Material-Microbes Interactions: Environmental Biotechnological Perspective

Material-Microbes Interactions: Environmental Biotechnological Perspective brings great insights into microbes-material interactions, biofilm formation and emerging bioprocesses within the field of applied biotechnology. The book systematically summarizes the fundamental principles, the state-of-the-art in microbes-material interaction, and its application in bioprocess and environmental technology development. Understanding the fundamental processes of biofilm formation, the role of material to exchange the energy with microbes, biofilm matrix, and optimization of the biofilm formation process is useful to everyone involved with bioprocess development. This book will be of significant interest to environmental technology developers, researchers, university professors, policymakers, graduate and postgraduate students and other stakeholders.

Interestingly, academic institutions, wastewater treatment plants and research centers have upscaled biofilm-based environmental technologies, such as moving bed bioreactors, microalgae, tricking bed reactors, biofilters, and bioelectrochemical process as promising environmental technologies.

• Illustrates growing interest in biofilm-based technology development, either wastewater treatment using carrier materials or valorizing waste material into resources using biofilm-based bioprocess
• Focuses explicitly on the microbes-material interactions in various biotechnologies
• Covers a broad range of biofilm-based bioprocesses, including new and state-of-the-art options and trends within the field
• Includes photo-sets on biofilm development and bioreactor systems

• Language: English
• Publisher: Academic Press
• Release date: Jun 20, 2023
• ISBN: 9780323951258

Book preview

Preface

There is a significant shortfall between the amount of clean water, energy, and food available today and the amounts needed to sustain the world's population in 2050. Humanity is also facing multiple environmental challenges owing to the continued use of available resources and unsustainable manufacturing practices. Microbes, the first living creatures on earth, have the ability to bring new and better solutions to tackle these challenges. A better understanding of interactions between microbes and their surroundings is important to exploring their full potential for different applications. Among others, the interaction of microbes with compatible biotic or abiotic surfaces leads to the development of a matrix of cells living in close association and complex cooperation with each other, known as a biofilm. Biofilms growing on different material surfaces offer several benefits in industrial and environmental biotechnological processes. However, under certain conditions, they also have deleterious effects, leading to biocorrosion and biofouling, and may also cause antibiotic resistance. Therefore, knowledge of microbe–material interactions is essential not only in bioprocess development but also in corrosion control.

Environmental technologies have applied biofilm processes for wastewater or gas treatment and bioremediation. Biofilm-containing carrier material-based wastewater treatment is a commercially applied biotechnology that depends on microbe–material interactions. For instance, the capacity to control microbial communities in moving bed carrier materials has led to enhanced degradation of organics in wastewater treatment plants. Biogas produced from anaerobic digestion often contain H2S and CO2. Therefore, upgrading is necessary to enrich the methane content of the biogas, which can be done in trickling bed and biofilter reactor units. Advances in biofilm formation by electroactive microorganisms have been extensively investigated to develop microbial fuel cells and allied technologies in which different types of electrode materials significantly affect biofilm formation, which determines the performance of the technology. Furthermore, microbial electrosynthesis from CO2 is another emerging technology based on electrode–microbe interaction. Thus, understanding the fundamental processes of biofilm formation, the role of materials in exchanging energy with microbes, the biofilm matrix, and optimization of biofilm formation is highly desirable. Academic institutions, industries, and research centers have scaled up biofilm-based technologies, such as moving bed bioreactors, microalgae, trickling bed reactors, biofilters, and bioelectrochemical processes for wastewater treatment and other applications. Many private and public organizations are also engaged in biofilm-based technology development and implementation. Nevertheless, access to specific and comprehensive information on microbe–material interactions and biofilm-based processes in environmental biotechnology is limited. This book offers detailed insights into microbe–material interactions in terms of fundamental principles, state-of-the-art development, specific applications, and the sustainability aspects of biofilm-based environmental bioprocesses.

The book's contents are presented through three broad categories: fundamental and general aspects, specific applications, and scaled-up and sustainability aspects of biotechnologies based on microbe–material interactions. The first part provides basic information on biofilm formation and development, electron transfer processes between microbes and electrodes, biocompatible materials, and general applications. The second part comprehensively discusses various aspects associated with biofilm-based applications. These include wastewater treatment and energy recovery with anaerobic digestion, microbial fuel/electrolysis cells and constructed wetlands technologies, CO2 use with microbial electrosynthesis technology, biogas upgradation with different processes, and contaminated air cleanup with biofilters, bioscrubbers, and biotrickling filters. It is important to evaluate the environmental and social aspects besides the technoeconomic feasibility of developing sustainable biofilm-based processes. The sustainability assessment requires synergy among multidisciplinary subjects in which engineers cooperate with environmental scientists, public health experts, economics, and social scientists. Thus, the last part of this book covers the sustainability of a few technologies evaluated based on technoeconomic implications and life cycle assessment.

We believe this book provides comprehensive knowledge in the microbe–material interactions area by covering a broad range of biofilm-based bioprocesses, state-of-the-art technologies, and current trends. The book will be of significant interest to environmental technology developers, researchers, university professors, policy-makers, graduate and postgraduate students, and other stakeholders exploring microbe–material interactions for different applications. Although we have tried to be objective in our choice of topics discussed in this book, some themes not included here may become important in the future. We will try to cover them in the second edition.

Although the authors, editors, and many expert reviewers tried their best to check the contents extensively, mistakes might have crept in inadvertently. We would appreciate it if the readers would highlight these and make comments or suggestions to improve and update the book’s contents for future editions.

Nabin Aryal

Department of Process, Energy, and Environmental Technology, University of South-Eastern Norway (USN), Porsgrunn Norway

Yifeng Zhang

Department of Environmental & Resource Engineering, Technical University of Denmark, Lyngby, Denmark

Sunil A. Patil

Department of Earth and Environmental Sciences, IISER Mohali, Sahibzada Ajit Singh Nagar, Punjab, India

Deepak Pant

Separation and Conversion Technology, Flemish Institute for Technological Research (VITO), Mol, Belgium

Part A

Fundamentals and advances

Outline

Chapter 1. Environmental microbial biofilms: formation, characteristics, and biotechnological applications

Chapter 2. Microbe–material interactions for direct interspecies electron transfer in anaerobic digestion

Chapter 3. Electron transfer processes between microbes and electrodes in bioelectrochemical reactors

Chapter 4. Wastewater granules: material–microbe formation and arrangement lead to blurry metabolic boundaries and greenhouse gas emissions

Chapter 5. Recent progress in antibacterial membranes for water treatment

Chapter 1: Environmental microbial biofilms

formation, characteristics, and biotechnological applications

Sivakumar Krishnan ¹ , Sunil A. Patil ² , and Y.V. Nancharaiah ¹ , ³       ¹ Biofouling and Biofilm Processes Section, WSCD, Chemistry Group, Bhabha Atomic Research Centre, Kalpakkam, Kanchipuram, Tamil Nadu, India      ² Department of Earth and Environmental Sciences, Indian Institute of Science Education and Research Mohali (IISER Mohali), Sahibzada Ajit Singh Nagar, Punjab, India      ³ Homi Bhabha National Institute, Mumbai, Maharashtra, India

Abstract

In natural settings, microbes live mainly in interactive communities known as biofilms and aggregates. These microbial communities are associated with a copious amount of extracellular polymeric substances matrix and behave differently from their planktonic counterparts. Biofilms are a serious concern in the medical field because they tend to grow on tissues and implants and exhibit higher tolerance to antimicrobials. In industrial settings, biofilms profoundly affect material deterioration, heat transfer efficiency, maritime activities, aquaculture equipment, and sensors. Microbial biofilms also have beneficial roles in the environment in many ways, including primary productivity in aquatic environments, biogeochemical cycling of elements, natural attenuation of pollutants, water treatment, bioremediation, and wastewater treatment. Microbes inhabiting biofilms or aggregates/granules confer higher tolerance to harsh environmental conditions and toxic pollutants and thus are suitable for effective water and wastewater treatment. Microbial biofilms or aggregates have been successfully employed in engineered settings, including in the activated sludge process and in upflow anaerobic sludge blanket reactors, moving bed biofilm reactors, aerobic granular sludge reactors, and bioelectrochemical systems for wastewater treatment and resource recovery. In this chapter, several aspects of environmental biofilms, such as their formation, life cycle, and related attributes, their distribution in different settings and their impacts and their biotechnological applications for sustainable development are covered in detail.

Keywords

Aerobic granular sludge; Aquatic environments; Bioelectrochemical systems; Bioremediation; Marine biofilms; Microbial aggregates; Resource recovery; Wastewater treatment

1. Introduction

Microbial biofilms are microbial assemblages with a copious amount of extracellular polymeric substances (EPS) matrix and are ubiquitous in various environmental settings. It is important to study environmental microbial biofilms owing to their vital role in organic matter decomposition, the transport of energy and nutrients to higher trophic levels, the biogeochemical cycling of elements, and biotechnological applications. Biofilms exist in both natural and engineered environments. Typically, they are conceived as complex microbial colonies attached to abiotic or biotic surfaces with an embedded EPS matrix. Studies on biofilms can be traced back to 300 years ago to seminal microscopic observations on the plaque scraping of teeth by Antonie van Leeuwenhoek. Afterwards, notable observations made by Heukelenkian and Heller (1940) and Zobell (1943) highlighted the importance of biofilm growth. These include the bottle effect of marine microorganisms, which refers to enhancing bacterial growth and activity by incorporating a surface in the water bottle to which the microbes can attach and grow. Zobell found a remarkably higher number of bacteria on immersed surfaces than in the surrounding seawater, which led to the development of biofilm biology in subsequent decades (Zobell and Allen, 1935; Zobell, 1943).

The term biofilm was first coined by Bill Costerton in 1978. It refers to a heterogeneously structured bacterial community enclosed in a self-produced EPS matrix on inert (i.e., rocks, glass, plastic) and organic (i.e., skin, cuticle, mucosa) surfaces (Costerton et al., 1978). Microbial biofilms have been detected in the soil, on rocks, and in sediments of freshwater, marine, and terrestrial ecosystems, hydrothermal environments including hot springs, acidic pools, deep-sea vents, and in plants, animals, and humans (Rasmussen, 2000; Westall et al., 2001; Taylor et al., 1999; Reysenbach and Cady, 2001; Koerdt et al., 2010; Rinaudi and Giordano, 2010). The formation of microbial biofilms is a multistep process that starts with the attachment of microorganisms to the surface with the subsequent growth of colonies and the production of EPS composed of proteins, polysaccharides, extracellular DNA (eDNA), humic substances, and other signaling molecules (Peterson et al., 2013; Das et al., 2013).

Microbial biofilms were initially known for their adverse impact, and researchers mainly focused on developing suitable control measures for their prevention in industrial and medical settings. It is now well-known that biofilms are vital to maintaining healthy aquatic ecosystems and environmental well-being. The roles of biofilms in natural aquatic ecosystems are immense in supporting productivity, the decomposition of natural organic matter, energy and material transfer across trophic systems, and the natural attenuation of pollutants and biogeochemical cycling of elements. As a natural extension of these beneficial aspects, microbial biofilms are engineered to develop biotechnologies to achieve bioremediation of contaminated matrices and wastewater treatment. This chapter focuses on various aspects of environmental biofilms, including detrimental and beneficial aspects. Biofilms in different fields, including medicine, terrestrial, aquatic, and marine ecosystems, are described. Their beneficial applications in bioremediation, wastewater treatment, and agriculture are discussed in the context of sustainable development.

2. Brief history of biofilms

In 1684, Leeuwenhoek made the first observations on colonies of bacterial cells on hard surfaces (dental plaque). However, these did not receive much attention until research by Paul Zobell on marine bacteria. Zobell made a seminal observation that bacteria exist at a remarkably higher number on immersed surfaces than in the surrounding seawater (Zobell and Allen, 1933, 1935; Zobell and Anderson, 1936; Zobell, 1943). Subsequently, Characklis (1973) reported the occurrence of microbial slime in industrial water systems and their resistance to disinfectants such as chlorine. In 1978, Costerton et al. reported that microorganisms adhere to both living and nonliving surfaces. The term biofilm was coined by Bill Costerton's group to describe bacterial growth on surfaces. Costerton et al. (1985, 1987, 1995) also reported that the extracellular polysaccharide substances or the glycocalyx secreted by microbial cells have a major role in secondary colonization. The glycocalyx produced by microorganisms for building biofilms can act as an ion exchange matrix and trap several metal ions (Ferris et al., 1987a,b; Geesey and Jang, 1989). Some early studies reported that the physical, electrochemical, and relative hydrophobic characteristics of surfaces are important in bacterial adhesion (Fletcher and Loeb, 1979; Dahlbäck et al., 1981). For instance, rougher surfaces are more favorable for bacterial colonization, providing suitable niches against shear stress, turbulent flow, and biocidal activity (Lytle et al., 1989; Konhauser et al., 1994).

Boyle et al. (1991) stated that the mechanisms involved in bacterial adhesion mainly depend on the physiologic status of the microorganisms and the nature of the substratum. Jones et al. (2012) used scanning and transmission electron microscopy to examine biofilms grown on trickling filters in a wastewater treatment plant (WWTP). Over several decades, significant progress has been made in biofilm research through the application of various experimental systems and tools. Standard microbiological culture techniques, scanning electron microscopy, confocal laser scanning microscopy, genomics, and proteomics have advanced the fundamental and applied aspects of biofilms, including their microstructure, cell–cell interactions, chemical signaling, the chemistry of the biofilm matrix, and the physiology and identification of proteins and genes involved in cell attachment and biofilm formation (Relucenti et al., 2021). Mass spectrometry techniques have expedited knowledge about the metabolic profiling of biofilms. Omics tools, including RNA sequencing, microarrays, and reverse transcription quantitative polymerase chain reaction have improved the understanding of metabolic traits and gene expression in microbes residing in the biofilms. Attached growth systems such as integrated fixed film systems (IFAS), moving bed bioreactors (MBBRs), and membrane biofilm reactors have been developed using biofilms in water and wastewater treatment (Nancharaiah et al., 2019 ). Aerobic granular sludge (AGS), a distinct form of biofilm, is a breakthrough biotechnology developed for sustainable wastewater treatment (Nancharaiah and Sarvajith, 2019). Moreover, the capabilities of some microbes to form biofilms at the electrode surfaces in bioelectrochemical systems have been exploited to develop various microbial electrochemical technologies for wastewater treatment, resource recovery, bioremediation, biosensing, and bioproduction applications (Kiran and Patil, 2019).

3. Biofilm life cycle and attributes

3.1. Biofilm life cycle

Biofilm formation is a developmental process involving distinct stages dependent on microbial cells, surface characteristics, and environmental factors. Successful biofilm development involves the stages of reversible attachment, irreversible attachment, proliferation, maturation, and dispersal (Fig. 1.1). Primarily, surfaces immersed in aqueous media are conditioned by different inorganic and organic molecules present in the surrounding water medium. The attachment of microbial cells to the surface is aided by various interactions, including higher polar or hydrophobic interactions and less polar van der Waals forces (Busscher and Weerkamp, 1987). Initial attachment of microbial cells can be temporary, and attached cells detach from the surfaces owing to higher fluid shear forces (Allison and Gilbert, 1992; Characklis and Marshall, 1990). Then, microbial cells firmly attach to the surface in a reversible phase followed by irreversible attachment aided by cell surface adhesins and EPS. In this phase, attachment between microorganisms and the surface is weak, and microbes can easily be removed by mild treatment with cleansers and sanitizers. Hence, different short-range interactions such as dipole–dipole, ion–dipole, and hydrophobic interactions are involved in the attachment of microbes to surfaces.

Figure 1.1  Biofilm life cycle. Distinct stages of biofilm life cycle such as attachment, microcolony formation, development of three-dimensional structure, and dispersal.

Bacterial attachment to materials or surfaces involves the expression of certain genes, which influences cell morphology, motility, and EPS production. Reversible attachment is a rapid process and mediated by hydrodynamic and electrostatic interactions. Irreversible attachment of microbes is driven by van der Waals interactions between the hydrophobic region of the outer cell wall and the substratum (Tuson and Weibel, 2013). Bacterial cell envelope components such as proteins, lipids and exopolysaccharides, fibrils, and nonfimbrial structures are involved in bacterial attachment to surfaces (Berne et al., 2018) (Fig. 1.2). The pH and ionic strength of the surrounding medium influence the charge on the bacterial cell envelope, and thus attachment. Extracellular appendages have a vital role in the direct or indirect adhesion of bacteria to surfaces. In addition to flagella, filamentous appendages, including fimbriae, curli, fibrils, and pili, regulate bacterial attachment to surfaces.

In addition, attached cells start secreting EPS, which further aid in the development of stable microcolonies. The EPS matrix is composed of polysaccharides, proteins, lipids, and nucleic acids. All of these macromolecules determine the structure and mechanical stability of biofilms (Flemming and Wingender, 2010). Thus, the developed biofilms are rich in nutrients and act as a support system for the growth of microbes residing within them. Water channels or diffusion gradients of mature biofilms facilitate the transport of oxygen, nutrients, and other components for microbial growth, and aid in the release of waste products.

Under nutrition-deprived conditions and during shearing of biofilm aggregates in the environment, biofilms start shedding actively growing cells, which return to the planktonic growth mode and recolonize in the new niches to propagate and establish biofilms at other locations. The process of recolonization is species-specific. For example, Pseudomonas fluorescence recolonizes the surface after about 5 h. Recolonization of new surfaces by Vibrio harveyi and Vibrio parahaemolyticus occurs after 2 and 4 h, respectively (Donlan, 2002). In the case of fungi, mycelium undergoes differentiation and produces spores that spread into new locations.

Figure 1.2  Bacterial cell surface depicting cell surface appendages including fibrils, pili, curli fibers, flagella, and nonfimbrial adhesins that aid in attachment of bacterial cells on surfaces.

Figure 1.3  Three types of multispecies biofilm composed of distinct colonies (aggregates), coaggregates, and layered structures.

The established single species or multispecies microbial biofilm provides a basis for the colonization of higher organisms, leading to macrofouling (Flemming, 2002). In environmental habitats, microbes prefer to live in communities. Interactions among microorganisms and with the external environment regulate the formation, structure, and function of biofilms. Microbial diversity and hydrodynamics prevailing in the surrounding environment may affect the composition and structure of multispecies biofilms. Apart from cell–substratum interactions, cell–cell interactions among diverse group of microorganisms have a critical role in shaping complex biofilms (Fig. 1.3). Multispecies biofilms are common in natural environments and are composed of archaea, bacteria, fungi, and even microalgae, depending on environmental conditions (Donlan, 2002; Hall-Stoodley et al., 2004; Flemming et al., 2016; Flemming and Wuertz, 2019). These complex microbial communities are more resilient to phages, antibiotics, and toxins than the single-species biofilms (Wang et al., 2002; Molin and Tolker-Nielsen, 2003; Reisner et al., 2006). Microbes in a multispecies biofilm interact through different mechanisms, including autoaggregation, coaggregation, cross-species protection, cometabolism, quorum sensing (QS), and genetic exchange. Thus, synergistic and antagonistic interactions can influence and shape biofilms and their functionality and tolerance to stressful environmental factors. Synergistic interactions also aid in horizontal gene transfer (HGT) among different species via plasmid conjugation or DNA transformation, which is an important adaptation mechanism for microorganisms of multispecies biofilms (Zupančič et al., 2018; Joshi et al., 2021).

3.2. Environmental microbial biofilms: an important intrinsic factor of ecosystems

Biofilms are one of the most widely distributed and successful modes of life on the earth. They drive biogeochemical cycling of key elements in diverse environmental settings and are important components of food chains in all kinds of ecosystems. Environmental factors determine the attachment and formation of biofilm and their dispersal. The development of bacterial biofilms is a dynamic and complex process regulated by several factors including the incubation period, nutrient levels, temperature, pH fluctuations, nutrient status, ionic strength, salinity, and predation. The attachment of bacteria to surfaces in any environment is considered to give a selective advantage in terms of nutrition and protection from prevailing environmental conditions. Apart from others, bacterial cell surface appendages (i.e., pili, other proteins) and the surface chemistry of the substratum have important roles in biofilm formation and maintenance in environmental settings. Cell–substratum interactions and attachment are governed by electrostatic, van der Waals, hydrophobic and contact interactions (Huang and Gregory, 2011; Kurincic et al., 2016). Microbial biofilms are formed at interfaces as floating mats (e.g., pellicles) at the liquid–air interface, under submerged environments (solid–liquid interface), and on the surface of living and nonliving objects (Martí et al., 2011).

Modern industrialization has put pressure on ecosystems by continuously releasing toxic pollutants into the environment. These pollutants enter aquatic habitats through the discharge of domestic wastewater, industrial effluents, and surface runoff from urban and agricultural areas. Apart from the effect of polluting chemicals, such contaminated environmental niches may act as a reservoir for several pathogens and increase the risk for spreading infectious diseases in humans.

3.3. Extracellular polymeric substances (EPS) matrix

The EPS matrix is an important component of biofilms. It confers protection against harsh environmental conditions such as acid stress, shear stress, antimicrobial agents, UV radiation, desiccation, biocides, solvents, high concentrations of toxic chemicals and pollutants, and predatory protozoa (Flemming and Wingender, 2010; More et al., 2014; Davey and O'Toole, 2000; Mah and O'Toole, 2001). Important components of the EPS matrix include polysaccharides, proteins, eDNA, lipids, and other molecules (Fig. 1.4). Exopolysaccharides are major components of the biofilm matrix that constitute about 50%–90% of its total organic matter (Vu et al., 2009). The charges of these polysaccharides may be neutral and polyanionic in Gram-negative bacteria and cationic in Gram-positive bacteria. The anionic property is confirmed by the presence of uronic acids, including D-glucuronic, D-galacturonic, and D-mannuronic acid or ketal-linked pyruvate. Exopolysaccharides are long linear or branched molecules with molecular weights of 500–2000 kDa. For instance, Pseudomonas aeruginosa produces a minimum of three kinds of exopolysaccharides (alginate, Pel, and Psl) in biofilms. Among these, alginate is an unbranched heteropolymer with high molecular mass, which has an important role in establishing microcolonies and is responsible for mechanical stability in mature biofilms.

Surface proteins are found in significant amounts in the biofilm matrix and have a critical role in biofilm architecture and stability (Cucarella et al., 2001). Some of these proteins aid in biofilm formation and can be divided into enzymes and structural proteins. Many extracellular enzymes participate in degrading EPS, and the substrates of these enzymes are biopolymers such as polysaccharides, cellulose, proteins, and nucleic acids. EPS also act as a nutrient store. They are digested by extracellular enzymes during starvation. In addition, these enzymes are useful in the dispersal of established biofilms and the recolonization of new surfaces. Biofilm-associated proteins (baps) and homologous bap-like proteins are structural proteins that promote biofilm formation (Latasa et al., 2006). Amyloid is another protein mostly found in the biofilm matrix. It is reported in different environmental biofilms, including freshwater lakes, brackish water, drinking water reservoirs, and WWTPs (Otzen and Nielsen, 2008; Rajitha et al., 2020 , 2021). On the other hand, alginate-like exopolysaccharide is a dominant structural polymer in mixed microbial aggregates (Nancharaiah et al., 2019).

Another component of EPS is eDNA. The presence of eDNA was initially observed in the biofilms of P. aeruginosa, Streptococcus intermedius, Enterococcus faecalis, and Staphylococcus species. eDNA with high molecular weights (up to 30 Kb) can play an important role in the formation and structural stability of biofilms, gene transfer mechanisms and facilitates the self-organization of cells inside biofilms (Gloag et al., 2013; Tetz et al., 2009).

Figure 1.4  Different components of extracellular matrix of microbial biofilms. eDNA, extracellular DNA; QS, quorum sensing.

Lipids and alkyl group-linked polysaccharides such as methyl and acetyl groups contribute to the hydrophobic property of EPS recovered from biofilms (Neu et al., 1992; Busalmen et al., 2002). Microbes produce hydrophobic molecules that help in initial cell attachment and biofilm formation. For instance, Thiobacillus ferrooxidans produces lipopolysaccharides (Sand and Gehrke, 2006) and Serratia marcescens produces surface-active extracellular lipids (Matsuyama and Nakagawa, 1996), which are hydrophobic and aid in attachment and biofilm development. These molecules have surfactant-like properties and have a vital role in altering the surface tension between air and water (Leck and Bigg, 2005).

3.4. Physiologic characteristics of biofilms

Microcolonies are the basic structural and functional units that exhibit biofilm attributes such as water channels, different redox microenvironments, nutrient gradients, cell–cell signaling or QS, and HGT. Microcolonies of environmental biofilms are composed of multiple genera or species and microenvironments suitable for the biochemical cycling of carbon, nitrogen, sulfur, and other redox-active elements.

3.4.1. Quorum sensing (QS)

QS is a cell-to-cell communication that moderates cell density in biofilms before reaching an unsustainable level (Nadell et al., 2008). It depends on chemical signaling molecules known as autoinducers, which trigger several physiologic processes (Fig. 1.5). Acyl-homoserine lactone is a QS molecule produced by a diverse group of bacteria. Changes in the QS mechanism by the enzymatic degradation of signaling molecules may prevent the formation of stable biofilms or destabilize the three-dimensional structure of biofilms. At appropriate cell densities, signaling molecules reach a concentration required to activate certain genes involved in biofilm differentiation. Davies et al. (1998) stated that at least two different cell–cell signaling systems, such as lasR-lasI and rhlR-rhlI, operate in the biofilm of P. aeruginosa. Yung-Hua et al. (2001) demonstrated that the QS mechanism coordinates genetic transformation in Streptococcus mutans. They also reported that genetic transformation frequencies are 10–600 times higher in biofilms than among planktonic cultures. Conjugative gene transfer of plasmids occurs at up to 1000 times higher frequencies in bacterial biofilms (Hausner and Wuertz, 1999; Nancharaiah et al., 2003). This implies that biofilms are hot spots for HGT owing to the close proximity of cells as well as different physiologic conditions compared with those in planktonic cultures. The process of QS is not only responsible for controlling population density; it also spreads beneficial mutations and provides enhanced access to nutrients, virulence, antibiotic resistance development, and antibiotic tolerance to microorganisms (Hannan et al., 2010).

Figure 1.5  Overview of quorum sensing mechanism in bacteria and biofilm formation.

3.4.2. Horizontal gene transfer (HGT)

Biofilms are an ideal niche for exchanging extrachromosomal DNA (plasmids). The rate of conjugation is high between cells in biofilms compared to planktonic cells. Ghigo (2001) stated that medically relevant strains of bacteria with conjugative plasmids could readily form biofilms. He reported that the F conjugative pilus has a vital role in cell-to-cell adhesion interactions and the development of three-dimensional structures in biofilms of Escherichia coli. In contrast, E. coli strains lacking plasmid produced only microcolonies but not biofilms with a three-dimensional structure. Thus, the transfer of plasmid from donor strains to recipient strains in microcolonies enhances biofilm formation. Biofilm environments with minimal shear and closer cell-to-cell contacts facilitate enhanced gene transfer events through conjugation. Hausner and Wuertz (1999) demonstrated 1000-fold higher conjugative gene transfer frequencies in defined bacterial biofilms using in situ gene transfer quantification based on tagging plasmid with a green fluorescent protein gene. Subsequently, dual labeling of Pseudomonas putida with green and red fluorescent protein genes was used for cultivation-independent quantification of conjugal transfer of TOL plasmid in biofilm reactors (Nancharaiah et al., 2003; Venkata Mohan et al., 2009). Thus, tagging of plasmid and chromosomes of donor cells enabled HGT to be monitored and conjugation frequencies to be quantified by monitoring fluorescence in transconjugants without requiring their cultivation on agar plates. In natural microbial communities, HGT is an important process for evolution and genetic diversity, especially for adapting bacteria to a new environment and changing environmental conditions (Kokare et al., 2009). Mobile genetic elements, including conjugative plasmids, transposons, and bacteriophages are responsible for coordinating HGT among bacteria in biofilms (Koonin, 2001).

3.4.3. Predation and competition

In a particular environment, competition occurs among species of the same genus or different genera for substratum and nutrients. In addition, the presence of predators maintains the population of prey organisms in an environmental niche. For bacterial biofilms, free-living protozoa, Bdellovibrio spp., and bacteriophages act as predominant predator organisms. For instance, Hartmannella vermiformis, a free-living protozoan, colonizes and subsequently acts as a predator of heterotrophic biofilms (Donlan, 2002; Murga et al., 2001). Acanthamoeba spp. has been observed to be a predator of biofilms formed on contact lenses (Mc Laughlin et al., 1998). James et al. (1995) reported that competition between biofilms of P. putida and Hyphomicrobium sp. in P. putida dominated biofilms during the invasion of Hyphomicrobium sp. biofilms. In some cases, two bacteria can coexist in a particular environment. Stewart et al. (1997) demonstrated that Klebsiella pneumoniae and P. aeruginosa can coexist in the same biofilm community. Dominance of a particular microorganism in a multispecies biofilms is governed by several factors including cell interactions, the biofilm formation potential, and its effects on neighboring microbes.

3.4.4. Antibiotic resistance

The complex architecture of biofilms is suitable for availing the microenvironment with limited oxygen and nutrients for cells and permits the development of persister cells (Brown et al., 1988). Microenvironments in biofilms favor slow bacterial growth and the development of antimicrobial resistance or tolerance (Brown et al., 1988; Field et al., 2005). Decreased rates of respiration and metabolic activities have been observed in microbes residing in biofilms. In addition, a dormant or persister state in bacteria is a mechanism by which a certain fraction of cells exhibits tolerance and is protected from antimicrobial agents. The antimicrobial resistance of biofilms is accomplished by the diffusive barrier properties of the EPS matrix, the slow growth rate of microbes, inactivation of antimicrobials, and the formation of spores. Bacteria in the form of biofilms are more tolerant to antibiotics than their planktonic forms (Stewart and William Costerton, 2001; Luppens et al., 2002; Davies, 2003). For instance, antibiotic-susceptible bacterial strains often become resistant to antibiotics when they are in the biofilm mode, and antibiotic susceptibility is quickly restored to these cells when they enter the planktonic mode (Anderl et al., 2000). Bacterial antibiotic resistance can be divided into innate resistance owing to growth in a biofilm and induced resistance acquired as a result of antimicrobial treatment (Costerton et al., 1999; Lewis, 2001; Donlan and Costerton, 2002; Dunne, 2002; Stewart, 2002; Hoiby et al., 2010). Novel antibiofilm compounds including ionic liquids and antimicrobial peptides are investigated for effectively controlling microbial biofilms in medical and engineered settings (Reddy and Nancharaiah, 2020a,b, 2021; Nancharaiah et al.,