatively pronounced increase in biofilm accumulation. Examination of the tubes after the tests showed that some fibres had become embedded in the residing biofilm. A major problem with the use of fibres to control biofouling would be the need to remove fibres before disposal of the water back to the environment or into a waste-water circuit. Another potential problem would be the likely settlement of the fibres onto the packing of a cooling tower, if it were employed to reduce the temperature of the cooling water before reuse.

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1.0 control test

Velocity 1.6 m/s

ABSORBANCE

0.8

0.6

0.4

0.2

0.0 0

72

144

216 HOURS

288

360

432

Figure 4.24 The effect of 100 ppm polymer fibre on microbial accumulation. (Courtesy I. Chem. E.)

1.0 Velocity 1.6 m/s

control test

ABSORBANCE

0.8

0.6

0.4

0.2

0.0 0

72

144

216

288

360

432

HOURS

Figure 4.25 The effect of 200 ppm polymer fibre on microbial accumulation. (Courtesy I. Chem. E.)

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Some very preliminary studies in a small-scale cooling tower suggest that this did not appear to be a problem (Private Communication, 2003). More research in this aspect of fibre use is required.

4.4.2. The Use of Ice Crystals An associated technique used as an alternative to the use of polymer particles is the possibility of circulating ice crystals. After passing through the equipment (if not before!) the crystals would have melted, so that deposition in a cooling tower would not be a problem. There would be an increase in operating costs due to the production of the ice crystals but their melting would help to reduce the temperature of the cooling water. Ainslie, Quarini, Herbert et al. (2009) have investigated what they describe as ‘ice plugging’. Although the experiments involved a sand deposit, the technique would seem applicable to biofilm control. The process involved pumping a particulate ice slurry, a mixture of ice particles and liquids containing a freezing point depressant through the fouled duct or equipment. Among the points made as a result of the experiment were: 1. The ice plug has the ability to flow through various geometries, maintaining its plug-like formation. These geometries include multiple path, static mixers, and changes in diameter and plate heat exchangers. 2. The ice slurry is capable of applying a high shear at the pipe wall, removing fouling that water flowing at the same flow rate was unable to remove. 3. Through ‘tuning’ the production of the ice slurry in terms of the freezing point depressant and ice formation required, a particular fouling situation can be successfully met and the fouling effectively removed. It would appear that large-scale successful trials have been carried out on water supply mains with a large range of pipe diameters and lengths. Materials of construction included asbestos, concrete, cast iron and PVC, representing the majority of pipe materials used in the water industry.

4.4.3. Biofouling Control Using Ultrasound Ultrasound has been used successfully to control fouling in combustion systems where mixed deposits usually at very high temperatures (often in the region of 1000  C) exist. The action of the ultrasound is to make the unwanted deposit vibrate, break up and become dislodged. In view of the successful application in mineral deposit removal it was thought that this could also be applied to biofouling removal.

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Brizzolara, Welch, Mazzole, Burnett, and Licina (1999) demonstrated with a field test that significant biofouling control could be provided on a titanium pipe with a ‘strap-on’, pulsed, acoustic source. The test provided the following assessments: 1. Visual observations of pipe sections indicated thinner, less developed biofilm on the pipe treated with the pulsed acoustic than on the untreated pipe. 2. Analysis of the test coupons showed that that there were fewer microorganisms on the treated pipe than on the untreated pipe. 3. The untreated pipe had acquired a substantial amount of both biological and inorganic fouling at the end of a 17-day test. Brizzolara et al. (1999) hypothesised that the acoustic wave from the emitter caused a short-lived disruption to the boundary layer, resulting in a shear force sufficient to dislodge any microorganisms that had colonised the pipe surface before they could attach on a more permanent basis. Using a specially designed test circuit that simulated a cooling water system, in which two tubes were fitted with 600 W ultrasonic processors (Vibracell, Sonics and Materials Inc.), the effectiveness of biofouling control was tested (Bott, 2000). The ultrasound generator converted the 50 Hz frequency of the normal power supply to a high frequency electrical energy at 20 kHz, which was passed to a transducer where mechanical vibrations were generated. The ultrasonic probe focused the energy and created a concentrated zone of high intensity. The result was a multitude of small, highly agitated bubbles that scoured the surface of the tube on which a biofilm was being formed. The assessment of the biofouling accumulation was made using the infrared device described in Bott (2003). In simple terms, the difference in absorbance of energy by biofilm at the end of a test period and before colonisation by the microorganisms, is a measure of the biofilm accumulation. It is possible to correlate experimentally the infrared absorbance with the amount of biofilm present (Bott, Gonzalez, and Llovet 1998). Fig. 4.26 compares the thickness of a biofilm developed with and without the application of ultrasound. It would apper that control was only achieved when the biofilm thickness had reached about 20mm. Some data presented in Table 4.6 give an indication of this particular control technique. As would be anticipated, both the intensity of application of the ultrasound and the frequency of treatment influenced the quality of biofilm control. The use of an amplitude of 20% was less effective than using the higher amplitude of 40%. There was around 90% reduction in biofilm

Biofouling Control

Biofilm thickness m

Treatment 3

40

145

30 s once per day

Control

30 Ultrasound applied at 20% amplitude

20 10

0

40

80

120

160

200

240

280

320

300

Time h

Figure 4.26 The effect of ultrasound once per day at 20% amplitude. (Courtesy A. I. Chem. E.)

Table 4.6 Control of biofouling with different ultrasound treatments. % Reduction in biofilm growth Ultrasound% Ultrasound Frequency of compared with Amplitude Treatment Treatment per day control

20 20 20 40 40 40 40

3 3 1 3 3 1 1

30 s 30 s 30 s 30 s 30 s 30 s 30 s

1 6 8 10 1 24 8

20.0 65.5 40.3 88.0 72.7 92.6 91.9

growth with the amplitude of 40% as compared to the control test without any ultrasound. Only 40% reduction was obtained for an amplitude of 20%. These data were the result of applying one 30-second burst of ultrasound at three hour intervals. Although ultrasound technology seems effective for biofouling control, there are likely to be some negative aspects to its application in large-scale operations. First there is the capital cost of the ultrasonic units and their effective installation into the intended recipient equipment. Nevertheless the equipment and its operation could be regarded as environmentally friendly, although the fact that electrical power is required may offset that advantage if the origin of the power is obtained via combustion of fossil fuel with associated emission of CO2.

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4.4.4. High Voltage-capacitance Technology The attachment of microorganisms described in Chapter 3 involves the interaction of physicochemical forces such as the DLVO and Van der Waals forces in relation to the surface of the particles (microorganisms) and the surface to which they attach. Rodrigo, Romo, Pitts, and Handagama (2007) have discussed the possibility of using a technique based on high capacitance technology to modify this interaction so that the accumulation on the receptive surface is reduced. It is concluded that the three phases of biofouling, i.e., attachment, growth and detachment may all be modified by the application of high voltage-capacitance technology to reduce biofouling potential. These authors provide examples of biofouling control in an array of industrial cooling applications using this technology involving: 1. Removal of mature biofilm from evaporative cooling media. 2. Comparison study of biofouling, scale formation and corrosion in a condenser-cooling tower water system. 3. Biofouling and biocorrosion control in a cooling tower water system. 4. Biofouling control in a power utility steam plant piping system. Quoting from the paper of Rodrigo et al. (2007), these examples provide reliable data which support the theory that high voltage–capacitance systems can significantly reduce or even eliminate biological deposits in cooling water systems. Several conditions are cited ranging from mild to severely aggressive accumulations. It remains to be seen in the light of further investigation, whether the general applicability of this technology and the associated cost, will be acceptable to the relevant industries.

4.4.5. Reciprocating Scraped Surface Heat Exchanger Solano et al. (2009) described a novel anti-fouling heat exchanger used in the food and waste-water treatment industries The technique involves the use of a reciprocating scraping rod inside each of the tubes of a multi-tube heat exchanger that keeps the inside surface of the tubes free of deposit. Furthermore the scraping action of the rods increases the heat transfer coefficient on the inside of the tubes due to the disturbance of the laminar sublayers. The scraping action can be either continuous or intermittent. These authors suggest that the scraper should work intermittently for cleaning operations or continuously with minimum scraping frequency, to minimise power consumption.

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The initial capital cost and the maintenance costs of the technology could be prohibitive for very large scale operations; although this might be offset by not having to install duplicate equipment to maintain operation when off-line cleaning becomes necessary.

4.4.6. Physical Controldconcluding Remarks In very general terms, physical methods of control of biofouling are less complex than the use of biocides (except where there is a combination of the technologies), particularly in the disposal of residues. On the other hand the capital cost and possibly the operating costs are likely to be higher for the physical methods. Furthermore, there are in general, fewer health and safety problems to overcome. What might be considered to be a drawback to physical control however, is that once it has been incorporated into the process plant, there would be great resistance because of the investment costs, to replace it if it proved to be inadequate or difficult to operate. A modification that might be considered under these circumstances is a combination of this technology with a suitable biocide. In contrast, the use of chemical treatment does provide the opportunity to substitute another chemical if the one in current use proves to be unsatisfactory.

4.5. COMBINED USE OF PHYSICAL AND CHEMICAL TECHNIQUES Although the control of biofouling has been discussed as two separate technologies i.e., chemical and physical, it is logical to think in terms of linking the two to enhance effectiveness. The concept will be illustrated by two different combinations.

4.5.1. Potential Combination of Ultrasound and a Biocide The opportunity to enhance the effectiveness of biocides for biofouling control was discussed by Bott and Tianqing (2004). Experiments had been carried out to illustrate the effects of combining ultrasound and the biocide ozone (Tianqing and Bott, 1998). The basis of the work was to carry out tests over a four week period using Pseudomonas fluorescens as the test microorganism in simulated cooling water. There were six test sections consisting of a glass tube 1 m length and 18 mm internal diameter. At the

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bottom of two tubes, ultrasound probes were located. The other four tubes were used as the controls. The operating conditions were: Temperature Fluid velocity Cell concentration in the water

25e30  C 1 m/s 1  109 cells/ml

The extent of the biofouling thickness was determined using infrared absorbance converted to biofilm thickness using an experimentally determined correlation. The first week of the test allowed the biofouling to develop under the set flow conditions on the inside of tubes without any treatment. The thickness achieved by the biofilm under these conditions was about 50mm (in the range of 45–60 mm). During the second week water containing ozone (2.2 mg/1) was pumped into the circulating water for three hours each day, followed by ultrasound application at 20% amplitude (see earlier section) in two tubes for three minutes each day. In the fourth and fifth week the ozone concentration was raised to 2.8 mg/1. The results of the test are summarised in Fig. 4.27. From these admittedly limited results it would appear that the combined use of ozone and ultrasound is more effective than either ozone or ultrasound alone. Although there is a reduction of biofilm thickness by the application of ozone, the removal of biofilm is enhanced by the application of ultrasound.

Biofilm thickness m

70

without any treatment

60 50 40 30 20

only with ultrasound

only with ozone treatment

combined use of ultrasound and ozone

10 0

Figure 4.27 Comparison of bioflim thickness under different control regimens and water velocity 1 m/s.

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4.5.2. Combination of Inserts and Biocides Again using Pseudomonas fluorescens as the biofouling organism and the use of Hitran wire-wound inserts and dosing with a proprietary biocide based on tetrakis hydroxymethyl phosphonium sulphate (THPS), tests were carried out to reveal the effectiveness of their combination (Wills et al., 2002). High and low density inserts were used in the experiments. The results are recorded in Fig. 4.28. As indicated elsewhere, absorbance of infrared was used as a measure of biofilm accumulation. Two water velocities were used (0.86 and 1.27 m/s) with an initial THPS concentration of 15 mg/l. From the results it would appear that biofilm development was controlled with 15 mg/l of THPS. For this reason it was decided to investigate, reducing the concentration to 10 mg/l. There was some biofilm growth particularly at the lower velocity of 0.86 m/s but in effect, the growth was effectively zero with no apparent significant difference in biofilm development between the different insert dimensions. After about 500 hours, the biofilm in the control sections showed some evidence of oscillation in mass accumulation, which is a feature of biofilms that have reached their potential maximum thickness. In comparison with the results without inserts, their presence reduced considerably the concentration of THPS required for control.

2.5 0.86 m/s - Test Low Density 1.27 m/s - Test Low Density 0.86 m/s - Test High Density

2

1.27 m/s - Control Low Density

Absorbance

0.86 m/s - Control Low Density

1.5

1 15 mg/l 0.5 10 mg/l 0 0

100

200

300

400

500

600

700

800

Time (hours)

Figure 4.28 Comparison of bioflim development at two water velocities. Test ¼ insert þ THPS, Control ¼ insert only. THPS (tetrakishydroxymethylphosphonium sulphate).

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4.6. POTENTIAL COMBINED PHYSICAL AND CHEMICAL CONTROLdCONCLUDING REMARKS Although potential combined physical and chemical control would seem attractive, the total cost of the equipment required may be a deterrent. It is quite possible however, that the technology could be acceptable in certain industrial operations, particularly where it is possible to extend the interval between off-line cleaning operations.

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Bott, T. R. (2000). Biofouling control with ultrasound. Heat Transfer Engineering, 21(3), 40. Brizzolara, R. A., Welch, M., Mazzole, M. S., Burnett, E., & Licina, G. J. (1999). Pulsed acoustics for biofilm control in heat exchangers and piping systems. In T. R. Bott (Ed.), Proceedings of the International Conference. Mitigation of heat exchanger fouling and its economical and environmental implications (pp. 153). New York: Begell House Inc. Cal Gavin Ltd. Minerva Mill Innovation Centre, Station road, Alcester, B49 5ET, U.K. Casanueva-Robles, T., Nebot, E., Casanueva, J. C., Ferna´ndez-Baston, M. M., & Sales, D. (2005). Economic and performance evaluation of two oxidising biocides in sea-water cooling systems, Conf., Heat exchanger fouling and cleaning- challenges and opportunities, Kloster Irsee. Germany: ECI, Brooklyn. Chen, M. J., Zhang, Z., & Bott, T. R. (2001). The resistance of Pseudomonas fluorescens biofilms to H2O2 biocide, Pro. 6th. Melbourne: World Congress of Chemical Engineering. Cheung, D. T., & Nimni, M. E. (1982). Mechanism of crosslinking of proteins by gluteraldehyde. Connect Tissue Research, 10, 201. Cowbourne, M. A., Lester, D. A., & Bott, T. R. (2001). The effectiveness of environmentally friendly biocides and biodispersants. In H. Mu¨ller-Steinhagen (Ed.), Heat exchanger fouling fundamental approaches and technical solutions. Essen: Publico Publications. Denyer, S. P. (1990). Mechanisms of action of biocides. International Biodeterioration, 26 (2–4), 89. Eager, R. G., Leder, J., & Theis, A. B. (1986). Gluteraldehyde: factors important for microbicidal efficacy, 3rd. Conf. on progress in chemical disinfection. Binghampton: Copyright, Union Carbide. Edwards, H. B. (1983). The economical advantage of ozonating the cooling tower water system, Proc. 6th Ozone world conference. International Ozone Association. Elmir, K. (1985). Recommendations for the optimum cleaning frequency of the Taprogge tube cleaning system Tech Report 85-26. Taprogge Gesellscaft, Wetter. Ferreira, C., Simo˜es, M., Pereira, M. C., Bastos, M. M. S. M., Nunes, O. C., Coelho, M., et al. (2009). Control of biofouling of industrial surfaces using microparticles carrying biocide. In Mu¨ller-Steinhagen. (Ed.), Heat exchanger fouling and cleaning. Austria: Eurotherm Conf., Pichl/Schladming. Fo¨rster, M., & Rumpf, B. (2003). Low fouling heat transfer surfaces for industrial applications. In P. Watkinson, & H. Mu¨ller-Steinhagen (Eds.), Heat exchanger fouling and cleaning fundamentals and applications, Conf. Santa Fe: ECI, Brooklyn. Gilbert, P., Collier, P. J., & Brown, M. R. W. (1990). Influence of growth rate on susceptibility to antimicrobial agents; biofilms, cell cycle, dormancy, stringent response. Antimicrobic Agents Chemotherapy, 34, 1865. Gorman, S. P., Scott, E., & Russel, D. (1980). Antimicrobial activity, uses and mechanism of action of glutaraldehyde. Journal of Applied Bacteriology, 48, 161. Gough, M. J. & Rogers, J. V., (1987). Reduced fouling by enhanced heat transfer using wire matrix radial mixing elements, AICh.E Symposium Series, 257, Vol. 83, 16, Pittsburg. Grab, L. A., Emerich, D. E., Baron, S. J., & Smolik, N. A. (1990). Glutaraldehyde; a new slimicide for paper making. Papermakers Conf., printed by. Atlanta: Tappi Press. Grab, L. A., & Rossmore, L. A. (1992). Biocide efficiency vs acid producing and iron oxidising bacteria, Paper TP 92-13. Houston: Cooling Tower Inst., Meeting. Grant, D. M., & Bott, T. R. (2005). Biocide dosing strategies for biofilm control. Heat Transfer Engineering, 26(1), 44. Grill, A. (1993). Review of the tribology of diamond-like carbon. Wear, 168, 143. Henry, B. D. (1993). Union Carbide’s gluteraldehyde based microbicides for industrial applications. South Africa: Union Carbide. 25th Oct.1993. Hu¨ttinger, K. J. (1987). Surface bound biocides–a novel possibility to prevent biofouling. In L. F. Melo et al. (Eds.), Fouling science and technology. Dordrecht: Kluwer Academic Publishers.

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Kaur, K., Bott, T. R., & Leadbeater, B. S. C. (1992). Effect of ozone as a biocide in an experimental cooling water system. Ozone Science and Engineering, 14, 517. Kaur, K., Curan, A., & Bott, T. R. (1991)The effect of ozone on Pseudomonas fluorescens. Proceedings 10th of Ozone World Congress, Vol. 1. Monaco: International Ozone Association. 347. Klaren, D. G., & de Boer, E. F. (2007). Achievements and potential of self-cleaning heat exchangers using untreated sea water as coolant, Proc. Conf. Heat exchanger fouling and cleaning. Tomar: ECI Brooklyn. Klaren, D. G. (2003). Improvements and new developments in self-cleaning heat transfer leading to new applications, Conf; Heat exchanger fouling and cleaning. Engineering Conference International, 6, Metro. Tech. Centre, CI., Brooklyn, NY 11201. Krueger, A. W., & Pouponnot, F. (2009). Heat exchanger performance enhancement through the use of tube inserts in refineries and chemical plants–successful application examples: Spirelf, Turbotal and Fixotal systems, Eurotherm Conf. Heat exchanger fouling and cleaning. Austria: Pichl/Schladming. 8973. Le Chevallier, M. W. (1988). Inactivation biofilm of bacteria. Applied and Environmental Microbiology, 54, 2492. Lowe, M.J., (1988). The effect of inorganic particles on the development of biological fouling films, Ph.D. Thesis, University of Birmingham. Malchesky, P. S. (2000). In S. S. Block (Ed.), Disinfection, sterilisation and preservation (pp. 979). Baltimore: Lippincott, Williams and Wilkins. Marshall, A., & Bott, T. R. (1987). Effectiveness of biocides. In L. F. Melo et al. (Eds.), Fouling science and technology. Dordrecht: Kluwer Academic Publishers. Mauermann, C., Bellmann, C., Eschenhagen, U., Bley, T., & Majschak, J. P. (2009). Influence of surface charge and surface ageing on fouling and cleaning properties of modified and unmodified stainless steel, Eurotherm Conf., Heat exchanger fouling and cleaning. Austria: Pichl/Schladming. 8973. McDonnell, G. (2007). Peroxygens and other forms of oxygen: their use for cleaning, disinfection and sterilization. In P. C. Zhu (Ed.), New biocides development: the combined approach of chemistry and microbiology. Washington: American Chemical Society. McDonnell, G., & Russell, A. D. (1999). Antiseptics and disinfectants activity action and resistance. Clinical Microbiology Reviews, 12, 147. McNair-Scott, D. B., & Lesher, E. C. (1963). Effect of ozone on survival and permeability of E. coli. Journal of Bacteriology, 85, 567. Mitchell, P. K. (1985). Bromination: Two methods available for sanitizing, Pool and spa news, April, 15th. 128. Munton, T. J., & Russell, A. D. (1973). Interaction of glutaraldehyde with spheroplasts of Escherichia coli. Journal of Applied Bacteriology, 36, 211. Nesaratman, R.N. & Bott, T.R., (1984). Effects of velocity and sodium hypochlorite derived chlorine concentration, on biofilm removal from aluminium tubes, Process Biochemistry 19, Nov, 14 Payne, K. R. (Ed.). (1988). Industrial biocides, critical reports in applied chemistry. London: John Wiley and Sons. Patching, J. W., & Fleming, G. T. A. (2003). In P. C. Lens et al. (Eds.), Biofilms in medical, industry and environmental biotechnology. IWA Publishing, 12, Caxton Street, London SW1H 0QS. Petrucci, G. & Rosellini, M., (2003). Chlorine dioxide in sea water for fouling control in power station and petrochemical plant, Conf. Heat exchanger fouling and cleaning fundamentals and applications, Brooklyn. Eng. Conf. Intl. Puckorious, P. R. (1991) Ozone use in cooling tower systems - current guidelines - where it works. Toronto. Presented for the International Ozone Association.

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Pugo, M. (1993). Effects of hydrodynamic conditions and biocides on biofilm control. Ph.D. Thesis University of Birmingham. Pujo, M. & Bott, T. R., (1991). Effects of fluid velocities and Reynolds numbers on biofilm development in water systems, In J. F. Keffer, R. K. Shah, & E. N. Garnic, (Eds.), 2nd. World Conf. Exptl. Heat transfer, fluid mechanics and thermodynamics, 1358. Railkin, A. I. (2004). Marine biofouling. Boca Raton, London: CRC Press. New York, Washington, 179. Rice, R.G. & Gomez –Taylor, M., (1986). Occurrence of byproducts of strong oxidants reacting with drinking water contaminants – Scope of the problem, Environ. Health Perspectives, 69, Nov, 31. Rice, R.G. & Wilkes, J.F., (1991). Fundamental aspects of ozone chemistry in recirculating cooling water systems. Paper 205, in Conf. Corrosion 91, Nat. Assn. Corrosion Engrs. Rodrigo, F. V., Romo, M., Pitts, M., & Handagama, N. B. (2007). Biofouling control in heat exchangers using high voltage capacitance based technology, Proc. Conf. Heat exchanger fouling and cleaning-VII. Tomar: ECI, Brooklyn. Rossmore, H. W., & Sandorsi, M. (1988). Applications and mode of action of formaldehyde condensate biocides. Advances in Applied Microbiology, 33, 223. Russell, A. D. (2003). Bacterial resistance to biocides; current knowledge and future problems. In P. Lens, A. P. Moran, & T. Mahony et al. (Eds.), Biofilms in medicine, industry and environmental technology. London: IWA Publishing. Santos, R. C. C., (1993). Ph.D. Thesis, Polymer coatings in relation to single and mixed population biofilms. University of Birmingham. Sim oes, M., Pereira, M. O., & Vieira, M. J. (2003). Effect of different concentrations of ortho-phthalaldehyde on biofilms formed by Pseudomonas fluorescens under different flow conditions. Biofouling, 19, 287. Sinha, J., (2000). Treatment of poultry waste water with ozone, Ph.D. Thesis, University of Birmingham. Solano, J. P., Garcia, A., Vincente, P. G., & Viedma, A. (2009). In H. Mu¨ller- Steinhagen (Ed.), Conf. Heat exchanger fouling and cleaning. Austria: Pichl/Schladming. Taylor, R.J. (1995). Efficacy of industrial biocides against bacterial biofilms, Ph.D. Thesis, University of Birmingham. Tianqing, L. & Bott, T. R. (1998). Combined use of ultrasound and ozone for biofouling control in heat exchangers, In J. S. Lee (Ed.), Proc. 11th Int. Heat Trans. Conf. Kyongju, Korea Vol. 6, 221. Videla, H. A., Viera, M. R., Guiamet, P. S., & Staibano-Alais, J. C. (1995). Using ozone to control biofilms. Journal of Materials Performance, 34(7), 40. Wills, A., Bott, T. R., & Gibbard, I. J. (2000). The control of biofilms in tubes using wirewound inserts. Canadian Journal of Chemical Engineering, 78(1), 61. Wills, A., Bott, T. R., & Gibbard, I. J. (2002). The effect of tube inserts on biocide efficacy. In H. Mu¨ller-Steinhagen (Ed.), Heat exchanger fouling fundamental approaches and technical solutions. Essen: Publico Publications. Youngblood, J., & Stratton, T. (2009). A large task for large molecules,. The Chemical Engineering Issue, 821, 36. Zhao, Q., Liu, Y., Wang, S., & Mu¨ller-Steinhagen, H. (2005). Development and evaluation of Ni-Cu-P- PTFE composite coatings to minimize microbial adhesion in Conf. Proc. Heat exchanger fouling and cleaning-challenges and opportunities. Engineering Conference International Brooklyn. Zhao, Q., Su, X. J., Wang, S., Zhang, X., Navabpour, P., & Teer, D. (2009). Development and evaluation of modified DLC coatings to minimise fouling adhesion. In H. Mu¨llerSteinhagen (Ed.), Heat exchanger fouling and cleaning, Eurotherm Conf. Austria: Pichl/ Schladming.

CHAPTER

5

Cleaning Off-line Contents 5.1. Introduction 5.2. Application of Cleaning Technologies 5.3. Concluding Remarks on Off-line Cleaning References

155 155 158 158

5.1. INTRODUCTION Although steps are taken to control biofouling in industrial equipment, it is likely to become necessary to clean equipment off-line from time to time to remove persistent residues. The frequency of cleaning will depend on the local conditions and there will be considerable variations. Under the special hygienic conditions necessarily imposed on food processing the maintenance of clean operating conditions is vital. It may even be necessary to clean more than once per day! Cleaning times reported by Mauermann, Bellman, Eschenhagen, Bley, and Majschak (2009) suggest, quoting references, that in the dairy industry 15% of the production time is taken up with cleaning surfaces. In other less stringent industrial conditions, cleaning may only be necessary at the annual shutdown for maintenance purposes. It is important that at the design stage of an industrial process that the material of construction is chosen not only in respect of the anticipated biofouling, but also in respect of the necessary cleaning that is likely to be required. This is of particular importance where physical techniques are being considered, which may involve the dismantling of equipment that might result in damage to the equipment in the long term. Chemical methods of cleaning are generally more versatile. If it is anticipated that chemical cleaning will be a required option, it will be necessary to choose a material of construction that will withstand any potential corrosive action of the chemical additive.

5.2. APPLICATION OF CLEANING TECHNOLOGIES The range of cleaning techniques is vast; from totally manual to completely non-manual. It is probably true to state that no two cleaning situations can be regarded as identical. It is only possible therefore, to give a very general summary of the methods available. Industrial Biofouling ISBN 978-0-444-53224-4

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Wilson (2003) has set out the challenges posed in the cleaning of process equipment. He points out that there is considerable scope for optimisation of methods by effective management of cleaning activities. It is of particular importance to plan the cycle of operation and cleaning to avoid the sudden imposition of necessary and vital cleaning as a result of unacceptable plant operation that could be costly in terms of lost production. It is important to be able to identify the nature of the deposit and its consistency throughout the process equipment and over time, so that the methods adopted for cleaning will be suitable. The emphasis placed on any of the factors will be determined by the application, the stated cleanliness aims, reproducibility and reliability. There are a number of options available for off-line cleaning, making effective management vital for satisfactory operation. Care has to be taken not only in the choice of cleaning method but also in the implications of its introduction, particularly in respect of the probability that its use may be sporadic. The first important criterion is that the choice of cleaning method must not risk damage to the equipment to be cleaned. Of similar importance is the protection of all employees involved in the cleaning process against injury and health risks, and to protect the environment from any potential detrimental effects imposed by the method adopted for cleaning. Vigilance is required, since the cleaning process may be on an irregular basis and may therefore be considered as only a temporary exercise. Excessive pressure on operators for a quick ‘turnaround’ must not compromise safety. Wilson (2003) points out that knowledge of the chemical and physical nature of the biofoulant is required to identify appropriate cleaning methods. The cleaning will require bond rupture within the biofouling layers and at the biofouling/surface interface. The techniques that can achieve rupture, some of which have already been mentioned in connection with techniques for biofouling control, include erosion or thermal shock by the application of shear or normal forces or by chemical conversion of the fouling species to a less cohesive or less adhesive material. Factors to be considered in the selection of cleaning technologies include: 1. Effectiveness and efficiency. 2. Extra equipment requirement. 3. Cost. 4. Verifiability. 5. Scope for contaminants or compatibility. 6. Cost of disposal of deposit material and cleaning chemicals.

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The last point, which has implications that are growing in importance, includes health and environmental risks and the cost of cleaning materials. Recycling of the cleaning reagents employed is gaining in importance due to these concerns regarding the cleaning processes. Another aspect is the search for solutions that can be effective at similar temperatures to the process, thereby reducing the time taken to introduce the cleaning procedure and so reducing downtime and possibly reducing energy requirements. Methods available for off-line cleaning include: 1. Manual cleaning, which may be carried out ‘dry’ or ‘wet’, is a relatively simple and familiar technique involving wiping, brushing, and scraping. Because of its labour-intensive nature the cost of manual cleaning could be high. Access may also be a problem but it is particularly possible where the equipment to be cleaned is relatively easily dismantled e.g., plate heat exchangers. 2. Jetting that may involve the use of high pressure water, air or steam lances. Access may be a difficulty with this method. 3. Drilling and rodding is a possibility for tenacious deposits but this is not usually the case with biofouling. The drilling may be accompanied by the inclusion of compressed air or a water supply to take away the deposit. The technique is particularly relevant to the inside of tubular equipment, such as shell and tube heat exchangers. 4. High temperature sterilisation or heat shock often referred to as ‘steam soaking’, usually involves the admission of steam into the equipment to be cleaned. If the temperature of the biofilm is raised sufficiently and the steam applied for a suitable time based on experience, thereby killing the microorganisms, subsequent removal by water flushing may be effective. However, there is the possibility that large volumes of dead material and other debris may present difficulties of removal, thereby causing blockage, partial or complete, that may necessitate a shut down of the process. Increased frequency of cleaning is most likely the best solution to overcome this particular problem. 5. Osmotic shock that upsets the metabolic processes of the microorganisms and results in lysis of the cells, is a means of controlling biofouling but it may also be employed for cleaning purposes. Cleaning is achieved by passing salt water through the system containing the unwanted biofouling. The process of removal may be prolonged and the salinity may induce corrosion of the processing equipment that could represent a problem in its own right in the longer term. It may also be considered as relevant to an off-line clean-up procedure.

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6. Chemical cleaning is, in essence, simply an extension of the biocide treatment already discussed in Chapter 4 but it is likely to involve much greater biocide concentrations as well as chemicals not generally regarded as biocides. Bott and Taylor (1997) discovered the difficulty of total removal of established biofouling with chemicals, including chlorine, hydrogen peroxide, peracetic acid and potassium permanganate (see Figs. 4.12 to 4.17). The technique may introduce disposal problems and operating conditions that could be difficult to accommodate. It would also be necessary to take account of the material of construction of the equipment to be cleaned to avoid corrosion problems. Knowledge of the volume of the equipment would be necessary so that the amount of chemical required could be estimated. Any particular hazards of the potential cleaning agent and associated safety requirements, should be critically considered. Particular attention must be given to ‘dead spaces’ in the plant being cleaned, where cleaning chemicals may accumulate causing subsequent operating problems if they are not removed before the plant is returned to its normal function.

5.3. CONCLUDING REMARKS ON OFF-LINE CLEANING There is a wide range of options that apply to cleaning of equipment subject to biofouling. In the final choice of the method of off-line cleaning, the prime considerations to be borne in mind are ease of application, the associated costs and safety. It is possible that two methods of cleaning may be required to be applied in sequence, such as chemical treatment followed by jetting to physically remove the treated biofouling. Heat shock, osmotic shock and the use of chemicals (discussed in Chapter 4) are in a sense attractive, because if the frequency of use is carefully chosen it may not be necessary to dismantle equipment. Clearly, this could be an advantage in reducing downtime, associated lost production and potential damage to the plant through repeated dismantling and reassembling. There are specialist organisations that have wide experience in the cleaning of process plants, who could be consulted in respect of particular problems.

REFERENCES Bott, T. R., & Taylor, R. T. (1997). The effects of velocity on biocide use for biofilm removal in flowing systems. In El-Genk. (Ed.), Heat transfer, Baltimore, AIChE Symposium series, 93 (pp. 314.1).

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Mauermann, M., Bellman, C., Eschenhagen, U., Bley, T. H. & Majschak, J. P. (2009). Influence of surface charge and surface ageing on fouling and cleaning properties of modified and unmodified stainless steel, Proc. Conf. Heat exchanger fouling and cleaning, Eurotherm, Pichi/Schlmayrgut, Austria. Wilson, D. I. (2003). Challenges in cleaning: Recent developments and future prospects, ECI Conf. Heat exchanger fouling and cleaning fundamentals and applications, Santa Fe, USA.

CHAPTER

6

Biofouling Monitoring Contents 6.1. Introduction 6.2. Off-line Monitoring 6.3. Process Plant Monitoring 6.4. Monitoring in the Laboratory 6.5. Biofouling Measurement Techniques 6.6. The Accuracy of Test Data 6.7. Concluding Remarks References

161 161 165 169 174 176 177 178

6.1. INTRODUCTION The presentation in Chapter 3 demonstrated the complex nature of biofilm formation and accumulation on surfaces. The extent of the likely biofouling in a particular industrial operation, is extremely difficult, if not impossible, to predict. Information on possible remedies, particularly the use of chemical additives, would also provide useful information. The equipment to be employed in industrial operation could be better designed to limit biofouling if some information on the effects of the operating parameters were available at the design stage and the potential for control investigated. Physical techniques described in Chapter 4 may be incorporated in the design but treatment with chemicals in general, is likely to be based on monitoring and experience during the commissioning of the process plant in its particular location. It is often the situation that chemical treatment is something of an afterthought, when the commissioning has taken place. Furthermore, it would be important to take account, as early as possible in the design and commissioning stages, of the legal requirements concerning the use of chemicals that are in place or are likely to be imposed in the future. Of particular concern as stressed elsewhere, is the discharge of cooling water back to the natural environment from where it was obtained, such as a river, lake or the sea.

6.2. OFF-LINE MONITORING It is clear that in order to avoid operational and legal problems effective monitoring of the effectiveness of the proposed control technology to be Industrial Biofouling ISBN 978-0-444-53224-4

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employed must be made. Some methods of monitoring have been alluded to in previous chapters and Flemming (2002) points out the limitations of conventional monitoring. Continuous monitoring would be desirable because of the changes that are likely to occur with time so that effective remedies may be tested. In addition reliable monitoring will make it easier to control costs i.e., to reduce or eliminate for instance, unnecessary or excessive use of chemical treatment. Inadequate dosing may be revealed that might allow excessive accumulation of biofilm with detrimental effects on production, including frequent ‘shutdowns’ for cleaning. The traditional method of investigating the formation of biofilms in the laboratory is to suspend ‘coupons’ in a static or possibly agitated suspension of microorganisms. Although this gives a rough indication of say, the effect of surface on adhesion or the efficacy of different chemical biocides, the data cannot be taken as absolute. In static tests the contact between the microorganisms and the surface is rather problematical, dependent on ‘Brownian’ motion or the ability of the microbes to motivate themselves. Although in some situations this may apply in process plants, it is more often the situation that the liquid phase is in motion with profound effect on the relationship of the microorganisms with the surface. It is possible to overcome the problem to some extent, by agitating the microbial suspension in some way, but again, it is unlikely that this will replicate the conditions in the process plant. Now however, in the laboratory the effects of operational conditions can be taken into account and evaluated in terms of a particular velocity of the fluid (usually water) across a replica of the host surface. In many full-scale operational conditions the biofilms although essentially consisting of microorganisms and the extracellular polymers they produce, may also contain particulate material and microbial wastes together with material resulting from process leaks and other debris. If the water originates from natural sources it will likely contain material emanating from living plants, animals and insects. All may affect effectiveness of control, particularly the use of biocides. The replication of these conditions is virtually impossible to create in a laboratory environment. It is therefore, wherever possible, necessary to utilise a side stream from the particular process to investigate different control techniques, particularly the use of chemicals and associated dosing strategies. Alternatively it may be possible to employ a pilot plant replica of the process alongside the full-scale plant (as described in Chapter 4). The cost of this kind of approach however, could be prohibitivednot least in the capital involved in the construction of the pilot plant.

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Monitoring can be considered to fall into two broadly based groups: 1. Direct monitoring of the biofilm itself e.g., number of cells or activity. 2. Indirect monitoring that relies on the effect of the biofilm on operational variables e.g., pressure loss in flowing systems, heat transfer restriction or local changes in pH. Bryers and Characklis (1992) advocate the application of reactor engineering fundamentals and set out the experimental parameters that are relevant to monitoring (reproduced as Table 6.1). It will be apparent that some of these variables are associated to process plant assessment i.e., those that are set by the process conditions. There are other variables however, that represent experimental conditions that can only be applied in laboratory investigations. Even in those special situations the opportunity to exercise complete control of conditions may be limited, particularly experimentation with mixed cultures and the presence of antagonist organisms. Table 6.1 Experimental variables Chemical Parameters Physical Parameters

Substrate type Substrate concentration pH Inorganic ions Dissolved oxygen Microbial inhibitors

Temperature Fluid shear stress Heat flux Surface composition Surface texture Fluid residence time

Biological Parameters

Microorganism type Culture type (mixed or pure) Suspended cell concentration Antagonist organisms

Sampling the biofilm for analysis can represent a serious challenge for the investigator. In order to obtain the data required, e.g., composition, cell numbers or protein content, it will be necessary to destroy the biofilm by removal from the surface to which it is attached. This is particularly the situation where the biofilm itself is the focus of interest. The design of the device for accurate sampling is often dependent on the ingenuity of the experimenter and the requirements of the particular set of experiments. The careful removal of the sample is a vital requirement for obtaining reliable data. Where the effects of the biofilm on process operation and the control of accumulation is required, in situ confirming estimation of some aspect of the biofilm is necessary i.e., change in thickness or some physical property. It is of course, essential that measurement is dependent on biofilm accumulation and it can be continuously registered over long periods of time. The so-called ‘Robbins device’ is often used for this purpose. It consists of a square or rectangular flow channel into which flat removable coupons are

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Aluminium block with tapered flow channel

TEST PLATE (x10) aluminium

11

m

1.4

cm

m

4c cm

m

5c

10

cm

2c

Gasket

1 cm 3 cm

m

1c

18

Perspex face screwed onto aluminium body

Flow

Figure 6.1 Tapered test-section with removable test plates (“coupons”). (Courtesy Elsevier.)

fitted with their surface flush with the flow channel (Fig. 6.1). If the coupon is not a perfect fit or not flush with the flow channel surface, erroneous results will be obtained due to the disturbance of the flow across the coupon that in turn, will influence biofilm growth and retention. The effects of flow can be studied with this device. A suitably tapered duct as illustrated on Fig. 6.1, can provide results that demonstrate the effects of different velocities. The biofilm accumulation can be obtained by weighing or electrical conductivity of the biomass on the individual test plates. An effective technique based on electrical conductivity, using a modified micrometer was described by Bott (1995). As the thickness of the biofilm is not uniform, it is necessary to take a suitable number of thickness measurements in order to obtain a reliable average biofilm thickness. It is possible, although tedious, to make an analysis of the biofilm deposit and use say, protein content for comparison purposes. The effects of the quality of the surface (e.g., roughness) and the material of construction can be obtained with these techniques. A technique that has proved extremely useful in the laboratory for the testing of biocides and other control techniques is the use of infrared absorbance referred to elsewhere in this book (Bott 2003). The technique requires a transparent material e.g., glass, for the test surface to allow the passage of the infrared radiation. This may be considered as a drawback, in that it imposes conditions that are different in respect of adhesion from those

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that might be encountered in process plant operation. Nevertheless it can provide valuable insights empirical though they may be, on the effects of changing conditions e.g., temperature, nutrient availability and fluid velocity across the surface in the particular situation, together with the effect of applying chemical or physical methods of control. It is interesting to note that a three-day conference ‘International Specialised Conference on Biofilm Monitoring’ was held in 2002 (IWA, 2002). The programme was devoted to techniques and methods of examining biofilm behaviour in industrial operations and in the laboratory. A number of the papers presented at the conference are discussed in this Chapter.

6.3. PROCESS PLANT MONITORING Flemming (2002) suggested that the control of biofouling could be regarded as ‘biofilm management’. The principle to be applied is that the occurrence of biofilm will happen. The aim of testing is to keep this development below a threshold of interference, which has to be defined for the particular system under review. He suggests that for a given system, the strategy must take account of the basic conditions, particularly addressing the availability and quality of the nutrients in the water phase. What is required in an operating process plant is an early-warning system, which provides rapid information online, in situ, in real time, nondestructively and is suitably designed for automated computer aided arrangements. The ideal situation would not require the removal of samples for specialist scrutiny so that operating personnel could make necessary adjustments as quickly as possible. Flemming (2002) recognised that three levels of information can be distinguished, by which current monitoring devices can be identified: 1. Systems which detect the kinetics of deposition of material and changes in thickness of the deposit layer but cannot differentiate between microorganisms and abiotic deposit components. There are a number of such devices, which have their individual merits. Online real time information about deposit formation on submerged surfaces is difficult to acquire, yet if available, provides valuable information. 2. Systems which can distinguish between biotic and abiotic components of a given deposit. A suitable way of accomplishing this is through specific detection of the signals of biomolecules. 3. Systems which provide detailed information about the chemical composition of the deposit or directly address the microorganisms.

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It is likely that the costs associated with techniques in level 2 and level 3 would be prohibitive, except for specialised research activities. It has to be recognised that for extensive studies, laboratory investigations are more than likely to be required. In a sense, the data gained in the control room of the process plant can be an empirical measure of biofilm accumulation. The obvious example would be the pressure drop through the equipment for a given flow rate, if suitable pressure tapping points were available or could be made available. Although if there are no suitable pressure points, the flow rate of the process cooling water achievable through the equipment will be a rough guide to the extent of biofilm growth. Temperatures are usually displayed in the control room, particularly where the process is temperature sensitive in some way. In general this would be applicable to heat exchanger fouling for example, the changes in the process fluid (gas or liquid) temperature. Where biofouling occurs the heat transfer will be impaired. It has to be appreciated however, that temperature changes across a heat exchanger may be in part, due to fouling on the other side of the heat exchanger, not in the passages where biofouling is occurring. Another method that is sometimes employed if there are no suitable data points, so that a pressure drop or temperature measurement cannot be taken, is the quality of the product, if for instance, the desired cooling to prevent deterioration is not achieved. In a very rough way, this is a measure of the heat transfer efficiency of the heat exchangers that may be subject to biofilm accumulation on the water side. The achievable product output in relation to the desired output may also indicate the extent of the biofouling, where its occurrence can influence the throughput either directly or indirectly. It will be recognised that these observations however, are not really acceptable methods of assessment. Even where more sophisticated methods of monitoring are employed, some of the characteristics of plant operation are used as pointers to the need for cleaning. In this context, an important decision at the design stage of a process plant is whether or not to duplicate (in parallel) items of plant such as heat exchangers so that one set can be cleaned while the other set is in normal operation. It will be appreciated that these techniques that appoint the process plant to be its own monitor can only be regarded as crude attempts at biofilm management. It has to be stated however, that many plant operators often in the light of experience, develop a ‘sixth sense’ of the extent of the biofouling that is occurring and make suitable adjustments to prolong the interval between successive essential cleaning operations. Despite the potential

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opportunity to use this skill however, it is preferable to take a more positive approach to biofouling control through suitable operational measurements using a monitor. Potentially there are three possible methods of utilising a monitor on site including: 1. A side stream in which water from the plant is passed through tubes or across surfaces similar to those in the plant in terms of material, roughness, diameter or other characteristic dimensions. The conditions in the side stream in terms of fluid velocity and temperature are the same as in the part of the process plant it is intended to simulate. Such a device can also be used as a test bed for experimental chemical or physical control techniques. The basic concept is essentially the same as the laboratory pilot plant (discussed below) except that instead of generating a biofilm with microorganisms grown in a fermenter, the biofouling stream is taken directly from the process plant itself. 2. A pilot plant, which simulates exactly as far as is possible on a relatively small scale, the process that allows the effects of changes in the process variables to be explored in some detail. The various streams where appropriate, are taken from the full-scale plant. It is not necessary to reproduce the process in its entirety but with particular reference to the part where the accumulation of biofilm is likely to occur or is occurring. It will be obvious that this is a relatively expensive way of tackling a biofouling problem. Despite the high cost, pilot plant studies have been made; for instance, a pilot plant to investigate biofouling on a 500 Mw power station has been employed by Casanueva et al. (2003) and Nebot, Casenueva, Casaneuva, Fernandez-Basto´m, and Sales (2006). The pilot plant was designed and operated to simulate the conditions that led to biofilm development in the tubes of the power station steam condenser system. In this particular plant, sea-water is used as the cooling medium. Table 6.2 makes a comparison of some of the details of the design and operation of the pilot plant and the full-scale plant. A flow sheet of the fully automated pilot plant is presented in Fig. 6.2. It was designed to be portable and built into a shipping container approximately 6 m long so that with suitable modification it could be used at other sites. The installation of the pilot plant in the container was carefully considered in order to facilitate the investigations. Studies could be made of different tube materials and diameters and different biocides and dosage patterns. Wireless remote control and data acquisition were installed so that continuous detailed monitoring could be

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Table 6.2 Comparison between the power station condenser and the pilot plant Variable Power station Pilot plant

Tube material Tube dimension Flow velocity through tube Temperature difference through tube condenser exchanger

ASTM-B111. 79; alloy 687 22.9  25.4  6058 mm 1.98 m/s 0.9e1.0  C/m

ASTM-B111. 79; alloy 687 12  15  3200 mm 1.98 m/s 1.0e1.2  C/m

ELECTRIC BOILER (CE) EXPANSION VESSEL THREE WAY REQULATING VALVE

MIXING TANK (TM)

PUMP (B-60)

PUMP (B-50)

PLATE HEAT EXCHANGER (ICP)

BIOCIDE TANK (TH-A)

PVC SHELL & TUBE HEAT EXCHANGER (ICCT) DOSAGE PUMPS B-30 D

BIOCIDE TANK (TH-B)

B-30 C

PUMP (B-40)

FLOW METER

B-30 B

CHLORINATION TANKS DC-A

B-30 A

DC-B

FEED TANK (TA) FEED PUMP (B-10)

DC-C DC-D S/W CIRCULATING PUMP (B-20)

SEA WATER SUCTION

FLOW CONTROL VALVES

Figure 6.2 Flow sheet of pilot plant. (Courtesy Elsevier.)

carried out. From the data obtained on pressure drop, flow rates and temperature distribution, it was possible to calculate the friction factor and heat transfer resistance. A specially made tool was used to collect the deposit from the inside of the heat exchanger tubes for examination. The design of the device is illustrated in the self-explanatory Fig. 6.3. 3. The monitor applied directly to the process plant itself. This would not be easy and straightforward to achieve so that reliable data could be obtained, which would be relevant to the plant operation as a whole. Nevertheless this would give precise data related to the part of the process plant to which the monitor was attached.

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Tube sheet

3 m heat exchanger tube

nut Flexible coupling

a Sleeve

Rubber piston

b 4 m rod

Compression washer

Figure 6.3 Fouling collecting tool. (Courtesy Elsevier.)

It may be necessary that in order to accommodate a particular method of gathering data, the surface on which the biofouling develops has to be different from the material of construction in the operational plant. For instance, it may be necessary to have a glass surface so that light can pass through it or non-ferrous material to avoid possible electromagnetic interference. These problems do not necessarily invalidate the data obtained but it would be necessary to appreciate the possible limitations of the data, although they would give an indication of the change that is likely to occur with changing operating conditions. An example of an online monitor was described by Mollica and Cristiani (2002). The monitor, named BIOX, is based on the ability of biofilms to change the electrochemical characteristics of the surface of passive metals in both aerobic and anaerobic environments. The mechanism involved is the modification of corrosion processes occurring at the metal/biofilm interface, which may be monitored by measurement of the galvanic current generated say, between a stainless steel surface and a sacrificial anode. An industrial version of the sensor was successfully tested to optimise chlorination treatments of sea water cooling systems in electrical power stations. The intermittent chlorination programme initiated by the sensor was sufficient to avoid completely any biofilm formation.

6.4. MONITORING IN THE LABORATORY In an industrial operation it is extremely unlikely that a pure biofilm is ever produced, i.e., one including only a single species of microorganism. To be realistic therefore, in laboratory investigations, mixtures of microorganisms

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would be required. It is well known however, that the consistency of such artificial mixtures is extremely difficult to maintain even under laboratory conditions. Nevertheless the use of a single species relevant to the full-scale plant conditions, to investigate the behaviour and the effects say, of a new chemical biocide, can provide valuable data. There are many useful ways of assessing the efficacy of chemical and physical methods for biofouling control in the laboratory. Because of the wide variability and diversity in bacterial attachment, depending on the particular system under investigation i.e., the microorganisms present, the surface (material and topography) and the environmental conditions, it is virtually impossible to extrapolate in detail from one system to another as each one is unique. To obtain satisfactory results therefore, it is important to test each system separately. As mentioned in Section 6.2, the laboratory method of evaluating biofilm formation and the effectiveness of biocides, based on the suspension of ‘coupons’ in microbial contaminated water has serious limitations, particularly in respect of flow conditions encountered in industrial operations. Despite these drawbacks however, it does give a comparison between alternatives with the implications that this has for full-scale operations. It would be much better to simulate as far as possible, the conditions in fullscale operation particularly in relation to liquid movement e.g., the velocity in relation to the surfaces as outlined in Chapter 2. The effects of different dosing strategies that might be employed could also be investigated. As with all investigations into the efficiency of different treatments the methods adapted can vary in detail. A test facility design (Fig. 6.4), which has been used for many years at the University of Birmingham to investigate the conditions that affect biofouling, has provided useful data. Studies have included the effectiveness of new biocides and surface active agents, the effects of changing operating conditions not only related to fluid flow across surfaces but the effects of temperature and materials of construction, the quality in respect of surface roughness, and the availability of nutrients. Some of the results of this work have been reported elsewhere in this publication. Tests have usually been confined to a single species of microorganism, usually the slime-former Pseudomonas fluorscens. Although the use of a single species of microorganism may be seen as a restriction on the universality of the data obtained, the experimental results do give very valuable insight into the effects of changing operating variables, in particular fluid velocity, temperature and microbial concentration, from which assessments and comparisons may be made (Bott, 2003 and Bott, 2007).

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Although there can be wide variations in the experimental conditions, typical basic conditions of fermenter operation include: Dissolved oxygen Air flow rate Temperature Concentration of cells

35e55% saturation at fermenter temperature 5.5 l/min 27  C 1  109 cells/ml

In many respects the laboratory equipment mimics the conditions that are likely to be found in an industrial cooling water system, such as on a power station. The experimental design and the details that follow are based on information presented by Taylor (1996). In the laboratory simulation (Fig. 6.4), a single species of microorganism is grown in the fermenter, which is carefully controlled in order to provide a constant concentration of cells. The microorganism concentrate from the fermenter is passed to a mixing vessel to provide by the addition of sterile water, a concentration consistent with those expected in cooling water on an industrial site. The simulated cooling water is passed vertically through the test sections to avoid any influence that gravity might have if they were not in the vertical position. Highly filtered air is injected into the fermenter and the mixing vessel to simulate conditions that would be relevant to an industrial

Air

Distilled or filtered tap water

Air Nutrients Fluid return

Nutrients Biocide Mixing vessel

Fermenter Overflow

Vertically mounted test sections

Overflow

Figure 6.4 Flow sheet of a laboratory simulated water circulation system for biofouling assessment.

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situation. It is into this mixing vessel that the controlling agent, such as a biocide, is introduced so that a well mixed consistent mixture is passed to the test sections to give the desired steady flow rate. It is in these test sections that the biofouling occurs either on the surface as a whole or on ‘coupons’ fitted flush to the test section surface to avoid disturbances to the flow as shown on Fig. 6.1 (Robbins device). It has to be emphasised that any extraneous effects on the flow across these coupons would give unreliable data in respect of biofouling under plant conditions. The assessment of the accumulation of biofilm on the surface could be made by a simple but accurate weighing procedure. The use of an in situ monitoring device however would be preferable since this would avoid the necessity of interrupting flow and removing the test surface in order to obtain data over an extended period of time without interruption. Factors that could influence the development of the biological fouling (e.g. pH, temperature and flow velocity) are carefully controlled at the desired values. It is prudent to use glass for vessels and interconnecting tubing since it allows visual observation and it is also biologically non-toxic and resistant to corrosion. The vessels and interconnecting tubing would most certainly need to be made sterile before a test was initiated otherwise erroneous data would be produced. The sterilising temperature should be relatively high, of the order of 120  C, therefore requiring a suitable autoclave. The fermenter must be carefully controlled in order to produce and maintain a consistent pure culture, to be passed through the test sections. The precise conditions will be dictated by the choice of microorganism. The composition of the nutrient for these experiments is given in Table 3.1. It has to be sterilised in order that unwanted microorganisms are not introduced. The glucose is sterilised separately to avoid colouration due to ‘browning’ reactions. Sterilisation of the nutrient is essential to avoid contamination that would affect the consistency of the results obtained. The build-up to constant conditions in the fermenter must be carefully controlled. The pH in the fermenter is maintained at neutral (i.e., pH ¼ 7) by the automated addition of alkali. The temperature is usually kept close to 27  C (i.e., between 26 and 28  C) by a tubular glass heat exchanger unit through which water is pumped from a thermostatically controlled water-bath, although the temperature may be dictated by the species of the microorganism. Aeration of the fermenter is achieved by the introduction of humidified compressed air. It is necessary to use humidified air to prevent water being lost from the fermenter. The air is filtered down to 10 mm to

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remove any particulate material present and finally down to 0.2 mm to remove possible contamination from microorganisms. The main air admission is through a stainless steel sparger but a small fraction of the air supply is introduced to the fermenter through the nutrient line so that it does not become blocked. Excess air in the fermenter is relieved through the overflow and vent. Agitation of the fermenter is achieved by the use of a magnetic stirrer. The conditions (i.e., pH, temperature and dissolved oxygen) are measured and controlled by suitable instrumentation. Assessment of biofilm accumulation under the various conditions of operation of the test facility and the effectiveness of biofilm control requires a suitable monitoring device. The methods discussed in Section 5.1 could be employed for these measurements. A specially developed infrared absorption device has been used for this purpose (Bott, 2007). It was for this reason that glass tubes were used in the experiments to provide the surface on which the biofouling occurred. It is well appreciated that glass is not the normal material of construction of cooling water circuits but the use of glass enabled the infrared system to be employed. It provided the opportunity without interruption, for the effects of different operating variables to be recorded and comparisons made, although the absolute values of the biofouling that would be accumulated in a full-scale plant, would not be obtained. It is possible however, to carry out experiments that provide a correlation between infrared absorbance and mass or thickness of the associated biofilm for instance, using the coupons from a Robbins device. In Chapter 3 the importance of the conditioning layer in connection with the biofouling process was emphasised. Therefore in order to simulate industrial conditions, it would be necessary to provide a conditioning layer on a virgin test surface before the commencement of a test. This can be achieved by circulating water from the town’s supply prior to the sterilisation of the equipment for an experiment. It has been established that once a test section has been used, the conditioning layer from the previous test remains and so it is possible to carry out experimentation immediately without a need to establish a new conditioning layer. Sterilisation of the equipment may be achieved by the circulation of low-pressure steam for a suitably long period of time (of the order of twenty-four hours) to ensure that all microbial contaminants that might be present in the test facility have been made inactive. Where experiments are to be carried out to investigate the effectiveness of biocides two mixing vessels will be required; one to provide data on biofouling growth without biocide and the other to include biocide so that

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comparisons may be made. These two parallel investigations are essential to provide data based on water streams of exactly the same quality. If the tests were carried out at different times, despite suitable precautions there may be some differences in basic water quality that could affect the value of the comparison.

6.5. BIOFOULING MEASUREMENT TECHNIQUES The estimation of the number of cells attached to a surface represents a serious challenge to the investigator. Furthermore, the number of cells generally only represents part of the assessment. In industrial conditions the biofouling layer will contain not only cells but associated extracellular polymers, particulate matter and other contaminants. For process plant assessment, the monitors referred to earlier may be used. In the laboratory however, (including laboratory pilot plants) because the work is likely to be more fundamental, detemining the actual numbers of cells may be part of the objective. The traditional method is actually counting cells with the use of a microscope. Removal of the cells from the surface to which they are attached can be difficult and is very susceptible to the introduction of errors. The use of scanning electron microscopy (SEM), transmission electron microscopy (TEM) and scanning controlled laser microscopy have been of considerable assistance in this respect. Structural components of cells may be utilised as measures of cell quantity on surfaces. These include protein, carbohydrate, adenosine triphosphate (ATP), lipopolysaccharide (LPS) and other suitable techniques. Radiotracer techniques may also be used to make biofouling assessments by labelling the cells and subsequently measuring the radioactivity from the biofouling layer produced on a test surface. Potential techniques recently reported include photoacoustic spectroscopy and the utilisation of quartz crystal surface sensitivity. Photoacoustic spectroscopy is based on the absorbance of electromagnetic radiation inside a sample, where non-radiative relaxation processes convert the absorbed energy into heat. As a result of thermal expansion, a pressure wave is produced that can be detected by microphones or piezoelectric transducers (Rosenewaig, 1980). The technology has been used to monitor biofilm formation and various antifouling strategies. (Schmidt, Panne, Haisch, & Niesser, 2003). Rudth (2003) stated that surface-sensitive quartz crystal microbalance with dissipation monitoring has been widely used to characterise molecular-surface interactions ranging in size from small molecules to large proteins or polymers. According to this author, the technology

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offers strong advantages for formation characterisation of biofilms, although as yet, it has not been used extensively. A method described by Yang Shanrang et al. (2009) is based on the relationship between AC impedance and fouling thermal resistance. It would seem to be applicable to the assessment of biofouling but an investigation into the possibilities would be required. Tests were carried out by Yang Shanrang et al. (2009) to measure fouling thickness in examples of crystallisation and particulate fouling. The tests revealed the typical fouling accumulation versus time curves. Work by Pereira, Mendes, and Melo (2008) demonstrated the possibilities of using nano-vibrations for monitoring biofouling. It involved the use of a mechatronic surface sensor that used the analysis of vibration to detect and measure the amount and nature of a deposit attached to a surface. The basis of the device was that the adhesion or removal of a deposit affected the amplitude of the output signal, which was found to be inversely proportional to the amount of deposit attached to a monitored surface. The damping factor is also a variable that is complementary to the amplitude, which provides an indication of the structure of the deposit (Pereira, Rosmaninho, Mendes, & Melo, 2006). This work also indicated that the sensor was found to follow the build-up of a milk deposit and whey protein on a stainless steel surface through the analysis of the amplitude of the surface wave. It would appear however, that the quality of the fouling layer could also affect the data obtained, which might limit its applicability. The characteristics of the device demonstrate its versatility for wideranging applications: 1. It is non-intrusive. 2. The data gathered corresponded to an integrated measurement over a significant area. 3. It can be applied to different types of surface materials. Tests carried out by Pereira, Melo, Martins, and Freire (2009) revealed that the mechanistic surface sensor was able to: 1. Quantitatively assess the amount of deposit attached to a surface. 2. Produce semi-quantitative information that distinguishes different types of attached layers. 3. Monitor cleaning operations and determine the respective end point in real time. 4. Monitor distinct processes from milk pasteurisation to cooling water systems and cleaning procedures. 5. Field tests confirm its suitability for industrial applications.

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6.6. THE ACCURACY OF TEST DATA Clearly, any measurements made in respect of biofouling need to be reliable, repeatable and accurate as far as is possible so that they may be applied to process plant operation with confidence. Much will depend on the quality of the experimental design employed. Glen, Howarth, and Jenkins (1997) made the following points in respect of fouling assessment in an investigation of heat exchanger fouling using cooling water. Although this may appear to be a particular industrial application, namely heat transfer, the recommendations are generally relevant to all aspects of industrial biofouling. The suggestions are essentially a specialised adaptation of general scientific principles of investigation. They are: 1. The geometry of any system and the physical and chemical conditions used for making fouling measurements should be as close as possible to those of the system which it models. 2. Where possible, measurements using sections of heat exchangers are to be preferred. This is particularly important if the data are to be used for design purposes and for the investigation of compact heat exchangers. 3. Where fouling is measured by an analysis of overall heat transfer, the achievement of a full heat balance is an essential prerequisite to obtaining reliable data. 4. Where data are required for the optimisation of a mitigation strategy, the use of one or more side stream fouling monitors in which the thermodynamic parameters characteristic of the heat exchanger are reproduced, is recommended. If possible, tests with and without the implementation of the strategy should run concurrently; if not, three or more sequential tests should be run over a short time period. 5. In view of the small temperature increase between the inlet and the outlet of small-scale monitors, it is recommended that the temperature difference between the two temperatures should be measured with a sensitivity of þ/ 0.005  K. 6. The following errors can be achieved in the fundamental measurements associated for determining fouling resistances based on a heat balance and higher values should be regarded as unacceptable. Volume flow Density Temperature rise Test surface area

0.3% 0.01% 5.0% or 0.0050  K 0.5%

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7. Whatever approach is adopted, an assessment of the errors of measurement should be undertaken and included in the reporting of the results. All interpretations under different conditions should be made with reference to this error analysis. 8. Where measurement of changes in heat transfer are being made to provide reference fouling data, an indication should be given of the method used to produce an initial clean surface and of its reproducibility; they should be supplemented by one or more type of measurement related to the amount and nature of the deposit. This may not be necessary if the measurements are only being used for control purposes. 9. Changes in pressure drop are much more sensitive to fouling of small channels than changes of heat transfer coefficient and can be related more unambiguously to the fluid causing the fouling. Differential pressure sensors offer better sensitivity than two separate sensors. 10. In systems where carbonate scale formation is possible, good control and accurate measurement of pH is essential. This can be particularly important in the laboratory where a model cooling water may not have the same degree of buffering as industrial waters. 11. The use of positive displacement or gear pumps with speed control are recommended to minimise flow velocity variations to 2–3%. These authors suggest that there is a need to establish the relationship that exists between the extent of the fouling observed and assessed under well defined but limited test conditions and the overall effect of a wider range of operating conditions that prevails in the full-scale industrial equipment.

6.7. CONCLUDING REMARKS Although the investigation of biofouling in the laboratory pilot plant would seem attractive and certainly, the work would give valuable background information and help to develop control techniques, it does not give data relevant to a particular process plant. Not only in an industrial process are there likely to be many different species of microorganisms but there are other components and conditions that affect the final outcome. Although in general terms the fouling layer is called ‘biofouling’, the other components present will affect the quality of the deposit and hence its likely influence on the efficiency of the operation. A major problem at the design stage of a water cooling system is that of assigning a value for a heat transfer resistance

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to take account of any biofouling that may occur. The value chosen, although often based on experience, may be regarded as little more than an educated guess. Since the biofouling is likely to represent the major contributor to inefficiency, there is the risk that an incorrect choice may be made. As a result, the design based on these data may not be strictly applicable. It is almost certain therefore, that when the plant is commissioned tests will be necessary to maximise the operating time between offline maintenance cleaning through the effective use of control techniques. Although the necessary simplification of laboratory investigation provides useful data, it is apparent that a pilot plant that mimics the full-scale operation or a side stream put through a laboratory type pilot plant, are the best options for process plant evaluation. It is under actual plant operating conditions that reliable data will become available so that alternative techniques of biofouling control can be investigated, potential difficulties realised and the likely costs involved can be established. A most important aspect is the composition of the microbial population.

REFERENCES Bott, T. R. (1995). Fouling of heat exchangers. Amsterdam: Elsevier. Bott, T. R. (2003). Monitoring biofouling using infrared absorbance. In P. C. Lens, A. P. Moran, T. Mahon, P. Stoodley, & V. O’Flaherty (Eds.), Biofilms in medicine, industry and environmental biotechnology (pp. 461). Tunbridge Wells: IWA Publishing. Bott, T. R. (2007). Biofilms and biocides: growing consistent monoculture biofilms for biocide assessment. In P. C. Zhu (Ed.), New biocides development: the combined approach of chemistry and microbiology. Washington: American Chemical Society. Bryers, J. D., & Characklis, W. G. (1992). Biofilm accumulation and activity: process analysis approach. In L. F. Melo, T. R. Bott, M. Fletcher, & B. Capderille (Eds.), Biofilms-science and technology (pp. 221). Dordrecht: Kluwer Academic Publishers. Casanueva, J. F., Sa´ndez, J., Garcia-Maralis, J. L., Casanueva-Robles, T., Lo´pez, J. A., Portela, J. R., et al. (2003). Portable pilot plant for evaluating marine biofouling growth and control in heat exchanger-condensers. Water Science Technology, 47(5), 99. Flemming, M. C. (2002). Role and levels of real time monitoring for successful anti-fouling strategies – an overview. Proceedings of International Conference on biofouling monitoring. Porto, Portugal: IWA. Glen, N. F., Howarth, J. H., & Jenkins, A. M. (1997). The estimation of errors and uncertainty in the determination of fouling resistances. In T. R. Bott, L. F. Melo, C. B. Panchal, & E. F. C. Somerscales (Eds.), Understanding heat exchanger fouling and mitigation. New York: Begell House, Inc. Publishers. IWA, (2002). International Specialised Conference on Biofilm Monitoring, Porto, Portugal. Mollica, A., & Cristiani, P., (2002). On-line biofilm monitoring by electrochemical probe “BIOX”, in International Specialised Conference on Biofilm Monitoring, Porto, Portugal.

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Nebot, E., Casenueva, J. F., Casanueva, T., Fernandez-Basto´m, M., & Sales, D. (2006). In situ experimental study for the optimization of chlorine dosage in seawater cooling systems. Applied Thermal Engineering, 26, 1893. Pereira, A., Rosmaninho, R., Mendes, J., & Melo, L. F. (2006). Monitoring deposit buildup using a novel mechatronic surface sensor (MSS). Transactions of the IChemE Part C: Food and Bioproducts Processing, 84(C4), 5. Pereira, A., Mendes, J., & Melo, L. F. (2008). Using nanovibrations to monitor biofouling. Biotechnology and Bioengineering, 99(6), 1407. Pereira, A., Melo, L. F., Martins, J., & Freire, M. (2009). Fouling and cleaning monitoring, using the MSS – industrial perspective, in Proceedings of Conference. Heat exchanger fouling and cleaning. Austria: Pichl/ Schladming. Rosenwaig, A. (1980). Photoacoustics and photoacoustic spectroscopy. New York: Wiley. Rudth, M. (2003). Quartz crystal microbalance with dissipation monitoring: a new tool for studying biofilm formation in real time. In P. Lens, A. P. Moran, T. Mahony, P. Stoodley, & V. O’Flaherty (Eds.), Biofilms in Medicine, Industry and environmental biotechnology. London: IWA. Schmidt, T., Panne, U., Haisch, C., & Niesser, R. (2003). Biofilm monitoring by photoacoustic spectroscopy. In P. Lens, A. P. Moran, T. Mahony, P. Stoodley, & V. O’Flaherty (Eds.), Biofilms in medicine, industry and environmental biotechnology. London: IWA. Taylor, R. J. (1996). Efficacy of industrial biocides against bacterial biofilms, Ph.D. Thesis, University of Birmingham. Shanrang, Y., Shengxian, C., Yanling, C., Hua, Y., Kangle, Z., & Zhiming, X. (2009). An online real-time monitoring of fouling layer thickness based on AC impedance technology, in Proceedings Eurotherm Conference Heat exchanger fouling and cleaning. Austria: Pichl/ Schladming.

CHAPTER

7

Biofilms in Industry Contents 7.1. Introduction 7.2. The Water Industry 7.2.1. Water Treatment 7.2.2. Groundwater improvement 7.2.3. Water Distribution Systems 7.3. Food Industry 7.4. Paper Manufacture 7.5. Cooling Water 7.6. The problem of Legionella pneumophila References

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7.1. INTRODUCTION Industries that handle large quantities of water are clearly vulnerable to the effects of biofouling, although in certain special situations the presence of a biofilm is beneficial. The methods adopted to combat the problem will often be process specific. For instance, the use of biocides may not be acceptable in certain processes due to difficulties associated with health and safety requirements. In other examples a physical method of control may not be a feasible proposition due to the investment required or operating costs or it may simply not be compatible with that particular mode of operation. On the other hand, industries that utilise biofilms to advantage need to provide conditions that facilitate their application.

7.2. THE WATER INDUSTRY The water industry in general, is based on the need for a nation’s population to have access to clean water for domestic purposes, particularly for drinking and for the disposal of human waste in a sewage system. Water is also needed for the activities around the home such as cleaning and gardening. At the same time industry of various kinds, at least in most developed countries, has access to this water. It is probably true to say that the water industry along with food provision is at the heart of modern western-style human society, although it has to be recognised that in many Industrial Biofouling ISBN 978-0-444-53224-4

Ó 2011 Elsevier B.V All rights reserved.

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parts of the world the provision of clean water is still only a dream. Therefore a major task of the water industry is to provide safe water for human consumption, chemically and biologically. Water quality is usually subject to legal safeguards in order to ensure that the water is safe to drink without any health risks. The origin of the raw water will clearly direct the extent of the required treatment to maintain quality. The treatment of waste water including sewage is a major concern in order to prevent contamination of water resources. There are two principal sources of raw water: 1. Surface water Surface water is taken from natural or man-made lakes, reservoirs and rivers which may be contaminated by the ‘run off ’ water from roofs, roads and fields, resulting principally from human activity of one sort or another. The contamination may include dissolved substances, colloids and particulate matter, with concentrations of organic material from the degradation of formerly living plants and creatures that often give the water colour. The presence of suitable nutrients encourages the growth of microorganisms. The extent of the treatment required and the processes needed to carry it out are clearly dependent on the quality of the raw water. Man-made lakes and reservoirs tend to be better managed than natural sources (Scholz, 2006). 2. Ground water As the name suggests, ground water originates underground and is usually obtained from boreholes. The extent of any contamination is usually much less than that experienced with surface water. The contamination is generally in the form of dissolved inorganic salts from the rock and soil with which the water comes into contact through seepage, together with some microorganisms and organic residues from agricultural activity, e.g., animal waste and residues of composting. Some examples of both water sources may be contaminated with residues in the soil at the sites where industrial operations have taken place in the past, particularly chemical processing. Modern health and safety regulations are likely to prevent that kind of contamination occurring in current industrial operations. Desalination of sea water may be a source of water for domestic use but is unlikely to be the source of water in the amounts required for industrial operations, due to the relatively high operating costs involved. In view of the varied origins of water, including recycled water used in industry, it is not surprising that many problems are encountered in its use

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due to impurities, particularly in respect of corrosion, deposition of particulate material and microbiological fouling.

7.2.1. Water Treatment In summary, conventional water treatment to produce town’s water, involves aeration (principally for ground water treatment), coagulation, flocculation settling, sorption, final filtration and disinfection, usually employing chlorine, chlorine dioxide or sodium hypochlorite. Interest in the use of ozone is increasing since it does not create unwanted residues (see Chapter 5). However, the use of chlorine does leave a beneficial disinfecting residue, reducing the potential for microorganism activity. The emphasis of the following discussion is in terms of wastewater that requires intense treatment but the concepts discussed are relevant to water treatment in general. For the production of high purity water however, chemical/physical techniques discussed in Chapter 5 and in this Chapter are unlikely to prove satisfactory. Distillation of suitably prepared water is probably the only method that would produce a satisfactory product, although some of the techniques discussed could be utilised for preparing water for distillation. Mozes and Rouxhet (1992) drew attention to the benefits of biofilms in the natural environment. For example, they are responsible for the removal of dissolved and particulate contaminants in environmental water. Biofilms in the natural world are usually in the form of ‘mats’ that influence the quality of the water in which they exist. They may increase the oxygen content or act to remove toxic or hazardous material from the water, breaking it down to acceptable components. These characteristics are the basis for the employment of biofilms in the industrial world of water, either of waste water or for the improvement of the water for drinking purposes. Waste water cannot be discharged indiscriminately into the environment since it could pose serious threat to public health and indeed to the natural world in which water plays such an important part. Legislation increasingly demands improvements in the quality of drinking water supplied to the public. The undesirable material contained in many waste waters results from wide ranging industrial manufacturing and food processing in addition to domestic sewage. In very general terms, it is organic in character and represents potential nutrient for microbial activity as part of the biological, carbon, nitrogen, phosphorus and other biogeochemical cycles (O’ Flaherty, and Lens, 2003). The treatment of waste water often takes advantage of these

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characteristics and suitable biofilm systems are devised to convert the unwanted components to CO2, CH4 and H2O. Two principal microorganism activities are generally involved: Aerobic reactions that produce CO2 and H2O Anaerobic reactions that produce CO2 and CH4. These products (CO2, H2O and CH4) are naturally present in the environment so that they are acceptable products of treatment processes but there could be some concern expressed for their contribution to global warming. Anaerobic reactions form the basis for the production of biofuels to supplement energy demand. O’Flaherty and Lens (2003) pointed out that the formation of biofilms and microbial aggregates, as described in Chapter 3, are prerequisites for the successful operation of biological waste-water treatment and nutrient removal systems. These authors stated that there has been significant advancement in biofilm reactor design and operation. The most common and widely used aerobic fixed-film reactor is the percolating or trickle filter. The simplest form consists of a bed of graded hard material such as granite, up to a depth of 2 m. (Fig. 7.1) The wastewater is distributed evenly over the surface of the bed by the rotating distribution arm. The volume and frequency of application may be varied to accommodate optimal conditions for the particular filtering process. The ventilating system ensures that oxygen is available throughout the filter to

Plan

Direction of rotation Biofilm support media

Intermittent application of sewage

Air

Biofilm support media

Air

Section

Effluent

Figure 7.1 Schematic representation of a percolating filter. (Courtesy IWA Publishing.)

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maximise the biological activity. The treated effluent passes out from the bottom of the filter to a settlement tank. It takes some time, 2–3 weeks in summer and up to 3 months in winter, for the biofilm to be established. If the filter is treating domestic sewage it is not necessary to inoculate the filter as the microorganisms to activate it are contained in the human effluent. In other systems it may be necessary to ‘seed’ the filter with the appropriate microorganism for the required activity. Uniformity of flow through the filter is essential for its effective performance. As might be envisaged, because of the natural basis of its activity, the biofilm is an extremely diverse entity. The mature biofilm contains a wide variety of microorganisms including bacteria, fungi, protozoa, nematodes and rotifers plus a diverse group of macroinvertebrates such as enchytraeids, lumbricids worms, dipterans, fly larvae and many other groups. Birds and rodents actively graze the film so that the microbial contents of their excreta will add to the biological varieties present. Scholz (2006) pointed out the activities of some of the live entities in the trickle filter: 1. Bacteria are active in the degradation of soluble organic material. Nitrifying bacteria convert ammonia to nitrate. In a low flow rate trickle filter there is a relatively higher nitrifier population and the effluent is well nitrified. Where there is a high flow rate there is more sloughing of the biofilm, which reduces the degree of nitrification. 2. Fungi also found in trickle filters, play an active part in their function but tend to dominate where the pH of the water is relatively low. They are usually associated with water contaminated with industrial waste. 3. Algae are active in the biofilm and produce oxygen during daylight hours. This activity helps to keep the uppermost layers of the biofilm aerobic in character. 4. Protozoa feed on the bacteria in the biofilm. This may seem to be a disadvantage but in effect, it helps to maintain efficiency by sustaining an active bacterial population. 5. Rotifers are predators present in the biofilm, feeding on the microorganisms; bacteria, algae and fungi. They are indicative of a high degree of treatment efficiency, serving to reduce the suspended solids in the effluent. 6. Macroinvertebrates in the filter are insect larvae that feed on the biofilm and help to control its thickness. In so doing they reduce the tendency for the pores of the filter to become clogged, thereby helping to maintain efficient operation. The larvae develop into adult insects in two to three weeks and can be a nuisance to plant operators or local residents who live close to the treatment plant.

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Because of the usual positioning of the biofilm support material, the flow through the filter, unlike the flow in a water-cooled heat exchanger, will affect biofilm thickness and hence its activity in different locations in the filter.. The diffusion of oxygen through the biofilm will be affected with the distinct possibility that at the lower layers, anaerobic activity will be promoted. In effect, this means that the surface area rather than the mass of biofilm is the criterion of effectiveness. The efficiency of trickle filters is very dependent on the operating conditions that affect the mass transfer to (air/water), through (water/biofilm) and from (biofilm/water) an active biofilm. The rate of reaction within the biofilm will also affect efficiency. Designs of biofilters that are increasingly used to overcome some of these operating restrictions in the simpler designs of trickle-filters include the rotating biological contactor, biological aerated filters and aerobic fluidised beds (O’Flaherty and Lens 2003). The rotating biological filter is essentially a series of discs which may be flat or corrugated (to give some additional area), usually fabricated from plastic material mounted on a horizontal shaft (see Fig. 7.2) The discs are made to rotate at right angles to the incoming wastewater so that 30–40% of a disc surface is immersed at any one time. Biofilm accumulates on both sides of the disc and the organisms are alternatively exposed to the waste water and to the atmosphere. The rotation of the discs ensures effective mass transfer from the liquid to the biofilm, thereby improving the efficiency of the operation. In biological aerated filters the biofilm grows over a support medium completely submerged in waste water with oxygen supplied by diffusers at

Rotating biodisk tank Effluent Setting tank M

Discharge

Disposal

Figure 7.2 Schematic representation of a rotating biological contactor. (Courtesy IWA Publishing.)

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the base of the reactor. As a result, a large surface area of the medium is used and a considerable quantity of biofilm is developed making the filters very efficient. The rapid growth of biofilm due to the small shearing effects of the slow moving water means that regular back washing is required to ensure that the filter does not become blocked. Fluidised-bed processes including the ‘airlift’ reactor, use an inert medium of small size e.g., sand particles (0.2–2 mm diameter), glass, anthracite, pieces of polyester foam pads or activated carbon granules to support active biofilms. Fig. 7.3 is a simplified diagram of the process plant. The medium contains an extremely high surface area compared to the media in static systems, allowing a large amount of biofilm to develop. Porous media, such as polyester foam, allow microbial growth both inside and outside the structure. In many reactors the oxygen to support the biofilm activity is provided by air injection. The high biomass density however results in a high oxygen demand that can only be satisfied by the

Biogas

Effluent

Fluidised bed

Waste Water

Air or Oxygen

Figure 7.3 Schematic representation of an aerobic fluidised bed contactor.

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Table 7.1 Summary of qualitative effects of tested parameters on detachment rate Parameter

Biomass decay Biofilm pellet batch Biofilm storage time Pellet morphology Pellet diameter Bare carrier roughness Bare carrier concentration Flow regime Superficial air velocity Reactor geometry

Effect

Negligible Not clear Not clear No effect observed, data limited No effect observed, data limited Important Important Important Within one flow regime no effect observed, data limited. Important

use of pure oxygen to replace the air injected at the bottom of the reactor. Control ensures that highly efficient wastewater contact with the biofilm is achieved. Fluidised-bed contactors are very compact but relatively expensive to operate due to the need for pure oxygen and the pumping requirements. There will be a delay before the reactor begins functioning effectively as the suspended carrier particles will take time to become completely covered in active biofilm. Under these conditions, clearly the quality of the surface of the particles i.e., the roughness will influence the length of time before the reactor can be said to be operating effectively. It is possible to shorten this time however, by pre-coating the particles. The biofilm accumulation is also strongly affected by abrasion processes in the reactor, largely due to particle interactions. Gjaltema, Tijhuis, van Loosdrecht, and Heijnen (1995) examined the mechanism of detachment of biomass in airlift reactors. In qualitative terms their observations are presented in Table 7.1, which indicates (although some of the data admittedly are limited) the care needed in the design, choice of particle and operating conditions. In the light of these unavoidable delaying factors it would be extremely difficult to predict the time scale before the reactor could be said to be fully operational. However practical experience would be helpful in estimating the necessary time to be allocated before full operational output can be achieved. Anaerobic wastewater treatment is gaining popularity for two reasons: 1. Difficulties such as instability, biofilm loss and slow establishment of the process have been overcome through improved understanding of the process principles.

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2. During anaerobic digestion, up to 80% of the energy content of the waste constituents is converted to methane and as referred to earlier, this has attracted attention as a potential addition to energy availability. O’Flaherty and Lens (2003) described anaerobic granulation as a unique example of biofilm formation. These authors discussed several theories to explain the formation of anaerobic granules. Immobilisation of the bacteria into dense, easy-settling, particulate biofilm is essential for reactor operation in waste water treatment, considerably reducing biofilm loss. Figure 7.4 is a schematic diagram of an up-flow, anaerobic, sludge blanket reactor, one of the various designs for anaerobic waste water treatment processes. Immobilisation of bacteria into dense particulate biofilms is essential for efficient operation. Poor granulation is a common cause of reactor instability and consequent inefficiency. Figure 7.5 illustrates anaerobic granules removed from an up-flow anaerobic sludge blanket reactor. The bases of theories to formulate anaerobic granulation are physical, ecological and thermodynamic (O’Flaherty and Lens, 2003). It is likely that Biogas

Effluent

Fluidised bed

Waste water

Figure 7.4 Schematic representation of an anaerobic fluidised bed contactor.

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Figure 7.5 Anaerobic granules from a laboratory-scale upflow anaerobic sludge blanket reactor. Scale bar ¼ 5 mm. (Courtesy IWA Publishing.)

some aspects of each of these theories plays a part in the granulation process, although at present the technology is more empirical. In the light of current research further improvements will be made. The biogas emitted from the reactors includes CO2 and H2O from the aerobic treatment and CO2 and CH4 from anaerobic contactors. Clearly there are implications in respect of the environmental problem of global warming. As anaerobic treatment is the basis for ‘biofuels’, it can be reported that a process has been developed in the U.K. to utilise the biogas products to power the treatment plant (Anonymous, 2010). There is increasing interest in the technology of high pressure membrane filtration processes for the production of high quality water ( Judd, 2004). The technique is particularly relevant to the removal of Cryptosporidium oocysts (a pathogen). Clearly this has important relevance to the task of producing water for human consumption. High pressure membrane filtration includes reverse osmosis and nanofiltration to provide drinking water of high quality, virtually free of pathogenic microorganisms. Because of the decreasing cost of membrane technology this development is gaining importance in the water industry. It is aided by operating experience and fabrication improvements. The fact that water is not sterile means that it is certain the membrane will become contaminated by biofouling. It is to be expected that the problem of biofouling of porous surfaces will be more complex than that of continuous surfaces. The build-up of microorganisms is not only on the surface but also within the pore structure. Nutrients in the water will enhance this biofouling. The presence of such biofouling will inevitably lead to production difficulties and the associated costs. The consequences of

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biofouling of membranes are summarised by Flemming, Schaule, and Mcdonogh (1992): 1. Increased membrane resistance by the biofilm including decrease of permeate production, increase in energy consumption and an increase in pressure drop across the membrane. 2. Formation of a gel phase between the water being processed and the membrane surface, resulting in the inhibition of conventional mass transport adjacent to the membrane, an increase in concentration polarisation due to a lack of tangential flow (see Chapter 2) and a decrease in salt rejection. 3. Damage to plant and product that involves microbial attack on the membrane, (e.g., cellulose acetate), microbial contamination of the permeate and a decrease in the life of the membrane (particularly due to damage as a result of the requirement for more frequent cleaning). 4. Increased operating costs due to reduced permeate production and quality, higher energy requirement, more frequent cleaning requirement and short lifespan of membranes. In order to anticipate likely problems in a membrane process used for water purification so that treatment programmes can be devised, an experimental biofouling simulator has been developed (Vrouwenvelder, van Paassen, Wessels, van Dam, & Bakker, 2006). The advantages claimed for this simulator are: 1. In comparison with the spirally wound membrane module, both showed: a. The same development of pressure-drop in time. b. The same concentration of active biomass. 2. It can characterise and quantify fouling. 3. Practical assets include a. It simulates the spiral wound elements in regard to material of construction, height of the spacer channels and hydrodynamic behaviour. b. It is suitable for in situ, real time and non-destructive (visual) observations, movement of pressure drop and analysis of membrane coupons taken from the simulator. c. It is easy to handle, well-defined and small, requiring small quantities of water and chemicals. 4. Within one week, the dosage of a tap-water supplemented with nutrient to the simulator, promoted sufficient biofilm accumulation to be readily detected by visual observation, pressure drop increase and analysis of coupons taken from the simulator.

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There are two basic methods for employing membrane filtration: 1. Dead-end filtration where all the water is forced to pass through the membrane; in time, a residue of the material builds up on the membrane. 2. Cross flow filtration in which the feed water flows along the membrane surface resulting in two streams; the concentrate is the stream rejected by the membrane and the permeate is the fraction of the total water flow that passes through the membrane (the treated water). The biofouling that is associated with membrane filtration is complex and very different from that associated with cooling water for instance. In water treatment the biofilm will be interspersed with solid particles including inorganic salts. Where the ultimate destination of the water is for human consumption, say from a reverse osmosis plant, biocide treatment to regenerate filter efficiency is not realistically an option. The ultimate method of fouling removal is by the process of backwashing i.e., reversing the permeate flow through the membrane, chemical cleaning using caustic solutions or the use of oxidants such as chlorine or hydrogen peroxide. Chemical cleaning however, can reduce the life of the membrane. There are techniques for lengthening the time between ‘cleans’ for instance, by using a membrane that has a reduced bacterial affinity or membranes that can more easily be cleaned (Hilal, Al-Khatib, Atkin, Kochkodan, & Potapchenko, 2003). Pretreatment of the membrane itself to change the interaction between the membrane and the foulant is another alternative. The pre-treatment of the feed water to the membrane is also a possibility to minimise biofouling (Solomon, Summers, and Speth, 1993; Suratt, 1993). It is common practice in the water industry. The addition of powdered activated carbon to water with a high naturally occurring organic material with coagulation, may also be employed for the reduction or prevention of the fouling of membranes. The choice of any pre-treatment will naturally depend, not only on the quality of the feed water but also on the nature of the membrane system, together with the ultimate destination of the water. Mandiralioglu (2004) studied the pre-treatment alternative of coagulation before ultra-filtration with different coagulants and different operational parameters, such as are commonly used in the water industry to minimise potential fouling in dead-end and cross-flow filtration, different trans-membrane pressures and different membrane modules. It was observed that the structure of the layer formed on the membrane has an impact on water production. The use of a coagulant always resulted in the

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production of high quality water with total removal of humic acid and a low coagulant residual. The use of aluminium sulphate instead of ferric chloride proved to be more effective. If the pre-treatment was applied, the fouling problem could be controlled completely in most situations.

7.2.2. Groundwater improvement As discussed earlier in this Chapter, groundwater after purification is also one of the major sources of potable water for domestic use. As the name implies, water after falling as rain or snow can be in contact with soil or rocks in the ground for long periods of time before it percolates into an aquifer, river or lake. As a result it will become contaminated with soluble substances. Much of this material will have to be removed in order to produce potable water. A full description of the technology available for the removal of contaminants in relatively small concentrations is outside the scope of this discussion of biofouling but an example of the utilisation of a suitable biofilm for the reduction of manganese will be described. Manganese is along with iron the most abundant heavy metal in the earth’s crust and as a result it is found regularly in the soluble divalent (Mn2þ) or quadrivalent (Mn4þ) form in ground water. Although ingestion of manganese through drinking water with concentrations up to 500 mg/l has no harmful effects on human health, (World Health Organisation 1993), high concentrations above 100 mg/l are likely to adversely affect the taste of tea and coffee and are undesirable due to the potential to stain laundry. The traditional method of reducing the manganese content of drinking water is to use a sand filter containing the appropriate microorganisms. Sand filters with pre-aeration but without pre-chlorination have been set up for biological manganese removal. Leptothrix discophora forms a large proportion of the manganese oxidising biomass (Vandenabeele, de Beer, Germonpre´, & Verstreate, 1992). This microorganism oxidises iron and manganese, both extra-cellulary and inter-cellulary by catalytic action of excreted enzymes. The development of a suitable mass of this microbe in sand filters is slow, sometimes taking months for the bed to ‘ripen’dthat is, to be fully active. Other organisms may interfere with the activity of Leptothrix discophora so that even a mature filter may not be effective in manganese reduction. It is possible to accelerate the establishment of an active bed by seeding with material from active filters. (Bourgine, 1994) This concept was developed further by Hope and Bott (2004). Using specially designed laboratory and pilot scale biofilters with monocultures of Leptothrix discophora, they demonstrated that it would be possible to seed a new manganese biofilter

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with recirculating batch cultures of Leptothrix discophora. This would reduce filter maturation times from months to days with a preponderance of the active microorganisms, thereby improving filter efficiency in terms of manganese removal.

7.2.3. Water Distribution Systems Block (1992) summarised the problems in distribution systems for drinking water and possible solutions to these problems. The effects on water quality due to microbial growth are clearly of major concern to the providers of the service, due to the potential for infection of customers. Because of the vigilance of the distributors, many customers accept the good quality of water without giving much thought to how it is achieved and maintained. The range of microorganisms encountered covers the whole spectrum of aqueous entities, bacteria, fungi and protozoa. Biofilms may be produced on the walls of distribution pipes in addition to ‘free swimming’ cells in the water itself. The removal of the biofilm and control of microbial growth are not so straightforward as they are in industrial systems, because of the overriding concern for the health of the consumers. The problems presented by the accumulation of biofilm have been tabulated by Block (1992). They include: 1. Bacteria can be the starting point of a tropic food web leading to the proliferation of undesirable higher organisms. 2. Specific bacterial species generate turbidity, taste and odours in drinking water. 3. High counts of heterotropic bacteria interfere with the detection of coliforms or sanitary indicators. 4. Accumulation of attached biomass provokes bacterial corrosion. 5. Biofilms increase frictional resistance to the flow of water, which reduces the capacity of the system to meet any sudden increases in demand, such as for firefighting. 6. Continual failure of the distribution system to meet all the established water quality criteria for coliforms or heterotrops. In general water distribution systems are very large. Actual human consumption, however, is a relatively small portion of the total supply. Yet all the water must be of the quality to meet the rigid health requirements. This does not mean that the water has to be sterile. Provided that it has no unpleasant taste or odour and meets the health standards, it is acceptable. Nevertheless, the retention of very low concentrations of a biocide, for

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example, chlorine or chloramine is frequently used to maintain microorganism control.

7.3. FOOD INDUSTRY The control of biofouling in the food industry is essential in order to prevent food contamination and the risk to consumers’ health. Surfaces are probably the most important route to infection, both directly via product contact surfaces i.e., production equipment or indirectly, as reservoirs of infection for product contamination, such as drains, floors and washroom surfaces (Timperley, Thorpe, & Holah, 1992). Fundamental to the control of contamination of food processing equipment is hygiene design. Effective hygiene design prevents the retention of microorganisms and other unwanted contaminants on surfaces during operation. As discussed in Chapter 3, rough surfaces and crevices facilitate the retention of microorganisms, so the first step in the total control of microbial accumulation problems in food processing is to ensure that equipment design and manufacture eliminate the ‘hide-aways’. In their paper Timperley, Thorpe & Holah (1992), illustrate in great detail, how careful design and thoughtful use of components that make up food processing equipment can reduce the incidence of biofouling. The list includes permanent and dismountable joints, fasteners such as screws, nuts and bolts and internal angles and corners. Furthermore, the absence of dead spaces facilitates cleaning. Relatively frequent cleaning is often employed in food processing and together with smooth surfaces, helps to maintain the desired hygienic conditions. Probably because the extent of fouling in food processing, is relatively small due to the care taken over design, operation and frequent cleaning, it is often referred to as ‘soil’. Because of the generally short intervals between cleans and the need to maintain good hygiene conditions, it is very unlikely that biofilms with the characteristics of those described elsewhere in this book are likely to exist. The soils will be principally food or residues from food material. The accumulations of living microorganisms in the food soil will depend very much on temperature; many food processing operations are at relatively high temperature, which would preclude the existence of living microorganisms. Nevertheless, there are likely to be regions and processes where the temperature allows living microorganisms to exist. Day to day variations in the food material being processed could affect the amount of soil produced. The extent of the fouling may also be dependent on other mechanisms that lead to fouling and

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hence the retention of microorganisms, such as chemical reaction, crystallisation, corrosion and solidification. Graham (1997) made some very valid points in connection with sterilising activities in the food industry to avoid contamination from microorganisms. Many of the disinfection agents that have been used, represent a potential hazard in themselves and Graham suggests that ozone (discussed in Chapter 4) is an excellent alternative choice. The following are some of the points that make the use of ozone attractive for food industry application: 1. It has been shown to be a powerful disinfectant for deactivation of a very large number of different organisms. 2. It has been used for the treatment of bottled water. 3. It has been used for increasing storage life and ozone dissolved in water has been used for sanitising surfaces of vegetables, fruits and other agricultural products. 4. It does not remain in water for a very long period. 5. Any byproducts that ozone may produce are similar to normal oxidation products. Because of the complexity of the food product, the technique of cleaning food processing equipment can be very wide ranging and aggressive. Holah (1992) emphasises the importance of cleaning as part of the sanitation of food processing equipment. The cleaning phase can be divided into three sections. 1. Wetting and penetration by the cleaning solution of both soil and equipment surfaces. 2. The reaction of the cleaning solution with both the soil and surface to facilitate peptization of organic materials, dissolution of soluble organics and minerals, emulsification of fats and dispersion and removal from the surface of solid soil components’. 3. The prevention of redeposition of the dispersed soil and removal from the surface of solid soil components.

7.4. PAPER MANUFACTURE In paper manufacture a very dilute suspension of wood fibre in water is passed through a machine whereby a continuous mat of the fibre is made by the removal of some of the water by suction. The mat is then drawn through ovens to evaporate the remaining water and leave a continuous sheet of paper. The water is recycled from various points in the system and mixed with the condensate from the drier to produce water, which is ideal for

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biological growth in terms of temperature and nutrients, unless biocidal control is employed. The ‘wet end’ of a paper or board machine has conditions that favour biofouling. The biofouling may ‘seed’ the paper with microorganisms that cause discolouration and may even create odours. Should biofilm growth in the machine be allowed to continue, ‘streamers’ may be produced and pieces may break away to cause spots and blemishes on the paper so that it has to be rejected. The problem of product damage of this kind is usually overcome by the application of a suitable biocide. Kemmer (1998) points out that the tolerable microorganism count in a paper mill varies with the type of paper being made and the paper machine operating conditions such as temperature and pH. Careful attention is particularly required where the destiny of the product has a connection with the food industry, such as for product wrapping, in order to avoid any contamination of the food.

7.5. COOLING WATER Cooling water has been the basis of the discussion of biofouling in this book because it represents the simplest and widespread example of industrial biofouling. Much of the background to the formation and control of biofilms in cooling water systems has already been covered. However a brief review of the use of cooling water is relevant to this chapter. Two major problems in cooling water systems as a result of biofouling on the surfaces of the associated heat exchangers are: 1. Increased pressure drop through the system. The presence of the biofilm represents a narrowing of the flow channel through which the water is flowing. For a given flow rate this means that the water velocity is increased. Since the energy required for pumping is related to the square of the velocity the increased energy requirement could be quite large. Furthermore the surface of the biofilm will be rougher than the surface of the clean heat exchanger. As a result of these two effects a relatively small amount of biofouling could produce a relatively large increase in the energy required for pumping, with the associated increased cost and emissions if the energy was derived by combustion. 2. Reduced heat transfer efficiency. A biofilm on a heat exchanger surface will act as a resistance to heat transfer thereby reducing the cooling efficiency of the heat exchanger. A wide range of industries employ the medium of water to remove heat from products or intermediates in their operations. The incidence of

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biofouling in heat exchangers handling cooling water will vary widely according to the industry involved. Major biofouling problems are likely to occur in steam condensers in the power industry, whereas in the paper and pulp industry for instance, the incidence of biofouling although still a nuisance particularly in respect of product quality, is relatively low. Other industries that use cooling water are likely to fall between these extremes. Three types of cooling water systems employing fresh water are used in industry: 1. Closed recirculating systems: As the name implies, in these systems the water re-circulates through a totally enclosed system so that it is not exposed to the atmosphere. Prior to enclosure the water is usually made sterile in some way. Under these circumstances biofouling will not be a problem unless leakage brings about microbial contamination. The cooling water itself is often cooled by air-blown heat exchangers before being re-circulated in the system. 2. Once-through systems In once-through systems, the water is usually taken from a natural source, such as a river, a lake or the sea, passed through heat exchangers to effect the cooling and then discharged back to the source. Provided that the source is large enough, the thermal effects that could endanger wild life in the area will not present an environmental hazard. Problems that are encountered in open recirculating systems, including biofouling, are likely to cause difficulties in once-through systems and control using biocides with the associated environmental restrictions would be difficult to justify. 3. Open recirculating systems In open recirculating systems the water is usually obtained from the same natural sources as once-through systems. As a result the water will be contaminated with microorganisms and even macroorganisms. Town’s water is not usually employed on account of the costs involved, i.e., purchase and discharge charges. After the required heat removal has been achieved through suitable heat exchanger equipment, the recirculating water is itself cooled. Usually evaporation is employed using either a cooling tower or spray pond. Both methods rely on latent heat removal through evaporation thereby reducing the water temperature. Both methods of cooling are open to the atmosphere and this allows further inoculation with microorganisms dependent on the local atmospheric conditions.

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Evaporation E Windage W Cooling tower

Make up

Heat exchange system Blowdown B

Water M Basin Leakage L

Figure 7.6 Water loss in a cooling water system with a cooling tower.

Figure 7.6 illustrates the components and streams of an open recirculating cooling system. The following water streams may be identified: Makeup (M): water to replace that which is lost from the system in the following ways: Blowdown (B): as water vapour is lost through evaporation, the concentration of dissolved substances increases. This effect cannot be allowed to continue since it is likely to cause its own fouling problems. Contaminated water is removed from the system to be replaced with fresh water. Evaporation (E): water lost through heat removal as latent heat in the cooling tower. Windage (W): water lost as droplets into the atmosphere. Leakage (L): inevitably there will be leaks of cooling water through faulty joints and flanges. The water balance is : M ¼ W þ E þ L þ B It will be apparent that without some sort of intervention the system will allow biofouling to occur, particularly on the heat transfer surfaces. Algae growth could be promoted in the cooling tower basin and other collection points and fungi could exist where there is a suitable substrate. It is probably true to say that most industrial biofouling occurs in cooling water circuits, where temperatures and nutrient levels are ideal for microbial growth. Steps have to be taken to control it (See Chapter 4).

7.6. THE PROBLEM OF LEGIONELLA PNEUMOPHILA This microorganism was discovered to be the cause of the outbreak of the fatal disease amongst attendees at an American Legion convention in Philadelphia in 1976; hence the origin of its name. It is a gram-negative rod bacterium.

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Since its discovery it has been identified in many places where large volumes of water exist, such as natural locations and cooling water circuits. The bacterium is active over a relatively wide range of temperatures between 20 to 45  C but it is dormant at lower temperatures. As with microorganisms in general, it is killed at higher temperatures. It thrives in biofilms and on surfaces that are exposed to a bulk water flow. In industrial conditions Legionella is a potentially serious problem in relation to cooling towers and evaporative condensers. Where ‘mists’ are discharged to the atmosphere there could be a source of infection to anyone who happens to be in the vicinity. The risks are exacerbated where there is an opportunity for this mist-laden air to enter an air conditioning plant and infect those people who are in contact with the circulating air. There are strict safety requirements in connection with such systems, enforceable by law. It is imperative that cooling towers are fitted with effective high-efficiency ‘drift’ eliminators to prevent the discharge of water droplets into the surrounding atmosphere. Biocide depletion due to blowdown and other causes also needs careful attention to ensure continual biocide effectiveness, particularly in respect of Legionella.

REFERENCES Anonymous. (2010). C. Flavell-While, (Ed.), The Chemical Engineer. 831, 21. Block, J. C. (1992). Biofilms in drinking water distribution systems. In L. F. Melo et al. (Eds.), Biofilms – science and technology (p. 469). Dordrecht: Kluwer Academic Publishers. Bourgine, F. P., (1994). Biological processes at Saints Hill water treatment plant, Kent. J. IWEM, 8, 379. Flemming, H. C., Schaule, G., & Mcdonogh, K. (1992). Biofouling on membranes- a short review. In L. F. Melo, T. R. Bott, M. Fletcher, & B. Capdeville (Eds.), Biofilms-science and technology (p. 487). Dordrecht: Kluwer Academic Publishers. Gjaltema, A., Tijhuis, L., van Loosdrecht, M. C. M., & Heijnen, J. J. (1995). Biotechnology and Bioengineering, 46, 258. Graham, D. M. (1997). Use of ozone for food processing. Food Technology, 51(6), 72. Heinen, J. J., van Loosdrecht, M. C. M., Mulder, A., & Tijhuis, I. (1992). Formation of biofilms in a biofilm air-lift suspension reactor. Water Science Technology, 26, 647. Hilal, N., Al-Khatib, L., Atkin, B. P., Kochkodan, V., & Potapchenko, N. (2003). Photochemical modification of membrane surfaces for (bio)fouling reduction: a nanoscale study using AFM. Desalination, 158(1–3), 65. Hope, C. K., & Bott, T. R. (2004). Laboratory modelling of manganese biofiltration using biofilms of Leptothrix discophora. Water Research, 38, 1853. Judd, S. J. (2004). The main data base. Cranfield: Membrane Academia Industry Network Conference. Kemmer, F. N. (Ed.) (1988). The Nalco water hand book (2nd ed). New York: McGraw-Hill Book Co. Mandiralioglu, A., (2004). Control of fouling in membranes used for drinking water treatment, PhD. Thesis, University of Wolverhampton.

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Mozes, N., & Rouxhet, P. G. (1992). Modification on surfaces for promoting cell immobilization. In L. F. Melo et al. (Eds.), Biofilms-science and technology (p. 69). Dordrecht: Kluwer Academic Publishers. O’Flaherty, V., & Lens, P. (2003). Biofilms in wastewater treatment systems. In P. Lens, A. P. Moran, T. Mahoney, P. Stoodley, & V. O’Flaherty (Eds.), Biofilms in Medicine, Industry and environmental biotechnology. Tunbridge Wells: IWA Publishing. Scholtz, M. (2006). Wetland systems to control urban runoff. Amsterdam: Elsevier. Solomon, T. L., Summers, R., & Speth, T. F. (1993). The reduction of membrane fouling by physical, chemical and biological pretreatment. Baltimore: AWWA Proceedings Conference Membrane Technology. Suratt, W. B. (1993). Pretreatment for membrane processes. Baltimore: AWWA Proceedings Conference Membrane technology. Tijhuis, L., van Benthum, W. A. J., van Loosdrecht, M. C. M., & Heijnen, J. J. (1994). Solid retention time in spherical biofilms in a biofilm airlift suspension reactor. Biotechnology Bioengineering, 44, 867. Timperley, D. A., Thorpe, R. H., & Holah, J. T. (1992). Implications of engineering design in food industry hygiene. In L. F. Melo et al. (Eds.), Biofilms – science and engineering (p. 379). Dordrecht: Kluwer Academic Publishers. Vandenabeele, J., de Beer, D., Germonpre´, R., & Verstreate, W. (1992). Manganese oxidation by microbial consortia from sand filters. Microbial Ecology, 24, 91. Vrouwenvelder, J. S., van Paassen, J. A. M., Wessels, L. P., van Dam, A. F., & Bakker, S. M. (2006). The membrane fouling simulator: a practical tool for fouling prediction and control. Journal of Membrane Science, 281, 316. World Health Organisation, (1993). Guidelines for drinking water quality, (2nd ed.). Vol. 1, Recommendations, 50.

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Concluding Remarks It is not surprising that the attachment of biofilms to surfaces is an extremely complex phenomenon since it depends on aspects of physics, chemistry and microbiology. It is further complicated by the fact that in many industrial situations the wide-ranging composition of the population of microorganisms changes with time and is likely to affect the properties of the biofilm. The availability of suitable nutrients will also influence the quality of the biofilm. The conditions under which the biofilm develops e.g., fluid (usually water) flow velocity and the temperature prevailing in the industrial equipment will also affect biofilm development. The quality of the surface to which the microorganisms attach i.e., its roughness and surface charge will also have a profound effect on the growth and stability of the developing biofilm. It will be apparent therefore, from this wide range of factors which can randomly change with time and which influence the creation and retention of a biofouling deposit on industrial equipment that each example of biofouling is likely to be unique. For this reason, the application of control techniques is very empirical, and generally based on experience and preference rather than theory. As with any industrial activity, an important criterion is the cost of the technology involved in the chosen method of control and factors of health and safety of personnel, the local community and the likely impact on the environment. The technical skill of the operators of the process employed will also be an important factor in the effectiveness of the control. There is a wide range of industries that experience problems of biofouling, principally those that involve the use of water in some way. Many industries use water for cooling purposes, much of it emanating from natural sources, so that biofouling problems will occur unless steps are taken to eliminate or reduce its development. The character of a biofilm may change with time due to changes in the factors that influence its growth, such as changes in the prevailing season or the origin of the water. These changes could affect the efficiency of the method of control so that constant vigilance through effective monitoring is required. As many of the references quoted in this publication illustrate, much research work has been done or is in progress to provide a better Industrial Biofouling ISBN 978-0-444-53224-4

Ó 2011 Elsevier B.V. All rights reserved.

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understanding of the fundamentals of biofouling and hence the development of improved methods of control. It remains to be seen whether the emphasis of control shifts from chemical to physical techniques or a combination of the two. Much will depend on local legislation, which is likely to become more stringent and on the costs involveddboth capital and operating costs of the different technologies available for biofouling control.

INDEX A Air/gas injection, 133 Algae, 24, 35 Adhesion of microorganisms, 46–9 interacting forces, 49–55 quality of surface, 58–60 Adsorbed layers, 55–7

B Bacteria, 23–4, 36–8 aerobic, 36 anaerobic, corrosive, 24 classification, 37–8 colonies, 42–4 growth, 67–9 iron depositing, 24, 37 nitrifying, 24, 37 slime-forming, 23, 36 Spore-forming, 24, 36 sulphate reducing, 24, 37 Barnacles, 4 Biocide choice, 95, 96 classification, oxidising, 99–100 non-oxidising, 100–01 desired qualities, 88 dosing strategies, 89–94 immobilised, 121 interaction with microorganisms, 86–7 microbial response, 97–8 microbial transport, 122 removal of biofouling, 123 Biocides amines, 120 chlorine, 101–05 effect of pH, 102 effectiveness, 103 environmental problems, 104–5 storage, 104 chlorine dioxide, 105–07 qualities, 106

DBNPA, 2, 2- dibromo-3nitrilepropionamide, 118–9 formaldehyde, 117 glutaraldehyde, 114–17 effect of pH, 115 effectiveness, 115–7 hydrogen peroxide, 118 organo-sulphur compounds, 120–1 ozone, 107–14 advantages, 108 chemistry, 110–2 effectiveness, 113–4 Biodispersants, 127–8 Biofilms, 1, 23–76 corrosion effects, 73–4 establishment (summary), 62–3 formation, 29–31 industrial conditions, 61–2 influencing factors, 22 growth, 13–7, 67–9 industrial, 181–200 interacting forces, 49–55 interaction with particles, 64–7 metabolism, 41–3 modelling, 73 morphology, 98 removal, 17 stability, 69–72 structure, 69–72 surface importance, 45–6 problems, 2 removal, 17, 123–7 using chlorine, 124–5 using hydrogen peroxide, 125 using peracetic acid, 126 using potassium permanganate, 126 Bacillus, 28 Biofouling control, chemical, 84–128 requirements, 88–9 dosing, 89–94

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Index

Biofouling (Continued ) effectiveness, 95–8 physical, 129–47 combined physical and chemical, 147–50 costs, 4, 74–6 factors affecting, 22 interacting forces, 49–51 industrial problems, 2–3 measurement, 174–5 modelling, 73 Boundary layer, 8 Brownian motion, 11 Brush and cage system, 133

C Capital cost, 75 Chlorine, 101–5 Chlorine dioxide, 105–7 Cleaning, 3 off-line, 155–8 manual, 157 jetting, 157 drilling, 157 rodding, 157 high temperature sterilisation, 157 osmotic shock, 157 Clostridia, 28 Coating surfaces, 138–41 Cooling water, 197–9 Corrosion, influence of biofilms, 73–4

D

F Fick’s law, 12 Flagella, 27 Flexible tubes, 137 Fluid flow, 7–11 Reynolds number, 7–11 laminar flow, 8–10 turbulent flow, 8–10 Fluid head, 10 Fluidised beds, 137–8, 187–190 Food industry, 195–6 Forces, interacting, 49–55 van der Waals, 50–1 DLVO, 51–5 Friction factor, 10 Fouling Allowance, 21 measurement, 174–5 mechanisms, 20, 64 particulate, 64–7 Fungi, 24, 34–5

G Gamma rays, 129 Glutaraldehyde, 114–7 biocidal properties, 118 effect of pH, 115 Gram-positive bacteria, 25 Gram-negative, bacteria, 25 Ground water, 182, 193–4 Growth, 12

(DBNPA), 2, 2 Dibromo-3nitrilepropionamide, 118–9 Darwin’s theory, 26 Dechlorination, 105 Deposition, 12 Diffusivity, 12 Drag force, 10

H

E

I

Eddy diffusion, 11 EPRI, 61 Extracellular polymers, 28 EPS, 44

Ice crystals, 143 Inserts, 134–7 Industrial, problems, 2

Heat transfer, 18–21 resistance, 20–1 Housekeeping, 84 Hydrogen peroxide, 118 Hydroids, 4 Hypobromous acid, 109

Index

Industrial (Continued ) conditions, 61 biofouling, 181–200

L Laboratory monitoring, 169–174 Laminar flow, 8–10 Laminar sublayer, 13 Legioella pneumophila, 199

M Macroorganisms, 1 deposition, growth, removal, 12–8 Mass transfer, 11–2 Matrix formation, 44–5 Membrane filtration, 190–3 Mesosomes, 28 Metabolism, 41–3 Microorganisms, 1 adhesion to surfaces, 46–9 classification, 31 deposition, growth, removal, 12–7 interaction with biocides, 86–7 resistance to biocides, 86 pH effects, 39–40 Microbiology, 26–9 Microbial, accumulation, 2 adhesion, 46–9 appendages, 27 cell structure, 25–9 growth requirements, 38–41 interdependence, 33–4 nutrient requirements, 31–32, 41 metabolism, 41–3 matrix formation, 44–5 Monitoring off-line, 161–5 process plant, 165–9 laboratory, 169–74 measurement techniques, 174–5 accuracy, 176–7 Mussels, 4

N Nutrients, 14–6, 41

O Off-line cleaning, 155–8 Operating costs, 74–6 Organo-sulphur compounds, 120–1 Osmosis, 28 Osmotic shock, 157 Ozone, 107–14 cooling water treatment, 108 chemistry, 110–2 biocide, 112–4

P Paper manufacture, 196–7 Particle/biofilm interaction, 64–7 Percolating filters, 184–7 Physical control of biofouling, 129–47 air/gas injection, 133–4 brush and cage system, 133 coating, 138–41 flexible tubes, 137 fluidised beds, 137–8 gamma rays, 129 high voltage capacitance, 146 ice crystals, 143 inserts, static, 134–6 oscillating, 136–7 polymer fibres, 141–3 scraped surface, 146–7 sponge rubber balls, 130–3 ultrasound, 143–5 ultraviolet light, 129 Pseudomonas fluorescence, 18 Pili, 27 Pressure head, 10 Process plant monitoring, 165–9 Prokaryote cells, 25 Protozoa, 24

R Reciprocating scraped surface, 146–7 Ribosomes, 28 Robbins device, 163

207

208

Index

S Sea mats, 4 Sea squirts, 4 Serpulid worms, 4 Slimes, 1 Spores, 28–9 Stalks, 27 Steralisation, high temperature, 94 osmotic shock, 94 Surface water, 182 Surfaces, adsorbed layers, 55–8 importance, 45–6 microbial adhesion, 46–9 quality, 58–61

T Taprogge system, 130 Temperature, effect on biofilm growth, 19 Test data accuracy, 176–7

Thermal resistance, 20 Toxic metal ion release, 57 Trickle filters, 184–7 Turbulent flow, 8–10

U Ultraviolet radiation, 84, 129 Ultrasound, 143–6

V van der waals forces, 50–5 Velocity profile, 9 Viscous sub-layer, 8

W Water cooling, 197–200 distribution, 194–5 industry, 181–95 quality, 32–3, 95 treatment, 183–93 Wettability theory, 51–5