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In recent years, the formation and impacts of biofilms on dairy manufacturing have been studied extensively, from the effects of microbial enzymes produced during transportation of raw milk to the mechanisms of biofilm formation by thermophilic spore-forming bacteria. The dairy industry now has a better understanding of biofilms and of approaches that may be adopted to reduce the impacts that biofilms have on manufacturing efficiencies and the quality of dairy products.Biofilms in the Dairy Industry provides a comprehensive overview of biofilm-related issues facing the dairy sector. The book is a cornerstone for a better understanding of the current science and of ways to reduce the occurrence of biofilms associated with dairy manufacturing. The introductory section covers the definition and basic concepts of biofilm formation and development, and provides an overview of problems caused by the occurrence of biofilms along the dairy manufacturing chain. The second section of the book focuses on specific biofilm-related issues, including the quality of raw milk influenced by biofilms, biofilm formation by thermoduric streptococci and thermophilic spore-forming bacteria in dairy manufacturing plants, the presence of pathogens in biofilms, and biofilms associated with dairy waste effluent. The final section of the book looks at the application of modelling approaches to control biofilms. Potential solutions for reducing contamination throughout the dairy manufacturing chain are also presented.Essential to professionals in the global dairy sector, Biofilms in the Dairy Industry will be of great interest to anyone in the food and beverage, academic and government sectors.This text is specifically targeted at dairy professionals who aim to improve the quality and consistency of dairy products and improve the efficiency of dairy product manufacture through optimizing the use of dairy manufacturing plant and reducing operating costs.

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The Society of Dairy Technology (SDT) has joined with Wiley-Blackwell to produce a series of technical dairy-related handbooks providing an invaluable resource for all those involved in the dairy industry, from practitioners to technologists, working in both traditional and modern large-scale dairy operations. For information regarding the SDT, please contact Dr Liz Whitley, Larnick Park, Higher Larrick, Trebullett, Launceston, Cornwall, PL15 9QH, UK. email:[email protected]

Other volumes in the Society of Dairy Technology book series:

Probiotic Dairy Products (ISBN 978 1 4051 2124 8)Fermented Milks (ISBN 978 0 6320 6458 8)Brined Cheeses (ISBN 978 1 4051 2460 7)Structure of Dairy Products (ISBN 978 1 4051 2975 6)Cleaning-in-Place (ISBN 978 1 4051 5503 8)Milk Processing and Quality Management (ISBN 978 1 4051 4530 5)Dairy Fats (ISBN 978 1 4051 5090 3)Dairy Powders and Concentrated Products (ISBN 978 1 4051 5764 3)Technology of Cheesemaking, Second Edition (ISBN 978 1 4051 8298 0)Processed Cheese and Analogues (ISBN 978 1 4051 8642 1)Membrane Processing – Dairy and Beverage Applications (ISBN 978 1 4443 3337 4)Milk and Dairy Products as Functional Foods (ISBN 978 1 4443 3683 2)

Biofilms in the Dairy Industry

Edited by

Koon Hoong Teh

Massey University, Palmerston North, New Zealand

Steve Flint

Massey University, Palmerston North, New Zealand

John Brooks

Auckland University of Technology, Auckland, New Zealand

Geoff Knight

Food Process Hygiene Solutions, Melbourne, Victoria, Australia

 

 

 

 

 

 

This edition first published 2015 © 2015 by John Wiley & Sons, Ltd.

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Cover images: Top and bottom left photos by Sara Burgess with special acknowledgement to Manawatu Microscopy & Imaging Centre. Bottom right photo by Geoff Knight.

About the Editors

Dr Koon Hoong Teh

Dr Koon Hoong Teh graduated from Massey University in 2013 with a PhD in Food Technology. His work focused on the dairy biofilms found on milk tankers and their effect on the quality of dairy products. Prior to his doctorate study, his Masters research project was on biofilm formation by Campylobacter jejuni in a mixed bacterial population. He generated six scientific papers, five from his PhD and one from his Masters research project, and presented his works in national and international conference. Currently, he works as a rumen microbiologist. His research interest includes biofilms associated with food quality and safety, and culturing novel and previously uncultured rumen microorganisms. He is also a member of the New Zealand Microbiology Society.

Professor Steve Flint

Steve Flint is Professor of Food Safety and Microbiology and Director of the Food Division of the Institute of Food Nutrition and Human Health at Massey University, Palmerston North, New Zealand. Steve leads a team of postgraduate research students studying a variety of food safety and quality issues with an emphasis on understanding biofilm development and control. Approximately half these projects are associated with the dairy industry. Future research will focus on bacterial interactions in biofilms and mechanisms of biofilm dispersion. Steve has more than 100 scientific publications and more than 100 presentations at national and international scientific conferences. He lectures in food safety and microbiology and does consultancy work for food manufacturers. Steve is a fellow of the New Zealand Institute of Food Science and Technology, president of the New Zealand Microbiological Society and a certified food scientist with the Institute of Food Technology.

Professor John Brooks

John Brooks is a microbiologist, specialising in food microbiology. On graduation, he spent a period of time working at ICI UK, helping to develop the methanol-based Single Cell Protein process. He did a PhD in biochemical engineering at Sydney University, continuing his work on C1 metabolism. He then took up a position at Massey University, teaching food microbiology, and remained there for 30 years, eventually concentrating on biofilm research. John is now Adjunct Professor at Auckland University of Technology. He has consulted extensively for the food and process industries and is a Fellow of NZIFST.

Geoff Knight

Geoff graduated with a B. Appl. Sci. (Hons) in 1991 from La Trobe University (Bendigo) with a major in Microbiology. He initially worked on wastewater microbiology before moving to the food industry, where he developed an interest in biofilms. Geoff worked with The University of Tasmania and the Dairy Process Engineering Centre on research projects for the Australian dairy industry. In 1998, he joined CSIRO Division of Animal, Food and Health Sciences (formerly Food Science Australia), where he continued to study biofilms in food systems. His work at CSIRO has included investigating the impact of biofilm formation and cleaning-in-place procedures on the contamination of milk powders by thermophilic bacteria. More recently, his work has focused on the persistence of bacteria, including pathogens such as Listeria monocytogenes and Cronobacter species, in biofilms on environmental surfaces in dairy manufacturing plants.

List of Contributors

Editors

John BrooksSchool of Applied Sciences, Faculty of Health and Environmental SciencesAuckland University of TechnologyAucklandNew Zealand

Steve FlintInstitute of Food, Nutrition and Human HealthMassey UniversityPalmerston NorthNew Zealand

Geoff KnightFood Process Hygiene SolutionsMelbourne, VictoriaAustralia

Koon Hoong TehInstitute of Food, Nutrition and Human HealthMassey UniversityPalmerston NorthNew Zealand

Contributors

Rod BennettInstitute of Food, Nutrition and Human HealthMassey UniversityPalmerston NorthNew Zealand

Phil BremerDepartment of Food ScienceUniversity of OtagoDunedinNew Zealand

Sara BurgessInstitute of Food, Nutrition and Human HealthMassey UniversityPalmerston NorthNew Zealand

Michael DixonInstitute of Food, Nutrition and Human HealthMassey UniversityPalmerston NorthNew Zealand

Robin HankinSchool of Computer and Mathematical SciencesAuckland University of TechnologyAucklandNew Zealand

Norshhaidah Mohd JamaludinInstitute of Fundamental SciencesMassey UniversityPalmerston NorthNew Zealand

Isabel LiSchool of Applied Sciences, Faculty of Health and Environmental SciencesAuckland University of TechnologyAucklandNew Zealand

Kieran MellowInstitute of Fundamental SciencesMassey UniversityPalmerston NorthNew Zealand

Jon PalmerInstitute of Food, Nutrition and Human HealthMassey UniversityPalmerston NorthNew Zealand

Shanthi ParkarPlant & Food ResearchPalmerston NorthNew Zealand

Brent SealeSchool of Applied Sciences, Faculty of Health and Environmental SciencesAuckland University of TechnologyAucklandNew Zealand

Ben SomertonInstitute of Food, Nutrition and Human HealthMassey UniversityPalmerston NorthNew Zealand

Xuemei TangInstitute of Food, Nutrition and Human HealthMassey UniversityPalmerston NorthNew Zealand

Siti Norbaizura Md ZainInstitute of Food, Nutrition and Human HealthMassey UniversityPalmerston NorthNew Zealand

Foreword

Microbial biofilms have held a fascination for me since my first introduction to them as an undergraduate student. Since those early days, many scientific publications on this topic have shown the width and breadth of their complexity. Despite these extensive studies, so much still remains to be discovered. This is particularly true from a practical and manufacturing perspective. Publications in the scientific literature are one thing, but practical experiences in the real manufacturing environment often differ substantially from the sterile laboratory setting. Professor Steve Flint has had a long and successful career in both academia and in the dairy manufacturing industry, and has the ability to successfully translate academic biofilm studies and observations into practical applications for the industry. Professor John Brooks is one of New Zealand’s most respected food safety experts, and he and academics including Professors Phil Bremer, Brent Seale, Jon Palmer and a group of collaborative experts have meshed their experiences and expertise to create a comprehensive book on dairy biofilms. Thus, the theme of this book can best be described as an amalgamation of the available fundamental and theoretical science on bacterial biofilms and the practical experiences from a food manufacturing environment, specifically focusing on dairy production. Overviews on the roles that the microbial surface, the attachment surface and the composition of the growth medium (i.e. the dairy product) play in bacterial surface attachment and biofilm formation are presented. These concepts are interwoven with general theories on how bacterial biofilms form, and how control is maintained, especially for foodborne pathogens. Some practical examples of microorganisms in real dairy manufacture, e.g. Streptococcus and thermophiles, and in selected processes, e.g. ultrafiltration and dairy wastewater treatment, are discussed in detail. As a result, both academic and nonacademic audiences can learn greatly from these chapters.

Dr Denise LindsaySenior Research Scientist, Fonterra, New Zealand

Preface to the Technical Series

For more than 60 years, the Society of Dairy Technology (SDT) has sought to provide education and training in the dairy field, disseminating knowledge and fostering personal development through symposia, conferences, residential courses, publications and its journal, the International Journal of Dairy Technology (previously published as the Journal of the Society of Dairy Technology).

In recent years, there have been significant advances in our understanding of milk systems, probably the most complex natural food available to man. At the same time, improvements in process technology have been accompanied by massive changes in the scale of many milk processing operations, and the manufacture of a wide range of dairy and related products.

The Society has embarked on a project with Wiley-Blackwell to produce a Technical Series of dairy-related books to provide an invaluable source of information for practising dairy scientists and technologists, covering the range from small enterprises to modern large-scale operation. This thirteenth volume in the series, on Biofilms, provides a timely and comprehensive review of a natural threat to the integrity of manufacturing processes as well as the quality and shelf life of dairy products. These problems are not limited to dairy operations but are also found in other food manufacturing operations and much of the principles covered in the chapters can be applied elsewhere. Biofilms can also be used beneficially, for instance in the bioremediation of effluent streams.

Andrew WilbeyChairman of the Publications Committee, SDT

Preface

The dairy industry has grown in size, sophistication and quality to satisfy an international demand for food and food ingredients. The major risk to product quality and economic manufacture is microbial contamination, predominantly due to the release of microorganisms and their metabolites from biofilms forming on the surfaces of equipment used in the handling of milk and the manufacture of milk products. The ultimate origin of the microorganisms is the raw milk, but the conditions through the manufacturing process provide specific niches ideal for the propagation of biofilms. The composition of these biofilms varies according to the conditions at any particular point in the manufacturing process. Microbial groups from psychrotrophs to thermophilic spore-forming bacteria form biofilms at specific zones in the manufacturing process. In some situations, the conditions are so selective that only a single species is detected. In other areas, interactions between species that can enhance biofilm development, spore production and the production of metabolites such as enzymes occur, all representing a threat to product quality.

Our understanding of the factors involved in the development of biofilms in the dairy industry has focused on the processes leading to microbial attachment in a dairy environment, conditions supporting biofilm growth and potential damage to product quality, the release of microorganisms from biofilm communities and the effect of cleaning systems on controlling biofilms. This has led to engineering solutions to limit the amount of surface area available for biofilm growth, replicating key pieces of equipment (e.g. evaporators) to enable frequent cleaning without stopping manufacture, improved cleaning systems and changes in plant operation – especially temperature – to limit biofilm growth and prevent activities such as spore production.

This book represents the result of 15 years of research into dairy biofilms involving researchers across several universities and research organisations. The content covers methods used in the detection and analysis of the microflora comprising dairy biofilms, information on the environments within the dairy industry that support biofilm development and a critical analysis of control methods used for biofilm control. Dairy industry managers, researchers and students will find this book useful in providing a fundamental understanding of problems relating to biofilms in the dairy industry and in offering some solutions and suggestions for improvement in managing a dairy manufacturing plant.

Dr Koon Hoong TehProf. Steve FlintProf. John BrooksDr Geoff Knight

Acknowledgements

The editors and authors of this book wish to acknowledge Jessica Childs for her contribution in preparing graphics for several figures used in this book. Jessica was able to take our concepts and mould them into images that have added a unique aspect to this publication.

We also thank Matt Levin for setting up a virtual meeting room that enabled us to bring all the authors together for video conferencing during the preparation of this publication.

John Brooks and Geoff Knight deserve special mention for their proofreading of all of the chapters, which has provided some consistency and polish.

Owen McCarthy assisted in the final proofreading of Chapter 11.

This book was a true team effort from all concerned and could not have been achieved without the passion and dedication of everyone involved.

1Introduction to Biofilms: Definition and Basic Concepts

Phil Bremer1, Steve Flint2, John Brooks3 and Jon Palmer2

1Department of Food Science, University of Otago, Dunedin, New Zealand

2Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand

3School of Applied Sciences, Faculty of Health and Environmental Sciences, Auckland University of Technology, Auckland, New Zealand

1.1 Definition of biofilms

In 2012, the term ‘biofilm’ was defined by the International Union of Pure and Applied Chemistry (IUPAC), Polymer Division as an ‘Aggregate of micro-organisms in which cells that are frequently embedded within a self-produced matrix of extracellular polymeric substances (EPS) adhere to each other and/or to a surface’. IUPAC included the following notes after the definition:

Note 1:

A biofilm is a fixed system that can be adapted internally to environmental conditions by its inhabitants.

Note 2:

The self-produced matrix of EPS, which is also referred to as slime, is a polymeric conglomeration generally composed of extracellular biopolymers

in various structural forms.

The idea behind the development of this definition was to provide a terminology usable, without any confusion, in the various domains dealing with biorelated polymers, namely, medicine, surgery, pharmacology, agriculture, packaging, biotechnology and polymer waste management (Vert et al., 2012).

Bearing this definition in mind, in this book we use the term ‘biofilm’ to refer to ‘microorganisms attached to and growing, or capable of growing, on a surface’. This definition is broader than the IUPAC definition, as it includes cells or spores that are attached to a surface but have yet to produce a biofilm matrix. We have included attached cells not within a matrix in order to acknowledge that in many instances the act of attaching induces phenotypic changes to a cell. We have included the phrase ‘growing or capable of growing’ to reinforce the point that many of the unique features associated with biofilms arise as a result of the growth and replication of microorganisms on a surface, such as the production of EPS and the development of a complex three-dimensional structure.

In this chapter, we briefly discuss the importance of biofilms to the dairy industry, before introducing their general features, including their development, composition and structure, the advantages they confer to microorganisms living in them and how they may be controlled. This chapter serves as an introduction to the other chapters in the book, and includes cross-references to more detailed information on dairy-specific features in other chapters.

1.2 Importance of biofilms in the dairy industry

On a global basis, the dairy industry produces a wide range of perishable (milk and cream) and semiperishable foods (cheese, butter and yoghurt) and food ingredients (milk powders, whey protein concentrates and caseinates). Microbial contamination of dairy products is of great concern to the dairy industry. Strict adherence to microbiological guidelines is essential to maintain product quality, functionality and safety (see Chapter 4) and to allow companies to remain competitive in the international market.

Those microorganisms associated with bovine raw milk and dairy manufacturing plants that are of particular interest to the dairy industry can be divided into three major categories, namely, spoilage, pathogenic and beneficial microorganisms. Spoilage microorganisms can have an impact on the quality and sensory properties of milk and other dairy products, through the production of metabolic byproducts and/or extracellular enzymes. Pathogenic microorganisms (see Chapter 9) have the potential to cause human illness and to have significant economic repercussions. Beneficial microorganisms generally belong to a diverse group loosely termed ‘lactic acid-producing bacteria’ (LAB) and are used as starter cultures for the manufacture of cheese, yoghurt and other fermented dairy products. A subgroup of LAB that is becoming more commonly used in fermented dairy products, such as yoghurt, is the probiotic bacteria, which include strains of Lactobacillus and Bifidobacterium (Jamaly et al., 2011; Quigley et al., 2013).

Biofilms have become a major issue within the dairy industry and are now recognised as sources, or potential sources, of contamination by spoilage or pathogenic microorganisms, which can decrease product safety, stability, quality and value. Many manufacturing processes provide unique niches, within processing equipment, where bacteria are able to grow and survive. Examples are thermoresistant streptococci in pasteurisation equipment (see Chapter 6) and thermophilic spore-forming bacteria in milk powder production equipment (see Chapter 7). Within the last 2–3 decades the importance of biofilms in the processing environment has also been recognised, particularly around drains and other locations that are difficult to reach and where cleaning and sanitation applications may be inadequate to eliminate bacteria present within biofilms.

In dairy manufacturing plants, biofilms can be divided into two categories: process biofilms, which are unique to processing plants and form on surfaces in direct contact with flowing product; and environmental biofilms, which form in the processing environment, such as in niches where cleaning and sanitation is poor and around drains. Process biofilms differ from environmental biofilms in two key ways. First, in a process biofilm, one or a few species may dominate, as the unit operation employed (e.g. pasteurisation equipment) may select for particular groups of bacteria (e.g. thermoduric). Second, process biofilms are frequently characterised by rapid growth rates. An example of this is the increase in numbers from ‘not detectable’ to 106 bacteria per cm2 within 12 hours of operation that occurs in the regeneration section of a pasteurisation plant (Bouman et al., 1892). In contrast, environmental biofilms can take several days or weeks to develop (Zottola & Sasahara, 1994).

1.3 Biofilm formation

The development of a biofilm on a surface follows a logical series of steps, in which the first step is the initial contact of the free-living microorganism with the surface. The initial interaction of cells with a surface is influenced by a wide range of chemical, physical and biological cues, as outlined in detail in Chapter 2. In general, the initial interactions are influenced by: (i) the surface topography, chemistry (functional groups, surface charge, presence of antibacterial compounds) and free energy (hydrophobicity); (ii) environmental conditions, including temperature, pH, nutrients and the presence of other microorganisms, which can either inhibit or enhance contact; (iii) processing factors such as fluid velocity and shear force; and (iv) the various mechanisms employed by the cell (quorum sensing, nutrient sensing, production of EPS) and the cell surface structures (such as pili, flagella, fimbriae, adhesins) to interact with the surface (Figure 1.1).

Figure 1.1 Steps involved in biofilm formation over time (arrow) in a dairy processing plant under conditions of flow. (1) Cells and/or spores come into contact with a surface that may be fouled with protein, fat and salts. (2) Cells and spores attach to the fouled surface. (3) Spores germinate and cells grow, beginning to produce EPS. (4) Cells replicate, forming microcolonies enclosed in EPS. (5) Microcolonies increase in size and coalesce, forming complex three-dimensional aggregates of cells and EPS that may contain a variety of niches. (6) Dispersal of cells and spores from the biofilm occurs.

Once on or near a surface, a bacterium has to commit to adopting either an attached or a planktonic lifestyle based on a series of signals or cues it receives (Karatan & Watnick, 2009). An obvious cue for settlement is nutrient concentration, with high or low concentrations of nutrients promoting biofilm formation for different bacterial species. Bacteria, such as Salmonella spp., are more likely to join a multilayer biofilm in response to nutrient limitation (Gerstel & Romling, 2001), while for Vibrio cholera, the presence of glucose and other sugars induces production of a biofilm matrix and multilayer biofilm formation (Kierek & Watnick, 2003).

The second step in biofilm formation requires the cell to form at least a semipermanent association with the surface. This step is frequently referred to as the ‘attachment phase’. Many authors have broken this down into a reversible and an irreversible phase, but with increasing knowledge on cell dispersal, the term ‘irreversible attachment’ is proving to be overstated. In dairy processing plants, there is a wide range of different materials to which bacteria can attach, including 304 and 316 stainless steel, plastic, elastomer (rubber) materials, polyester/polyurethane (conveyor belt materials), epoxy surface coatings and tiles. Bacteria will attach at different rates and strengths to these materials. The ability of bacteria to attach to a surface and the rate at which they attach will, however, change as material (proteins, carbohydrates) from the processing environment comes into contact with the surface and modifies its characteristics. Such so-called ‘conditioning films’ (see Chapter 3) occur almost as soon as a clean surface comes into contact with a liquid. In addition, the rate of attachment and the ease with which bacteria can be removed from the surface will change as the surface material ages, becomes damaged through mechanical operation or is exposed to cleaning agents and sanitisers.

The effect of surface roughness on the propensity of cells to attach is unclear. Some research reports greater cell attachment on surfaces with high surface roughness, while other research reports that there is no correlation between surface roughness and cell attachment to inert surfaces (Vanhaecke et al., 1990; Flint et al., 2000; Mitik-Dineva et al., 2008, 2009; Truong et al., 2010). While there may be some debate about the influence of surface roughness on attachment, there appears to be general agreement about the importance of using surfaces with minimal cracks and crevices in order to reduce bacterial adherence and biofilm growth and to enhance cleaning effectiveness.

In the next step of biofilm formation, the cells on the surface begin to replicate and produce EPS, which can include polysaccharides, proteins, eDNA and lipids. The production of EPS and the incorporation of extraneous material from the environment, such as food residues (soil) and other microorganisms, into the biofilm, results in an increase in the biofilm’s bulk and complexity.

In the final stages of biofilm development, the growth and replication of the primary colonisers (the first cells to attach to the surface) lead to the formation of microcolonies on the surface. These microcolonies independently increase in size over time until they form a series of macrocolonies, which can eventually coalesce to varying degrees, forming complex three-dimensional aggregates of cells and EPS on the surface, variously described as being ‘mushroom’- or ‘pillar’-like. As the biofilm develops, the presence and metabolic activity of the bacteria within it, coupled with the production of EPS and its associated impact on the diffusion of compounds and gases into, out of and through the biofilm, can lead to the development of a wide variety of microenvironments or niches within the biofilm.

The ultimate structure of the biofilm is dependent on the bacterial species involved in its creation and the chemical and physical characteristics of its environment. Individual macrocolonies may merge together or may remain separated by narrow channels, through which nutrients and other molecules can readily diffuse. The developed biofilm is in a state of flux, where cells within it react to changes in the physical (flow rate, shear) and chemical (nutrient gradients, oxygen concentration) nature of the environment. The variety of conditions occurring within a biofilm can result in the development of phenotypically or genotypically distinct cell populations within it and can ultimately lead to the dispersion or release of cells from the biofilm.

Dispersal from biofilms may be either initiated by the bacteria themselves or mediated by external forces such as fluid shear, abrasion and cleaning. At least three distinct modes of biofilm dispersal have been identified: erosion, sloughing and seeding. Erosion is the continuous release of single cells or small clusters of cells from a biofilm at low levels, owing to either cell replication or an external disturbance to the biofilm. Sloughing is the sudden detachment of large portions of the biofilm, usually during the later stages of its growth, perhaps as conditions with it change or it becomes unstable due to its size. Seeding dispersal is the rapid release of a large number of single cells or small clusters of cells and is always initiated by the bacteria (Kaplan, 2010).

In the 1980s and 90s, interest in biofilms rapidly increased and there were many reports of biofilm formation and development following the generalised steps just described, leading to the proposal of a developmental model of microbial biofilms (O’Toole et al., 2000). This model received wide interest, but, 10 years after it was first proposed, Monds and O’Toole (2009) published a paper expressing concern that evidence in its support had not been forthcoming and that it should not be considered as dogma.

It is known that many, if not all, bacteria are capable of forming or at least living within a biofilm and that living within a biofilm is frequently their normal mode of existence in natural environments (Costerton et al., 1995; Stoodley et al., 2002). As living within a biofilm requires extensive changes in both cell form and function, this strategy entails a significant commitment (Monds & O’Toole, 2009). Once a cell is committed to a biofilm, the spatial stratification within the biofilm can drive an additional physiological differentiation of the population. However, rather than being seen as an indication of the presence of specialised developmental stages, this is increasingly being considered as simply a reflection of the microorganism’s response to the development of niches or a microenvironment within the biofilm. In short, it is the ability of bacteria to sense and to respond to their localised environment by regulating gene expression that leads to the development of a sustainable and complex biofilm, rather than an overarching bacterial community-focused goal.

1.4 Biofilm structure

While the structure of a biofilm is ultimately dependent on the species growing within it and the specific physical and chemical conditions in the environment surrounding it, a mature biofilm generally comprises clusters or layers of cells, which form a structure that can vary in thickness from a few micrometres to several millimetres. The cells are surrounded by EPS, which can contain up to 97% water (Zhang et al., 1998). In general, the bacterial cells within a biofilm make up only about 15–20% of its volume, with the remainder being taken up by EPS.

Based on modelling studies, classical porous biofilms containing channels and voids between the mushroom-like outgrowths are predicted to occur under a substrate-transport-limited regime, while compact and dense biofilms are predicted in systems limited by biofilm growth rate and not by the substrate transfer rate. Surface complexity measures, such as roughness and fractal dimension, will increase with increasing transport limitations, while compactness will decrease as the biofilm changes from being dense to being highly porous and open (Picioreanu et al., 1998).

Physical conditions, such as temperature, impact on the species composition (see Chapter 4) and growth rate of bacteria within a biofilm, while in pipelines, fluid flow dynamics can influence biofilm structure. Biofilms grown under laminar flow are reported to be patchy and to consist of aggregates of cells (mushrooms) separated by interstitial voids. Biofilms grown under turbulent flow may also be patchy but are characterised by the occurrence of chains of cells (streamers) that run from the biofilm surface into the bulk fluid phase (Stoodley et al., 1998a). The biofilm as a whole, and the streamers in particular, exhibits viscoelastic properties, which means that it elongates and deforms as flow velocity increases and retracts as velocity decreases (Stoodley et al., 1998b). Recently, it has been shown that the flow of liquid through porous materials, such as industrial filters, can stimulate the formation of streamers, which, over time, can bridge the spaces between surfaces and cause rapid clogging (Drescher et al., 2013).

For many years, it has been known that some bacterial species, growing either as free living cells or within a biofilm, produce or release diffusible signal molecules that increase in concentration as a function of cell numbers. In a process termed ‘quorum sensing’, bacteria communicate with each other via these signal molecules or autoinducers to regulate their gene expression in response to population density (Miller & Bassler, 2001). The role of quorum sensing in biofilm formation was first reported for biofilms of Pseudomonas aeruginosa growing in a flow-through reactor, where it was found that the quorum sensing signal molecule 3OC12− homoserine lactone (C12) was required for normal biofilm differentiation (Davies et al., 1998). The role of quorum sensing molecules in biofilm formation and differentiation has subsequently received considerable interest. While quorum sensing may not be significant in the structural development of all biofilms, there is evidence that for some species it can be important in events such as the attachment of bacteria to a surface, structural development and maturation and even the control of events leading to the dispersion or release of cells (Davies et al., 1998; Boles & Horswill, 2008; Periasamy et al., 2012; Lv et al., 2014).

1.5 Composition of the EPS

As previously discussed, as cells attach, replicate and grow on a surface they produce EPS. EPS is recognised as playing an important role in the formation and function of biofilms of many species in many different environments. In addition, EPS, which is usually the major component of biofilm matrix, can act as an impermeable or at least semipermeable barrier, limiting the penetration of compounds into and out of the biofilm, and thereby facilitating the establishment of ecological niches within the biofilm and protecting the cells against the actions of antimicrobial compounds.

The composition and structure of components within EPS is varied and complex, being dependent on the bacterial species involved and the environment (Sutherland, 2001; Flemming & Wingender, 2010). EPS compounds that originate from microorganisms include polysaccharides, proteins, lipids and extracellular DNA (eDNA) (Flemming & Wingender, 2010). Polysaccharides have been identified as one of the major components of EPS. However, in many cases, the biochemical properties and functions of polysaccharides remain elusive, due to their complex structures, unique monomer linkages and the fact that their composition and concentration can change over time. Most of the polysaccharides that have been described are long linear or branched molecules, with molecular masses of 0.5–5.0 × 105 Daltons, and they may be homo- or heteropolysaccharides and either polyanionic (e.g. polysaccharides, such as aliginate or xanthan) or polycatonic compounds (Flemming & Wingender, 2010).

The biofilm matrix can also contain a considerable number of proteins. A wide range of enzymes has been detected within biofilms. Many of these are reported to have bipolymer degrading ability, enabling them to break down complex compounds, such as polysaccharides, proteins, nucleic acids, cellulose and lipids, into nutrients that are more readily available to bacteria. Biopolymer degrading enzymes also play a role in the dispersal of cells from the biofilm. Nonenzymatic proteins in the EPS or biofilm matrix are often involved in the formation and stabilisation of the EPS matrix and are often therefore termed ‘structural proteins’. These include the cell surface-associated and extracellular carbohydrate-binding proteins, known as lectins, which form links between the bacterial surface and the EPS (Flemming & Wingender, 2010).

In addition to the obvious role of transferring genetic material between bacteria, via conjugation and DNA transformation, eDNA also appears to play a structural role in maintaining biofilms. The expression of conjugative pili has been shown to stimulate biofilm formation and can stabilise and influence the biofilm structure by forming connections between cells (Ghigo, 2001). The presence of eDNA has been shown to stabilise the young biofilms (Whitchurch et al., 2002). eDNA also has antimicrobial activity and causes cells to lyse by chelating cations that stabilise lipopolysaccharides in the outer membranes of bacterial cells (Flemming & Wingender, 2010).

Lipids, lipopolysaccharides and surfactants can also be found to varying degrees within some EPS, where they are believed to play a role in the initial attachment of the cell to the surface, the development of the biofilm structure and the dispersal of cells from the biofilm (Flemming & Wingender, 2010).

1.6 Composition of the biofilm population

Most biofilms found in nature comprise a range of bacterial species. However, in specialised niches within processing plants, especially in those areas subjected to extremes of temperature, or where the product has been treated to inactivate most microorganisms, it is possible for biofilms dominated by one or a few species to develop. An example of this is in the production of milk powder, where it is possible to find biofilms developing within the evaporators that are dominated by one or two species of thermophilic spore-forming bacteria (Burgess et al., 2010, 2013).

In general, biofilms are very heterogeneous environments characterised by a large degree of chemical, physical and biotic diversity. Variation in diffusion rates into and out of biofilms, as well as in the rates at which compounds are produced or metabolised, can lead to the development of concentration gradients for nutrients, oxygen, ions and signalling molecules. This can result in the creation of microenvironments and biotic diversity, even in monospecies biofilms, as cells adapt to changes in their local environment.

Like any other ecological niche, conditions within biofilms select for cells that are best suited to survive. This means that the resulting population is a reflection of the cells that come into contact with the niche, their ability to grow within the niche and the impact that cell growth and metabolism have on the niche. Based on the diversity of the planktonic population and the selective pressure at the surface and within the developing biofilm, biofilms can comprise one or a small number of species. In most instances, however, it is expected that a biofilm will contain a number of microbial species, with interactions occurring between them. In some cases, such interactions can facilitate the growth and survival of species that may be less suited to survival in a monospecies biofilm under the same environmental conditions (Bremer et al., 2001).

Biotic diversity therefore occurs through a number of mechanisms. In the simplest instance, phenotypic changes take place due to variations in the cell’s physiological status, dictated by nutrient or oxygen gradients (Stewart & Franklin, 2008). For example, cells located in the outermost layers of a biofilm that have a ready supply of nutrients and oxygen available can easily grow aerobically. The facultatively anaerobic cells in underlying layers may be oxygen-deprived and so will need to shift to an anaerobic metabolism in order to grow. This can encourage the growth of obligate anaerobic microflora. Cells at deeper layers within the biofilm may be nutrient-limited and have limited growth rates or be metabolically inactive. The response of individual bacterial cells to the local conditions drives phenotypic heterogeneity.

Phenotypic diversity may also arise due to variations in gene expression resulting from differences in transcription initiation or mRNA degradation. So-called ‘stochastic gene expression’ has been hypothesised to be a cell population’s insurance against potential dramatic changes in environmental conditions (Veening et al., 2008).

A third source of phenotypic heterogeneity is genetic mutations. Genetic variation occurring through point mutation, insertion or deletion can potentially increase the phenotypic variability within the biofilm. If such spontaneous mutants confer a significant selective advantage, especially in the presence of a stressor, they will confer a fitness advantage to the mutated cell and its offshoots and promote the survival of the cell population (Plakunov et al., 2010).

Gene transfer within biofilms is enhanced by the close proximity of cells and the ability of the biofilm matrix to trap gene products within the biofilm. Gene transfer occurs within biofilms by two main mechanisms: plasmid conjugation and DNA transformation. In conjugation, direct cell-to-cell contact is required for plasmid transfer. Therefore, while DNA transfer can occur at high rates within a biofilm (Hausner & Wuertz, 1999), the structure of the biofilm and the degree to which cells can move within the biofilm to establish direct contacts will ultimately limit the extent to which conjugation occurs (Molin & Tolker-Nielsen, 2003). DNA transformation occurs when DNA (chromosomal or plasmid) released by one cell is picked up by another. It has been reported that most, if not all, bacteria have the ability to release DNA (Lorenz & Wackernagel, 1994). Cells that have the ability to efficiently take up macromolecular DNA are defined as having developed natural competence. Transformation rates for Streptococcus mutans growing within a biofilm have been reported to be 10–600-fold higher compared to the rate in planktonic cultures (Li et al., 2001). Given that the presence of conjugative pili and eDNA, as discussed above, can stabilise biofilms (Whitchurch et al., 2002), it appears that efficient gene transfer is both a consequence of and a contributor to biofilm development (Molin & Tolker-Nielsen, 2003).

1.7 Enhanced resistance of cells within biofilms

A large number of authors have compared the resistance of bacteria within biofilms to their free-living counterparts and declared that the former are far more resistant to a wide range of stressors, including antibiotics, ultraviolet (UV) damage and sanitisers (Costerton et al., 1995; Elasri & Miller, 1999; Langsrud et al., 2003; Bridier et al., 2011). This protection has been postulated to result from a number of factors associated with living within a biofilm, including the binding of EPS to antimicrobial compounds, physical inhibition of the diffusion of antimicrobial compounds by the EPS or chemical reaction of antimicrobial compounds with components of the EPS matrix, all of which decrease the concentration of antimicrobial compounds reaching microorganisms within the biofilm (Thurnheer et al., 2003). For example, chlorine (in a 25 ppm solution), which chemically reacts with organic material, has been shown to only be able to penetrate to a depth of 100 µm into a complex 150–200 µm-thick dairy biofilm (Jang et al., 2006). In addition, chlorine concentrations within a mixed Pseudomonas aeruginosa and Klebseilla pneumoniae