Biofilm-mediated bioremediation of pollutants from the environment for sustainable development

Chapter 14

Biofilm-mediated bioremediation of pollutants from the environment for sustainable development Sangeeta Yadav and Ram Chandra Department of Environmental Microbiology, School for Environmental Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow, India

14.1

Introduction

A single species, or more than two species, of microorganisms may be involved in biofilm formation, which is a complicated process (Davey and O’Toole, 2000). During the development of a biofilm, microorganisms grow on a surface and secrete extracellular polymeric substances (EPSs). As a consequence, the phenotypes of microorganisms are modified in growth rate and gene transcription. In a biofilm, groups of microbial cells are very strongly (irreversibly) attached to surface. They are present in moist conditions where enough nutrients are available and surface attachment can be accomplished. Biofilm formation is initiated when bacteria that are present in a free-floating condition attach to a suitable living or nonliving surface. The use of EPSs as the adhesive substance plays a very important role during first step of attachment of cells on a surface. The EPS is composed of sugars, proteins, functional groups that participate in the diffusion process, and extracellular DNA (nucleic acid). The EPSs assist the microorganisms to adhere to each other in a biofilm, which leads to the creation of a bulbous and complex 3D structure known as a biofilm. The extent to which a biofilm grows (thin layers or thick layers of cells) is determined by multiple environmental conditions. Microorganisms that have capacity to produce large amount of EPSs can form thick layers of biofilms even if nutrients are present in smaller amounts. In addition, the biofilm formation in aerobic microorganisms (depend on oxygen) is dependent upon how much microorganisms can grow. Other environmental factors such as shear stress also affect biofilm formation. The biofilm is usually fairly thin if the flow of water is high. Biofilm becomes very thick when water flow is slow. The last phase of biofilm growth cycle is seeding dispersal. In the seeding dispersal process, the cells of the biofilm can leave the surface as a clump of cells breaks or individual cells that burst from the mature biofilm. These cells then adhere to a new surface to create a new biofilm. A biofilm can be formed by a single bacterial species or it can also formed by various bacterial species, such as fungi, algae, and protozoa. A biofilm is made up by microbial cells, water, and the biofilm matrix. A biofilm matrix contains the absorbed nutrients, cell lysis products, particulate material, secreted polymers and metabolites, and other unknown factors of the surrounding environment. The major component of the biofilm matrix is water (approximately 97%). The physical and chemical properties of matrix (viscosity) are governed by the solutes dissolved in it. Biofilm formation is governed by a process known as quorum sensing (QS). QS is a type of communication between the bacterial cells at a threshold density, with the help of specific chemicals known as autoinducers, to initiate specific gene expression. Quorum quenching (QQ) is the process in which cells secrete chemicals, lead to the inhibition of the QS system and blocking gene expression for bacterial activities. In cells, QS activates the processes such as biofilm formation, competence, sporulation, and antibiotic production during plant-microbe interactions. N-Acyl homoserine lactones (AHLs) are an autoinducer in many Gram-negative pathogenic bacteria (such as Rhizobium radiobacter, Erwinia carotovora, and Pseudomonas aeruginosa) and plant growth promoting rhizospheric bacteria (PGPR) (such as Gluconacetobacter diazotrophicus and Burkholderia graminis), which can control a broad range of bacterial characteristics such as antibiotic production, biofilm formation, competence, conjugation, motility, pathogenicity, sporulation, symbiosis, and virulence. LuxI/ R QS systems are present in the majority of the Gram-negative bacteria for AHL production and control the expression of specific characteristics. Bacterial AHLs are also perceived by plants to activate the gene expression to maintain homeostasis and defense mechanisms.

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Specific gene expression by plants due to biofilm-forming bacteria performs a very significant role in the bioremediation of organic and inorganic waste. Different types of AHLs are secreted by different species of microorganisms. Interestingly, furanones secreted by some bacteria and some higher plants (rice, clover, and soy bean) can mimic the AHL molecule and put off the QS-regulated behaviors of the bacterial population. Furanones and other AHL mimic compounds can bind with AHL receptors (LuxR) due to their homology to AHLs, leading to incorrect AHL signaling. Plants can also protect themselves from pathogens by the secretion of AHL mimic compounds. Moreover, QS systems in Gram-positive bacteria are different from the Gram-negative QS system which is AHL-mediated. In Gram-positive bacteria, an autoinducing peptide (AIP) is used as autoinducer signal in place of the AHL signal used by Gram-negative bacteria. However, a common QS system mediated by autoinducer-2 (AI-2, i.e., furanone) is also present in most of the bacteria such as Gramnegative and Gram-positive bacteria. This type of QS is known as a hybrid QS. Biofilm formation bacteria have tremendous properties to adapt against various environmental stresses. A comparison of the performance of free-living planktonic bacteria and their biofilm counterparts for the remediation of pollutants or toxins showed different results, with biofilms showed more remediation. However, the decreased survival of free-living planktonic bacteria in stress conditions might be due to unavailability of the pollutants, decreased protection, and low metabolic activity. Sessile microorganisms of a biofilm provide structure and protection due to their complex polymeric matrix (Vu et al., 2009). In addition, genetic diversification, the presence of aerobic and anaerobic organisms, and different metabolization processes are survival strategies of cells in a biofilm. This diversity and metabolic range makes biofilm-forming bacteria hold more potential in the bioremediation of pollutants. Indigenous biofilm-forming microorganisms in the soils perform bioremediation, which is part of the nutrient cycle and global self-purification system. Interestingly, QS in bacterial populations can regulate antibiotic production, bioluminescence, biofilm formation, plasmid transfer, symbiosis, and motility and virulence factors. These processes are executed only if the bacterial cell density is present at a threshold limit. The QS system is common in soil bacteria that create associations with plants and are helpful during phytoremediation of different pollutants, mostly for metals, EPSs production, bacterial survival, nitrogen fixation, motility, diversity maintenance and nodulation. Various type of signaling molecules are produced by most of the rhizobium bacteria. The biofilm-forming bacteria and fungi have been implicated in a variety of health conditions. Approximately 80% of microbial infections in the body is due to biofilm-forming bacteria. Biofilms can also grow on medical devices such as catheters, joint prosthetics, pacemakers, and prosthetic heart valves, leading to infections. This phenomenon was first on intravenous catheters and pacemakers in the 1980s. Moreover, wastewater treatment also involves nitrification by autotrophic bacteria using biofilms. Furthermore, biofilm-forming bacteria are also involved in fuel generation by converting organic waste into electricity. Hence, a bacterial biofilm provides a low-cost source of power and clean sustainable energy. Similarly, biofilms are uses in treating wastewater, heavy metals, persistent organic pollutants (POPs), hydrocarbons and explosives (TNT), and radioactive substances (uranium) into simple and less harmful compounds. Microbes can either degrade the pollutants or change their mobility or toxicity and therefore make them less harmful to humans and the environment. In the past few decades, rapid industrialization has been a major source of environmental contamination. This created waste all around the world, resulting in contaminated soil, water, and air. The POPs and successive environmental problems lead to the development of environmental disasters. Therefore, various processes and technologies are being developed for environmental sustainability. Bioremediation is an environmental-friendly process for the detoxification of harmful pollutants from soil, water, and air using microorganisms. In bioremediation, the damaging effect of complex and hazardous chemicals on the environment is reduced. Bioremediation offers several advantages over other methods, including no or minimum disruption of land or wildlife surrounding the treated area, a reduction of noise and dust during treatment, as well as avoidance of harsh chemicals. Bioremediation is found more economical for degradation and detoxification of complex pollutants at large scale as compared to conventional treatment methods of pollutants. Bioremediation was first discovered by the Romans (around 600 BC) to clean wastewater. Bioremediation was officially invented by George Robinson in the 1960s. Bioremediation through biofilm-forming microorganisms is an efficient and safer alternative process than the use of planktonic microorganisms. This might be due the cells in a biofilm have better chance of survival and adaptation against antibiotics and abiotic stresses, due to the protection of the polysaccharide matrix. The utilization of xenobiotic is accelerated due to mutual physical and physiological interactions between cells in biofilms, hence biofilms are used by industries to degrade and immobilize complex persistent pollutants. Two types, in situ and ex situ, depending on the location of the pollutant treatment, are reported. In situ bioremediation refers to onsite treatment, whereas in ex situ remediation, the contaminated samples are treated offsite. In situ bioremediation is an attractive treatment technique because it minimizes transportation costs and site disruption. In situ remediation can be enhanced by the optimization of physical and chemical conditions (such as pH, aeration, and moisture) for bacterial growth along with supplemented nutrients.

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Recently, bioremediation of industrial waste with biofilms in reactors has evolved as a core area of interest for researchers. The bioremediation process includes various microbes for degradation or detoxification of xenobiotic compounds, polyaromatic hydrocarbons (PAHs), POPs, pesticides, herbicides, volatile organic compounds, heavy metals, crude oil, and petroleum products. Persistent complex pollutants in the environment are pentachlorophenols (PCPs), PAH, polychlorinated biphenyls (PCBs), 2-benzene, toluene, ethylbenzene, dichlorodiphenyltrichloroethane (DDT), xylene (BTEX), 2-methyl-4-keto-2-trimethylsiloxypentane, 4-ethyl-2-methoxyphenol, ethyl-2-octynoate, 2-methyl-4keto-2-pentan-2-ol, 3,7-dioxa-2,8-disilanonane-2,2,8,8-tetramethyl5[(trimethylsilyl)oxy], hexadecanoic acid, cis-9hexadecenoic acid, octadecenoic acid, and trinitrotoluene (TNT). The pollutants are nominated by the US EPA as a priority for removal as they may cause carcinogenic and/or mutagenic impacts. Microbes, especially bacteria, can be easily grown and change the texture and nature of the complex pollutants which makes them suitable for bioremediation. Besides bacteria, fungi are also used for removal of various pollutants. Heterogeneity and social interactions in biofilms are very helpful for degradation of complex pollutants due to diverse catabolic pathways that exist in diverse environments with microbes. Diverse catabolic pathways for degradation of complex pollutants are dependent upon enzymatic activities of microbes for the transformation and degradation of environmental pollutants into less toxic or harmless components such as CO2 or H2O. The transfer of electrons from electron donors to electron acceptors is essential for any metabolic pathways. The electron donors act as food for the microbes. Microorganisms can degrade pollutants in the presence and absence of oxygen as aerobic and anaerobic degradation, respectively. In aerobic degradation, microbes use O2 as the final electron acceptor to convert organic and inorganic pollutant into less toxic products, often CO2 and H2O. Conversely, to break down organic compounds into carbon dioxide and methane in anaerobic degradation, electron acceptors are manganese, nitrate, iron, and sulfate (other than O2). However, sometimes some microbes degrade contaminants through the process of fermentation. Generally, aerobic microbes degrade pollutants faster than anaerobic ones. Pollutants that have tendency to donate electrons may be degraded in presence of oxygen by aerobic microbes, whereas those contaminants that are poor electron donors may degrade under anaerobic conditions. Many redox reactions are also involved in the immobilization of trace elements found in the contaminated sites. A change in the oxidation potential of chemicals such as metals will change the solubility and toxicity. For example, sulfur-reducing bacteria removes sulfate from wastewater by converting sulfate to sulfide, leading to a reduction in toxicity. In this process, the oxidation potential of sulfur is changed which further leads to a change in solubility and facilitates immobilization. Biosorption (microbial- or plant cell-mediated) and biotransformation (enzyme- or metabolite-mediated) are the most widely explored biological metal and organic hazardous pollutants removal strategies of biofilm-forming bacteria. An alternative strategy for efficient pollutant degradation is to develop a consortium by isolating potential microbial strains that show diversity in enzyme activity. The biological processes for treating contaminated wastewater are more prominent and superior than physical and chemical methods in terms of their proficiency and economic benefits. Recently, it has been realized that the biofilm-mediated bioremediation is another potential process. Biological treatment of industrial wastewater is the high level of cooperation performed by many bacteria through synthesizing cell signaling molecules or regulating the expression of specific genes in response to changes in the population density. In addition, biofilms are playing a very critical role in the phytodegradation of organic and inorganic pollutants. During QS and biofilm formation, root exudates play an important role because exudates can chemotactically invite rhizobia towards the roots of leguminous plants, colonizing and expressing a nodulation gene that promotes the plant’s growth and its remediation potential. Overall, metal mobilization during phytodegradation takes place through the acidification, protonation, and chelation, while immobilization takes place through precipitation, complexation, and alkalinization. However, the detailed mechanisms of plant-microbe-metal interaction remain unknown. In this chapter, we detail biofilm formation, regulation and the factors that affect biofilm formation. Strategies of biofilm adopted during bioremediation and biotransformation of complex pollutants is also discussed.

14.2

Biofilm formation by bacteria

A biofilm is a community of microorganisms in which cells are embedded in a matrix of EPS. The microorganisms adhere to each other and to any humid solid surface. Antonie van Leeuwenhoek first observed the “animalcule” (a microscopic animal) as microbial biofilms on tooth surfaces using his simple microscope. A biofilm typically comprise many species and it is a complex system that can have high cell densities (108– 1011 cells/g wet weight). Biofilms may form on various surfaces including medical devices, industrial or potable water pipes, wastewater treatment system (activated treatment system, trickling filter system, biofilter and anaerobic sludge blanket reactor), and living tissues. The water system biofilm is very complex system containing filamentous bacteria, freshwater diatoms, and noncellular materials such as minerals, clay, and silt. On the other hand, biofilms on medical devices are composed of a single, coccoid organism or blood

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FIG. 14.1 (A) Different stages of biofilm formation; (B) epifluorescence microscopy view of biofilm grown on a stainless steel surface stained with 4,6diamidino-2-phenylindole (DAPI); (C) Biofilm formation of E. coli on titanium oxide surface through SEM; (D) confocal laser scanning microscopy image of Psl in hydrated P. aeruginosa PAO1.

components. Where bacteria exist in small amounts in nature, they may survive under stressful conditions by creating a biofilms. Biofilm formation takes place in sequential steps as described in Fig. 14.1: 1. 2. 3. 4. 5.

Bacteria reversibly or irreversibly attaches to a solid support. Bacterial cells become irreversibly attached to surface and form a matrix (expansion). QS begins and cells become layered (maturation phase). 3D structured colonies are developed and reach the maximum thickness. Dispersion of planktonic bacteria from matrix (dispersion).

Biofilm-forming bacteria have motility and anchoring appendages that are helpful for movement and attachment to an appropriate solid support. Other adhesion molecules such as pili (or fimbriae) and curli are also play an important role in biofilm formation by enabling active attachment. Once bacterial cells are attached to a surface, bacteria grow for colonization through the formation of cellular aggregates known as microcolonies. Under favorable conditions, cell numbers are increase and microcolonies form 2D structures on a solid surface. These microcolonies mature into a defined architecture, i.e., a 3D structure that has developed the capacity to grow in their particular environment. Mature biofilm shapes include flat monolayers, 3D structures, mushroom-shaped, tulip-like association with low surface coverage, and all contain intervening water channels for nutrient and waste exchange. The gelatinous material produced by biofilm-forming bacteria is the EPS in which cells are embedded in a biofilm. An EPS is made up of polysaccharides, lipids, proteins, nucleic acids, dead bacterial cells, and other polymeric substances secreted by cells themselves; it is hydrated up to 85%–95%. The EPS plays an important role in the attachment of cells to biotic and abiotic surfaces, provides resistance, facilitates the uptake of nutrients from the surrounding, and prevents desiccation. The final phase of biofilm lifecycle is the dispersal of cells to the surrounding environment. These detached cells come back to their planktonic state and, when conditions are again favorable, they form a biofilm on another surface. The passive detachment of cells during the dispersal stage of biofilm can occur due to environmental changes (movement of surrounding liquid, nutrient availability, and other external forces). Detachment of cells facilitates the spreading of biofilm into the environment to carry out another cycle.

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Various structures such as fimbriae, flagella, curli (a proteinaceous, nonflagellar structure), EPS and outer membrane proteins (OMPs) play an important role in biofilm formation. The functions of these structures differ at different environmental conditions and species. Flagellar motility reduces the force that inhibits bacteria to reach the solid surface. Pili help with cell-to-cell and cell-to-surface attachment. The OMPs and curli are involved in the transfer of genetic material when bacteria reach the solid surface. Most fimbriae have high amount of hydrophobic amino acid residues. Fimbriae reduce electrostatic repulsion between the cell and surface, to assist in hydrophobicity and attachment. During biofilm formation, type I and type IV pili are needed for host-cell colonization. Biofilm-forming cells attachment was found greater on hydrophobic materials rather than hydrophilic materials. Combaret et al. (2000) isolated the OmpR gene, which is responsible for curli production and is required for biofilm formation in nonmotile strains. Biofilm is one of the most successful modes of bacterial life on Earth and plays a very important role in biogeochemical cycles in soil and water. Biofilms can have both harmful as well as beneficial effects depending on the environment and situation. Biotechnological applications on the beneficial side of biofilms, including the bioremediation of hazardous and toxic pollutants, is becoming a promising and economically very feasible technique, along with the filtration of drinking water, biocatalysis, and production of biofuels. This might be due to diversities in bacterial communities and metabolic pathways. However, biofilms are also involved in pathogenicity of humans, animals, and plants. It is also responsible for deterioration of drinking water, resistance to UV light and antibiotics, biofouling and contamination of process water, and microbial-influenced corrosion. The biofilm survives under stress and desiccation environments and can be considered a habitat and matrix generator (Fig. 14.2). A biofilm matrix contains EPS which gives it stability and architecture. In addition, the spaces between the cells of a biofilm are involved in the localization of nutrients and waste. Biofilms can trap nutrients and other molecules by adsorption/absorption of EPS molecules and using the channels of the matrix. Biofilm-forming bacterial cells have several emergent properties that are absent in free-living bacterial cells. These properties of biofilm are due to habitat diversity and localized gradients, enzyme retention that provides wide range of digestive capabilities due to various enzymes, resource capture by sorption, adsorption of metals and organic matter, social interactions and the ability to survive under stress conditions. Enzyme retention of a biofilm that provides digestive capabilities leads to the biodegradation of complex pollutants, biodeterioration, and biocorrosion (Fig. 14.2). The microcolonies of biofilm grow in volume and the bacterial cells near the surface, i.e., inside the biofilm, have difficulties in absorbing nutrients from the environment. Conversely, the cells present on the surface of a biofilm are able to continue growing. Hence, a condition is created in which bacterial populations have different metabolic activities at different levels. This leads to the development of heterogeneity in biofilms due to gradients of nutrients, pH, waste products, electron donors and acceptors, and signaling factors. In addition, elements within a biofilm are interacting. Local conditions and coordinated lifecycles in biofilm govern the differentiation of cells in biofilm and lead to heterogeneity. A spatially heterogeneous ecosystem within the biofilm can be created due to stage-specific expression of genes and proteins. The matrix also provides spatial organization in a biofilm, they provide high gradients, biodiversity, and synergistic interactions (horizontal gene transfer and cell-to-cell communication). An EPS evolves in the formation of the biofilm architecture, which produces a spatial organization where cells in the biofilm cluster into microcolonies. The biofilm communities have novel structures, activities, patterns, properties and self-organization in complex systems that lead to biogenic habitat formation. The heterogeneous physiological activities of biofilms are shown in Fig. 14.3. Heterogeneous physiological activities of biofilms are not only observed in multilayer, dense biofilms, but it also observed in thin biofilms containing fewer Sorption (resource capture; adsoption of metals and organic maters) Cooperation (synergistic micro consortia) Competition

Enzyme secretion (biodegradation, biodeterioration, biocorrosion)

FIG. 14.2 Different processes involved in a fortress biofilm.

Matrix • Architecture • Stability • Pores and channels • Fills and forms the space between the cells • Localized nutrient and waste • Protection formation

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FIG. 14.3 Heterogeneity of biofilms. (A) Organisms are stratified according to oxygen availability; (B) nutrient consumption in aerobic oligotrophic biofilms by organisms in the upper layers results in the nutrient deficiency in the lower layers lead to the adoption of slow growth states, such as dormant cells or death cells; (C) signaling molecules gradients; (D) pH gradients. AHL, acyl-homoserine lactone; QS, quorum sensing; VBNC, viable but nonculturable cells.

cells adhered to the solid surface. Heterogeneity is also observed in monospecies biofilms, which is the result of phenotypic variation due to various gene expression in individual cells over time and different gene expression in different cells. This is mostly reported in multilayered biofilms (microbial mats or flocs) (Fig. 14.3). One very important gradient is also established due to the availability of oxygen as electron acceptor. The upper layer of the biofilm of aquatic ecosystem is aerobic due to the presence of oxygen. Aerobic microorganisms of a lower layer of biofilm can utilize oxygen faster which leads to formation of anaerobic zones in lower layers of the biofilm. A small distance within the biofilm creates a gradient of oxygen. Physiological layering, i.e., stratification and diverseness that creates heterogeneity in biofilms, are due to the organization of monospecies and mixed species in a biofilm. Phototrophic microorganisms (anoxygenic phototrophic bacteria, algae, and cyanobacteria) synthesize and secrete exudates that are beneficial for other neighboring species and enhance metabolic activity. In addition, nutrients are also utilized by microorganisms of the upper layer of biofilm, lead starvation conditions in lower layers of the biofilm. These conditions build up the dormant cells, viable but nonculturable cells (VBNC), persisters, and dead cells. VBNC are the living cells that are impotent when grown on the common media on which they are normally grown, however they are different from dead cells in many ways. Dead cells are metabolically inactive and do not express genes. Moreover, VBNC are metabolically active, have respiration capacity, and continuously produce mRNA through transcription. VBNC cells have many characteristics similar to viable cells such as the composition of cell walls and plasma membrane, cellular morphology, metabolisms, virulence potential, and gene expression. The strategy of VBNC cells in a stressful environment is to minimize energy requirements by reducing cell size and increasing the area and volume ratio. Moreover, a signaling molecule gradient is also present in biofilms: the concentration of QS molecules varies as distance increases from producing cells. In addition, pH gradients are also present in biofilm due to the heterotrophic metabolism of cells. The coordinated division of labor and complex network between close-proximity cells in the biofilm matrix encourages sociomicrobiology. The important features of sociointeractions in biofilms are intercellular signaling and metabolic activity. Due to high species diversity and cell densities, the exchange of metabolites between species is easy in a biofilm. Intercellular signaling and the exchange of metabolic byproducts are not present in planktonic cells. Cooperation or competition between the cells of biofilm develop the sociointeractions in biofilms (Fig. 14.4). Chemical communication (AHL) or electrical communications (nanowires) are involved in this cooperation. Moreover, social interactions are also involved in cooperative metabolisms such as the nitrification process in which ammonia is converted to nitrite by ammoniaoxidizing bacteria in the absence of urease (Fig. 14.4). Nitrite-oxidizing bacterium Nitrospira moscoviensis can convert nitrite into ammonia by using enzyme urease. The metabolites of ammonia and nitrite-oxidizing bacteria are exchanged with each other to reduce loss and increase the effective substrate uses. Another example of cooperation in biofilms is the synergistic degradation of linuron, a toxic herbicide found in some mixed-species biofilms (Hyphomicrobium sulfonivorans, Comamonas testosteroni, and Variovorax sp. WDL1). Mixed

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Communication

Competition

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FIG. 14.4 Social interactions in biofilms involve communication, cooperation, and competition between cells.

Nitrification Nitrite oxidizers A HL

Bioremediation

NH4+

NO2– NO3–

Ammonia oxidizers

• Inhibition of QS • Antibiotics • Bacteriocins • Biosurfactants

species biofilms showed more tolerance than the single species biofilms for linuron tolerance. Moreover, synergistic interaction is also observed in many biofilm-forming consortia containing fungi and cyanobacteria on buildings and rocks. Furthermore, negative interactions, i.e., competition, are also present in biofilms (Fig. 14.4). The production of antibiotics, bacteriocins, and extracellular membrane vesicles containing enzymes that reduce the growth or kill competing organisms are mechanisms biofilm cells that create competition. However, nutrient reduction and the inhibition of autoinducers is also a type of killing strategy that is adopted by bacteria present in biofilms. The synergistic multispecies consortia is most common when metabolic substrates and metabolites have low diffusion distances to minimize loss, which reinforces the importance of high cell densities for sociointeractions in biofilm. Moreover, QS is a type of cross-talk between bacteria for sensing the environmental changes that allow bacteria to express genes and respective characteristics in a density-dependent manner. QS controls the behaviors of bacteria under stressful environments for the survival of cells by regulating an array of genes, i.e., biofilm formation, siderophore formation, sporulation, pigmentation, N2 fixation, motility, intracellular dynamics, chemotaxis, clumping, biosurfactant production, exopolysaccharide production, bioluminescence, fruiting body development, cellular evolution, secondary metabolites or antibiotic production, cell division, plasmid transfer, exoenzyme production, production of VBNC cells, and virulence. Biofilm formation can be constructive or destructive for human beings. Biofilm-forming microorganisms are used for the degradation of organic and inorganic complex pollutants at optimum nutrient, pH, temperature, and redox potential through sessile and protective environments (Singh et al., 2006). Yang et al. (2004) reported a special type of biofilm for biological wastewater treatment in sequencing batch reactors (SBRs). A vast microbial population of oxygenic and anoxygenic bacterium, including dead microbial cells, exists in aerobic granules during wastewater treatment as a result of availability of nutrient gradients and oxygen. Several studies showed AHLs as signal molecules produced by several Gram-negative bacteria, i.e., Aeromonas spp., Pseudomonas spp., and Acinetobacter spp. (Chong et al., 2012; MorganSagastume et al., 2005; Yong and Zhong, 2013a). The autoinducers AHL and AI-2 were examined in the biomass during the period of aerobic granulation in a sequence batch fermenter (Zhang et al., 2011). The process of formation and structure of granulation is initiated by a very low concentration of AHLs in a granulation system. Mature granules and their extracts propagate a high adhesion capability of bacterial growth state, participate in the initial cell attachments and biofilm proliferation on solid surface, and hence increase the aerobic granules formation during an activated sludge process (Ren et al., 2010, 2013). This is only because of the increased activity of signaling molecules AHL in the aerobic granules (Li et al., 2014a). The relationship with signaling molecules and EPS production during formation of biofilm and aerobic granules was confirmed by several researchers ( Jiang and Liu, 2012; Li et al., 2014b). AHLs also control extracellular biosurfactants and protein protection (Daniels et al., 2006; Lv et al., 2014). Biofouling is the phenomenon which is created due to biofilm formation on various solid surfaces. Biofouling reduced the functioning of bioreactors and increased the bioreactors operational charges by 60% (Drews, 2010). AHL autoinducers from C4-HSL to C14-HSL as well as AI-2 have usually been recognized during biofouling in membrane bioreactors (Kim et al., 2013). The connection between autoinducers, EPS, biofilm and transmembrane pressure showed methods for controlling biofouling by inhibiting QS systems. Wastewater treatments by various bacterial communities involve the initiation and inhibition of QS (Song et al., 2014). Chemicals that are nonhalogenated, for example 2(5H)-furanone, (5R)-3,4-dihydroxy-5-[(1S)-1,2-dihydroxyethyl]furan-2(5H)-one, kojic acid, and 5-hydroxy-3[(1R)-1-hydroxypropyl]-4-methylfuran-2 (5H)-one are QQ molecules that control biofilm formation (Dobretsov et al., 2007, 2011; Ponnusamy et al., 2010). The role of QS in the aerobic granules formation was stabilized by using QQ. A QQ activity was observed in floccular sludge

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(Tan et al., 2014). When the floccular biomass was fully converted into granular sludge, QQ activity of the community was decreased by 30%, whereas the concentration of C4–C8 AHLs increased by 10–100-fold when granule formation was initiated. However, the amount of C6-HSL and C8-HSL was decreased as granules disintegrated (Tan et al., 2014). Several researchers have reported that the phenolic aldehyde, vanillin, decreases the production of autoinducer AHL and biofilm formation by Aeromonas hydrophila (Ponnusamy et al., 2009, 2013). D-Tyrosine is another substrate which acts as QQ molecule which decreases the microbial biofilm formation on a solid surface due to the inhibition of AI-2, eDNA, EPS, and protein production (Xu and Liu, 2011). Some enzyme (acylases) other than these chemicals are also used to reduce the biofilm formation which creates biofouling on reverse osmosis membranes (Kim et al., 2012). Immobilization of enzyme acylase is used for inactivation of AHL to reduce biofilm formation and increasing the permeability of the membrane is also a way to overcome the limitations of free enzymes, i.e., low efficiency and short lifespan (Yeon et al., 2009). Lactonase from Rhodococcus sp. BH4 inhibit the biofouling due to degradation of autoinducer (Oh et al., 2013). Pseudomonas sp. 1A1 possibly producing AHL-acylase to degrade AHLs was also encapsulated in the ceramic microbial vessel, as a result there was a significant reduction in polysaccharides and proteins. Immobilization based on encapsulation of effective enzymes is also a feasible and economic method for biofouling prevention.

14.3

Extracellular polymeric substances

An EPS as polymeric substances secreted by microorganisms that are involve in the formation of microbial aggregates. All polymers outside the cell that are not part of the outer membranes are considered as EPS (Nielsen and Jahn, 1999). Composition and surface characteristics of extracellular polymeric substances (EPS) secreted by biofilm forming bacteria are shown in Fig. 14.5. An EPS can be considered as the house of biofilms because it provides shade to bacteria from environmental stresses, e.g., physical, chemical, and biological challenges. The EPS is made up by carbohydrates, proteins, lipids, humic substances, uronic acids metabolites, extracellular DNA (eDNA), and some inorganic components. The EPS is generated due to cell lysis and breakdown of macromolecules. In general, the most important role of EPS is the fundamental construction of an EPS matrix formation. Polysaccharides and lectin-like proteins of EPS create 3D networks of EPS matrix by directly forming protein-polysaccharide cross-links, polysaccharide chains, and also indirectly through multivalent cation bridges. Hence, the physicochemical properties of microbial aggregates, i.e., structure, flocculation, adsorption, settling, dewatering, and degradation, is also affected by the EPS (Sheng et al., 2010). The EPS is divided in to two categories: bound EPS (attached organic materials, capsular polymers, condensed gels and sheaths), and soluble FIG. 14.5 Composition and surface characteristics of extracellular polymeric substances (EPS) secreted by biofilm forming bacteria. LB-EPS, loosely bound EPS; TB-EPS, tightly bound EPS.

Key components: Humic substances Major components: Carbohydrate, protein, and water Minor components: Lipids, eDNA, uronic acid, some inorganic acid Composition TB-EPS Cel

Cel

LB-EPS

Cell

S-EPS Cel

Surface characteristics Hydrophilic • Charged group of EPS such as carboxyl phosphoric, sulfhydryl, phenolic, and hydroxyl group can complex with heavy metals • Apolar group (aromatic, aliphatic in protein, and hydrophobic region in carbohydrates)

Hydrophobic • Adsorption of organic pollutants

Flocculating efficiency Treatment of wastewater

Aggregation ability Treatment of wastewater

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EPS which is soluble slimes and colloids macromolecules (Sheng et al., 2010). Bound EPS are further divided into tightlyand loosely-bound EPS. Tightly-bound EPS are closely adhered to the cells’ surface that has a certain architecture, is tightly bound and stable with the cell surface. Loosely-bound EPS are the outer layers of EPS that are loosely bound to cells without a defined edge (Sheng et al., 2010). The loosely-bound EPS in microbial aggregates is always present in a smaller amount than that of the tightly-bound EPS. The contents of the EPS influence the characteristics of the microbial aggregates. Soluble EPS are cellular components of EPS that are dissolved and release into surrounding liquid. This shows that interaction between cells and soluble EPS is weak. Different types of EPS show distinct chemical properties. The content of carbohydrates and proteins in tightly-bound EPS are not dependent on carbon sources or the C:N ratio. However, carbohydrate and protein content in loosely-bound EPS are dependent on the C:N ratio. The EPS also gives energy to bacteria and provide a good source of carbon. Centrifugation is the technique by which we can separate out the bound and soluble EPS. The pellets formed after centrifugation are considered as bound EPS and polymers that remain in the supernatant after centrifugation are known as soluble EPS. An EPS is physically and chemically different from the bacterial capsule. The EPS is highly hydrated because it binds with water through hydrogen bonding; hence it prevents the cells from desiccation. However, the chemical composition of an EPS is slightly different in different species. Moreover, the amount of EPS in a biofilm varies with age and different organisms. EPS production is also affected by nutrient status. EPS production is increased when carbon sources are present in excess amounts and nitrogen, potassium, or phosphate are present in limited amounts. Slow bacterial growth can also enhance EPS production. An EPS of a biofilm providing antimicrobial resistance might be due to the direct binding of an EPS to an antibiotic instead of bacterial cells. In most of the wastewater treatment systems, microorganisms are present in the form of biofilms, granules, and sludge flocs. Humic substances are also a basic component of the EPS (approximately 20% of the total EPS) of biofilms present in biological wastewater treatment systems. The EPS contains carbohydrates, proteins nucleic acids, lipids, uronic acids, humic substances, and some inorganic components. The EPS of biofilms has many functional groups responsible for binding and adsorption of metals and organic matters. There are many polar or charged groups (carboxyl, phenolic, hydroxyl, sulfhydryl, and phosphoric groups) and apolar groups (aromatics, aliphatics, and hydrophobic) as hydrophilic areas. The formation of hydrophobic areas in EPS could be beneficial for organic pollutant adsorption. The presence of both hydrophilic and hydrophobic groups in EPS molecules shows the amphoteric nature of the EPS. Carboxyl, hydroxyl, phenolic, phosphoric, and sulfhydryl groups of EPS react with positively-charged heavy metals, leading to their remediation. The presence of functional groups in the EPS shows its binding capacity. Carbohydrates, proteins, and nucleic acids in an EPS work as negative groups and have the ability to bind with positively-charged heavy metals. Moreover, soluble EPSs have more adsorption capacity for heavy metals than bound EPS. The binding between EPS and divalent cations (Ca2+ and Mg2+) plays an important role in maintaining the biofilm structure. The adsorption of heavy metals onto activated sludge is proportional to Ca2+ and Mg2+ release into the solution, indicating the involvement of ion exchange mechanisms during the remediation of heavy metals (Yuncu et al., 2006). An EPS also showed the adsorption of phenanthrene, benzene, humic acids, and dye, which might be due to the presence of hydrophobic regions (Liu et al., 2000; Esparza-Soto and Westerhoff, 2003; Sheng et al., 2008). Moreover, the binding capacity of protein is higher than humic substances. The fraction of proteins that is higher in a soluble EPS than a bound EPS. Hence, a bound EPS has a lesser binding capacity than a soluble EPS (Pan et al., 2010). eDNA, a common component of EPS, is different from chromosomal DNA due to its primary sequence. eDNA is release in EPSs through lysis of cells in bacterial populations through QS-dependent and QS-independent mechanisms. The eDNA release in the matrix of biofilms is a QS-independent mechanism which occurs via cell lysis. However, cell lysis is a QS-dependent mechanism which concurrently generates high amounts of eDNA. QS molecules such as AHL and pseudomonas quinolone signal (PQS) regulate the production of prophage and phenazine; these are cell lysis factors that induce cell lysis and lead to eDNA release. Microorganisms survive in harsh conditions by different mechanisms such as variation of some regulators, induction of SOS repair system, and increased eDNA uptake by cells to enhance genomic adaptability of the community. However, the exact role of eDNA is not yet defined. The EPS of biofilms in remediation of pollutants were dispersed in layers and their production varied along the biofilm depth. The distribution of various EPS components was also heterogeneous. The EPS in a core part of the granule was found to be five times higher than in the shell part of an aerobic granule (Wang et al., 2005). McSwain et al. (2005) also observed higher amounts of carbohydrates in the outer layer of aerobic granular sludge, whereas most of the proteins were found in the inner layer. Chen et al. (2007) conclude that the content of the EPS and their distribution depends on the environmental conditions. They found that b-D-glucopyranose polysaccharides and protein are formed at the core, while the cells and a-D-glucopyranose polysaccharides accumulated on the outer layers in the presence of acetate. In addition, proteins formed the core and a- and b-Dglucopyranose polysaccharides were accumulated at an outer layer in phenol-fed aerobic granules. The EPS present in

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the biofilm were directly observed by using a scanning electron microscope (SEM), environmental scanning electron microscopy (ESEM), and transmission electron microscopy (TEM). The distributions of carbohydrates, proteins, and nucleic acids of EPS can also be obtained by confocal laser scanning microscopy (CLSM) after staining with various fluorescence probes.

14.4

Factors affecting biofilm formation

Biofilm formation is affected by a variety of factors such as those discussed below.

14.4.1 Effect of nutrients, pH, and temperature on biofilm formation Biofilm formation depends on the nutrient conditions that can range from high to low concentration. Biofilm formation is found to be more promising in optimum nutrient conditions, while utilization of nutrients causes dissolution of cells from surfaces in a biofilm. Bacterial biofilms obtain nutrients through different means in which: (i) biofilm forming bacteria uses waste products of secondary colonizers of biofilm; (ii) the EPS concentrates the trace organics on surfaces; (iii) there is the screening of enzymes for food supply; and (iv) pH of the media. The pH of the media can disturb various processes of microorganisms. Bacteria quickly adjust to changes in pH conditions by synthesis of different type of proteins that are related with various cellular processes. For good bacterial growth, optimum pH varies from species to species, but it is around 7 for the majority of bacteria. In addition, microbial performances are also affected by temperature. For healthy growth of bacterial populations, optimum temperature is essential, whereas a slight change in optimum temperature may decrease bacterial growth. This might be due to the decrease in rates of enzyme activities. The optimum temperature is about 40°C for maximum growth in most bacteria.

14.4.2 Velocity, turbulence, and hydrodynamics Within the boundary layer, using velocity has failed to remove biofilms. The area outside boundary layer is a high level of turbulent flow and it affects the attachment of cells on the solid surface. The velocity of water also affects the size of the boundary layer. At high velocities, the size of boundary layer decreases and cells face high turbulence, leading to a decrease in biofilm formation. Hydrodynamic conditions can also affect the EPS production, structure, mass, thickness, and metabolic activities of biofilms.

14.4.3 Bacterial cells surface topography Generally, the cells’ surface topography plays an important role during biofilm formation. During the biofilm formation, surface roughness enhances attachment of bacterial cells to surface. A rough surface is most common in biofilms because it provides more surface and less shear forces. The surface for biofilm formation may be a living or dead tissue, or any inert substance. Microorganisms attach more promptly to hydrophobic surfaces than hydrophilic surfaces.

14.4.4 Production of EPS The EPS is a complex mixture of high molecular weight compounds (Mw ¼ 10,000) secreted by microorganisms, slimy in nature, and products of cellular lysis and hydrolysis of macromolecules from the surroundings environment. Mostly polymers present in an EPS include polysaccharides, proteins, glycoproteins, lipids, phospholipids, nucleotides, and humic acids. The EPS of biofilms requires a gel-like network formation in which cells are attached together due to hydrophobic interactions and multivalent cation bonding. Moreover, the EPS is plays a very important role for cells in regards to their surface attachment, granulation, flocculation, protecting bacteria from stress conditions, and facilitating bacteria for uptake nutrient from the surrounding area for survival. An EPS adsorbs large amounts of water by hydrogen bonding, creating a hydrated condition; hence it protects bacteria from desiccation. Different amounts of EPSs are produced from different biofilms and this increases with the age of biofilms.

14.4.5 Gene regulation and QS for biofilm formation Cell-to-cell signaling through autoinducers is known as QS. QS plays an important role in biofilms formation through cell attachment and detachment from surface. A chemical released by bacterial cells work as a signal is known as autoinducer (AI) which governs the growth and developments of biofilm on different surfaces in a density-dependent manner. After

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accumulation of sufficient amount of AIs, an array of gene expressions is initiated which leads to morphological differentiation and development of other characteristics. A number of genes that are upregulated and downregulated are involved in initial attachment of cells with a surface. Approximately 22% of genes of P. aeruginosa were upregulated and 16% downregulated during biofilm formation (Steyn et al., 2001). Enzymes involved in glycolysis or fermentation, such as phosphoglycerate mutase and alcohol dehydrogenase, were upregulated during biofilms formation in Staphylococcus aureus (Becker et al., 2001). In addition, algD, algU, rpoS genes are synthesis polyphosphokinases which regulate the biofilm formation in P. aeruginosa (Prakash et al., 2003).

14.4.6 Extracellular DNA eDNA, i.e., naked DNA, is an important component of several Gram-positive, Gram-negative, single, multispecies, and archaea biofilms. eDNA is a major component of various biofilms. eDNA is a central part of bacterial EPS and its primary sequences show a similarity with chromosomal DNA. The presence of eDNA in the matrix was initially described in P. aeruginosa in 1956. An EPS plays a significant role in various phases of biofilm formation in wastewater treatment; eDNA provides strength to biofilms facing various pollutants, physical stress, and antibiotics. eDNA also supplies nutrients to cells as carbon, nitrogen, and phosphorus sources. DNAse in P. aeruginosa is responsible for biofilm dispersal, nutrient acquisition, and horizontal gene transfer (Mulcahy et al., 2010). The antimicrobial properties of biofilm might be due to presence of eDNA which destabilizes the bacterial outer membrane and chelates cations.

14.4.7 Divalent cations The presence of divalent cations such as Mg2+ and Ca2+ may be one of the factors that bacteria sense during biofilm formation. eDNA chelates divalent cations that lead to modification in properties of cell surface, giving resistance to biofilms against inorganic and organic pollutants, detergents, and antimicrobial agents. More divalent cations increase the thickness of a biofilm, and as a result, the biofilm becomes more thick and stable. Some times Ca2+ work as a cofactor for certain proteins and play a significant role in QS, virulence, biofilm formation, alginate regulation, and extracellular product formation.

14.5

Biofilm regulation in bacteria

QS is a type of cross-talk between bacteria to sense the environmental changes that allows bacteria to express a specific gene in a density-dependent manner. Environmental changes can be detected and communicated by microbes with each other and they modulate their collective behavior for biofilm formation, environmental adaptation, and in-host colonization and virulence. Autoinducers are basically intracellularly synthesized then actively or passively interchanged with the surrounding. The concentrations of signal molecules are increased with an increase in bacterial population density. When the AI concentration reaches the threshold level, the respective receptors bind with the AIs and initiate signal transduction cascades, which lead to alteration in their gene expression. Three are three major types of QS system: QS in Gram-negative bacteria, QS in Gram-positive bacteria, and nonspecies-specific hybrid QS between Gram-negative and Gram-positive bacteria as a universal language. The signaling molecules secreted by most of the Gram-negative bacteria are AHL, while another type of signaling molecules are AIPs secreted by Gram-positive bacteria, and a third type of signaling molecules is the autoinducer-2 (AI-2). QS regulates the mass transfer performance in the surrounding environment and the spatial distribution of cells.

14.5.1 QS system in Gram-negative bacteria Most of the Gram-negative bacteria have LuxI/R QS systems. This type of QS circuit is shown in Fig. 14.6A and B. Vibrio fischeri is the first Gram-negative bacteria in which this type of system has been studied. To control the production of light, there are two proteins: LuxI for the synthesis of autoinducers, and LuxR for recognition of autoinducers. LuxI synthesizes an AHL as an autoinducer that can passively diffuse out from the cell. In case of V. fischeri, the signaling molecule is N(3-oxohexanoyl)-homoserine lactone. When the cell density of V. fischeri increases, the amount of N-(3-oxohexanoyl)homoserine lactone) also increases in both sides of the cell (Fig. 14.6B). When the autoinducer concentration reaches the threshold concentration, then the autoinducers are recognized and bind the receptor protein (LuxR). This binding activates and exposes a DNA binding domain of a receptor protein. A receptor protein has two domains, one for autoinducers and the other for DNA binding. The expression of luciferase genes for light production starts when an activated LuxR protein binds to the promoter region of the luxCDABE operon. The production and nonproduction of light is balanced

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New and future developments in microbial biotechnology and bioengineering

AHL AHL

AHL receptor

(A)

c Lux Lux I C D R No expression

A

B

c Lux Lux I C D R

E

A

B

E

Light

(B)

FIG. 14.6 Bacterial QS circuits. (A) QS in Gram-negative bacteria at low cell density and acylhomoserine lactone (AHL) results in no expression; (B) high AHL concentration leads to expression of genes.

by a positive and negative feedback regulation of the luxR gene. Gene LuxI is upregulated by the activated LuxR, hence QS enhances the production of LuxI and light. However, when LuxR levels decrease, then LuxI production and light production is also decreased due to the inhibition of luciferase gene (Fig. 14.6A). Most of the Gram-negative bacteria have a similar type of regulatory circuit (LuxI/LuxR). Gram-negative bacteria such as P. aeruginosa, an opportunistic pathogen, has four well-known QS pathways: LasI/ LasR, RhlI/RhlR similar to the LuxI/LuxR circuit of V. fischeri, a quinolone based system (PQS), and a 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde (IQS) system that functions under phosphate limiting conditions (Lee et al., 2013). Gene lasI encodes an enzyme autoinducer synthase (LasI) that leads to synthesis of autoinducer HSL, whereas the lasR gene encodes for LasR protein. Response regulator LasR binds to autoinducer LasI that initiates the expression of genes. QS systems LasI/LasR and RhlI/RhlR in P. aeruginosa regulate the production of virulence factor elastase and proteases and biofilm formation. The QS in P. aeruginosa has canonical-AHL and non-AHL autoinducers. Moreover, PQS, known as 2-heptyl-3-hydroxy-4-quinolone, also works as an autoinducer. PQS is produced by a genes pqsABCDH. Quinolones show antibiotic and anticancer activities. Furthermore, several Gram-negative bacteria, such as Burkholderia cepacia (lung pathogen), Yersinia enterocolitica (enteric pathogen), and Agrobacterium tumefaciens (plant pathogen) also have a luxI/ luxR-like QS system. Although QS systems have been reported in several other Gram-negative bacteria, their functions are currently unknown. Various types of signal molecules are produced by Gram-negative bacteria that activate the respective QS circuits. The diversity in signal molecules is only due to the presence of different groups in the acyl sidechain of HSL. The AHL as autoinducer signals are composed of two parts. One part is a core HSL which is conserved in nature, while the other part is a fatty-acid-based acyl chain attached to it by amide linkage. The chemical structures of specific QS signaling molecules are shown in Fig. 14.7. Different types of occurrences of odd-numbered acyl chains are also present but these are not yet well known. Interestingly, several reports have shown the presence of odd-numbered acyl chains C7-AHLs that have been extracted from pure cultures as well as natural environments (Poonguzhali et al., 2007; Decho et al., 2009). Autoinducers AHL are synthesized by the protein LuxI autoinducer synthase or its homologs in the presence of acyl-acyl carrier proteins and S-adenosylmethionine (SAM). An amide bond is formed between SAM and the acyl groups. Subsequently, lactonization takes place during AHL production of an inducer signal for stability under biological conditions. LuxR or LuxR-like proteins are made up of two separate domains. One domain, the amino terminal, binds to the autoinducer; and the domain of carboxy terminal binds to the promoter of the target genes. Polar residues of amino terminals of a receptor protein have three tryptophan residues which are highly conserved, being in contact with the HSL moiety of the autoinducer. In the meantime, van der Waals forces and hydrophobic binding with acyl chain moieties is less conserved. Moreover, the carboxy terminal of a LuxR protein has a helix-turn-helix motif and binds to the DNA. Presence of two separate domains in Lux R are innate properties of various bacterial transcription factors. Only approximately 76% of LuxR proteins have no accompanying LuxI synthases and belong to LuxR-solo class of transcription factors. This suggests that many autoinducers are present which are not synthesized by LuxI synthases or given by other bacteria. One remarkable exception is present in Rhodopseudomonas palustris (plant-associated photosynthetic bacterium) which synthesizes p-coumaroyl-homoserine lactone as a signal molecule in which p-coumarate gives the acyl group, LuxI type enzyme, and 4-coumaroyl-homoserine lactone synthase (RpaI) (Schaefer et al., 2008). Interestingly,

Biofilm-mediated bioremediation of pollutants Chapter

A

R1

O

O

O O

N

R2

O

O

N H

H

O

O

H

14

189

3-Oxo-C12-HSL

O

N H

C4-AHL

O

Basic structure of the AHL O

O

OH

HO B

O

O

O

HO O

HO

CH3

NH

OH

O

H2N

N H

O O HN

HO

HOOC

Autoinducer-2 (AI-2)

NH

Pseudomonas quinoline system (PQS)

O

AIP-1

HO OH B O O

HO

OH

HO O HO

HO C HO

OH

OH O

HN

O

O

H N

S

O NH HN

HO

O

S

O OOHL N H O H

O

R-THMF

S-THMF-borate OH O HO

G

G

OH

O O

C T

O

O

OH

T

OH

30 20

Rutin

G

G

T

O

O 50

G A

G

T C T

CC C

C

G

OH

HO

T A

G

G

G

O

HO

C

G

OH H3C

A

40 O

OH

T

T

O

G

C

G

C

C

G

5’

3’

O

S

Br

(Z)-4-((5-(Bromomethylene)-2-oxo-2,5dihydrothiophen-3-yl)-4-oxobutanoic acid)

Homoserine lactone (HSL)

S–Me

O

OH

S

O

Cis-11-methyl-2-dodecenoic acid diffusible signaling factor (DSF)

H-Tyr-Ser-Thr-HN

O NH lle Phe Asp

O S N H

O

O

Sericea aureas peptide thiolactone

N-(3-Oxo-acyl)-homocysteine thiolactone

FIG. 14.7 Chemical structures of specific QS signaling molecules.

a novel Gram-positive microorganisms designated as MPO (GenBank: JF915892) isolated from sea water showed the production of an AHL-like protein of C3-oxo-octanoyl homoserine lactone (OOHL).

14.5.2 QS in Gram-positive bacteria The QS systems of Gram-positive bacteria differ from the Gram-negative QS system. In Gram-positive bacteria, an AIP is used as autoinducer signal. First, precursor peptides are formed in Gram-positive bacteria, then after modification, AIP is exported from cells by using protein transport machinery because as AHL it is not freely diffused. Two component signal transduction systems are present in Gram-positive bacteria in place of direct binding with a cognate receptor as AHL. In a

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New and future developments in microbial biotechnology and bioengineering

Pro-AIP Process ATP

AIP

Histidine Kinase

AIP

ADP P

Processed

Autoinducer receptor complex

Response regulator P Pro-AIP

Pro-AIP Gene regulation Gene regulation

(A)

(B)

FIG. 14.8 Bacterial quorum sensing circuits. Autoinducing peptide (AIP) in Gram-positive bacteria by (A) two component signaling, (B) an AIP-binding transcription factor.

two-signal transduction system, AIP binds to histidine kinase sensor present in the cell wall, then the phosphorylation (addition of phosphoryl groups) of response regulator proteins take place. This process is regulated by transmitters in two ways. First, autophosphorylation in which regulator proteins have an autokinase activity in which phosphoryl groups from adenosine triphosphate (ATP) are attached to histidine residue (Fig. 14.8A). High energy intermediate is formed in which the phosphohistidine then phosphoryl groups are transferred to aspartate residue of regulator proteins. In the second type of phosphorylation, transmitter proteins have phosphatase activity for their respective receivers. Phosphorylated response regulator proteins are finally bound to the promoter region to activate the gene expressions as shown in Fig. 14.8B. To understand the QS in Gram-positive bacteria, Streptococcus pneumoniae is the best example. Similar to Gramnegative bacteria, S. pneumoniae upregulates the production of antimicrobial activity only when the concentration of the autoinducer becomes higher than the threshold limits. Five types of genes (ComC, ComAB, ComD, ComE, and ComX) are involved in S. pneumoniae for development of competition. In S. pneumoniae, the AIP signal is called the competence stimulating peptide (CSP). The CSP (which is 17 amino acids long) is synthesized by 41 amino acids-long ComC. The ComAB processes and secretes the CSP into the extracellular region. When the CSP concentration is present above the threshold concentration, a sensor kinase protein, ComD, detects the CSP. A membrane bound receptor, ComD functions with ComE to detect the CSP abundance. Higher concentrations of CSP promote the autophosphorylation of ComD by transferring the phosphoryl group to ComE. Phosphorylated ComE activates expression of the ComX gene for development of competence. Overall, in S. pneumoniae, comAB is involved in exporting peptide signals, comC produces AIP, comD functions as the AIP receptor, comE functions as the intracellular response regulator and comX is required for expression of genes. Similar systems have been also found in other Bacillus and Staphylococcus species.

14.5.3 Hybrid QS in Gram-negative and Gram-positive bacteria A common QS system that uses signaling AI-2 (i.e., furanone) is present in both Gram-negative and Gram-positive bacteria. This type of QS is known as hybrid QS. Chen et al. (2002) showed a novel furanosyl borate diester that uses AI-2. Autoinducer AHLs are a modified oligopeptide or quinolone. The presence of a boron atom is an important feature of AI-2. Activated methyl cycle (AMC) generates AI-2 and its modified product is 4,5-dihydroxy-2,3-pentanedione (DPD). The protein LuxS is requiring for AI-2 production. LuxS is the enzyme involved in the production of S-adenosylmethionine which is a methyl donor and DPD from S-ribosylhomocysteine (SRH) during the AMC pathway. Further, the DPD forms AI-2 after cyclization with boron. S-Ribosylhomocysteine is produced during AMC pathway which is toxic for bacteria. Hence, LuxS produces DPD and homocysteine from S-ribosylhomocysteine which is a detoxification process. Therefore, LuxS is the enzyme which involve in cellular metabolism as well as in QS. The cellular level of S-adenosyl-L-homocysteine (SAH) is major compound that plays a very important role in bacterial pathogenesis regulated by the AMC pathway (Fig. 14.9). Two types of AMC pathways take place in bacteria; one is two-step reaction pathway, the LuxS/Pfs pathway. In this pathway, S-adenosylmethionine is converted into S-adenosylhomocysteine which is subsequently hydrolyzed into adenine

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FIG. 14.9 The activated methyl cycle (AMC) pathway in bacteria for the production of autoinducer-2 (AI-2).

Methionine S-Adenoslyl methionine

Sah H

S-Adenoslyl homocysteine

Homocysteine Pfs Lux S Autoinducer-2

S-Ribosylhomocysteine

4,5-Dihydroxy2,3 pentanedione

and S-ribosylhomocysteine by the nucleosidase Pfs. LuxS and Pfs catalyze the conversion of SAH to homocysteine and AI2 which are involved in interspecies and intraspecies communication. The other pathway is the SahH pathway, a one-step reaction mediated by S-adenosyl-L-homocysteine hydrolase (SahH). In this cycle, AI-2 is not produced. Hybrid QS is detected in several bacteria such as Escherichia coli, Salmonella typhimurium, Helicobacter pylori, Vibrio cholerae, S. aureus, and Bacillus subtilis by using an engineered Vibrio harveyi biosensor. AI-2 was originally identified in V. harveyi and regulates light production with reference to cell density. Several authors have shown that boron-containing AI-2 is involved in bioluminescence in V. harveyi, a marine bacterium. The hybrid system has two QS systems. System one is primarily involved in intraspecies signaling and the signaling molecule is AHL (AI-1). The second system is involved in interspecies signaling and has a furanosyl borate diester that works as the autoinducer. AHL is produced by LuxLM. The hybrid sensor kinase for AI-1 is LuxN. Protein LuxS synthesizes the second autoinducer, AI-2. AI-2 is detected by protein LuxP and LuxQ together. Further, LuxN and LuxQ are hybrid response regulators that deliver instructions to LuxU which is a shared integrator protein. In the absence of signal, i.e., low concentration of the autoinducer, these proteins show intrinsic kinases activity and phosphorylate a complex phosphorelay system with LuxU and LuxO. Phospho-LuxO, in conjunction with s54, activates transcription of small regulatory RNA which inhibits information of the LuxR and stops the expression of the luciferase operon. While, when the autoinducers are present at a threshold limit, then sensors work as phosphatases and the system uses dephosphorylated acquiesce LuxR for expression of bioluminescence in V. harveyi. Several novel chemical signals, such as PQS, i.e., 3,4-dihydroxy-2heptylquinolone (PQS), diffusible signal factors (DSFs) and g-butyrolactones (GBLs), indole, butyrolactones and 3hydroxy palmitic acid methyl ester, are shown in Table 14.1 (Deng et al., 2010; Decho et al., 2011).

14.6

Biofilm mechanisms involved in bioremediation

Cost-effective and ecofriendly techniques for the detoxification of hazardous waste and metals containing wastewaters are essential to minimize the environmental hazard caused due to organic complex waste and metals. Varying treatment systems based on concentration, volume, and the nature of wastewaters makes it complex and unfit for treatment in conventional treatment processes such as activated sludge process, trickling process, chemical oxidation and reduction precipitation and sludge separation, reverse osmosis, ion exchange, coagulation, cementation, flocculation, electrochemical treatment, and/or evaporation. Most of these methods are either incapable of meeting treatment objectives or are very expensive. Hence, biofilm-mediated bioremediation is endorsed as a simple, efficient, cost-effective, and environmental-friendly process for sustainable development. Complete mineralization, detoxification, and low operational charges from contaminated sources are among the main advantages of biofilm-mediated bioremediation. Major characteristics of biofilms and their importance for bioremediation are shown in Table 14.2. As previously described, a biofilm is a community of microorganisms containing different microbial species attached to living and nonliving surfaces embedded in self-synthesized slime EPS matrix. It may be anticipated that the presence of various microbial species with different metabolisms play an important role for degradation of complex pollutants due to cometabolisms (Horemans et al., 2013; Gieg et al., 2014). Adaptation towards stress conditions, cometabolisms, and competition for oxygen and nutrients are important features of biofilm-mediated bioremediation. Biofilm is efficient for bioremediation because it immobilizes, absorbs, and degrades various environmental pollutants. Microbial populations of heavily contaminated sites mainly exist in the form of biofilms for better protection, persistence, survival, and handling

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TABLE 14.1 Quorum sensing signals in microorganisms and their characterization. QS signal

Autoinducer

Producing species

Ecological function

References

Autoinducers (AI-1) with short chain molecules

C4-HSL

Vibrio harveyi, Pseudomonas aeruginosa, Aeromonas hydrophila, Serratia marcescens, Erwinia stewartii, Vibrio fischeri, P. fischeri

Bioluminescence, biofilm maturation, biofilm formation, enzyme production, swarming, virulence

Papenfort and Bassler (2016)

C6-HSL

A. salmonicida, Chromobacterium violaceum, Yersinia enterocolitica, Y. pseudotuberculosis, Vibrio alginolyticus, Agrobacterium tumefaciens, Erwinia carotovara, P. aureofaciens, Nitrosomonas europaea

Biofilm formation, violacein, antibiotics, enzyme production, motility aggregation, settlement activity, ammonium oxidation

Miller and Bassler (2001), Huang et al. (2007), Burton et al. (2005)

C8-HSL

Roseobacter spp., Marinobacter sp., Agrobacterium tumefaciens

Potential regulate cells swimming, flocculant production

Miller and Bassler (2001)

C10-HSL

Vibrio anguillarum

Virulence

Defoirdt et al. (2004)

C12-HSL

Vibrio alginolyticus

Virulence formation

Defoirdt et al. (2004)

C14-HSL

Roseobacter spp.

–

Huang et al. (2007)

Furanosyl borate

Vibrio harveyi

Bioluminescence

Bassler et al. (1993)

Diester (boron containing AI-2)

V. cholera, V. harveyi, V. parahaemolyticus

Virulence

Chen et al. (2002), Henke and Bassler (2004)

2-Methyl-2,3,3,4tetrahydroxytetrahydro furan

V. cholera, Salmonella enterica

Virulence gene expression

Sperandio et al. (2001)

3-Oxo-C6-HSL

E. coli, Xanthomonas campestris

Biofilm formation, wastewater treatment, acid tolerance

Houdt et al. (2006)

AI-2 (S-THMFborate)

LuxS, presence of boron

Vibrio harveyi

4,5-Dihydroxy-2,3pentanedione (DPD) is synthesized by all LuxS enzymes and is thus the universal precursor to the widespread family of quorum sensing (S-THMF-borate), R-THMF)

Papenfort and Bassler (2016)

AI-2 (R-THMF)

LuxS, absence of boron

Salmonella typhimurium, E. coli

–

Papenfort and Bassler (2016)

Streptococcus Gordonii

Biofilm formation

Autoinducers (AI-1) with long chain molecules

AI-2

AI-3

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TABLE 14.1 Quorum sensing signals in microorganisms and their characterization.—cont’d QS signal

Autoinducer

Producing species

Ecological function

References

Polypeptide autoinducers (AIP-I)

Butyro lactonase, Thiolactone (diketopiperazines (DPKS), cyclo(L-Ala-LVal), and cyclo(L-Pro-L-Tyr))

Pseudomonas sp., Streptomyces griseus, Staphylococcus aureus

Activates AHL Biosensors: the induction threshold for DKPS is higher than AI-1, which indicates that DKPS may not have a significant role in the marine

Degrassi et al. (2002), Zhang and Dong (2004)

AIP-I

Staphylococcus aureus group I strains

Pathogenesis

Degrassi et al. (2002), Zhang and Dong (2004)

AIP-II

S. aureus group II strains

AIP-III

S. aureus group III strains

AIP-IV

S. aureus group IV strains

Others

Bradyoxetin, i.e., 2-{4-[[4(3-aminooxetan-2-yl) phenyl]-(imino)methyl] phenyl}oxetan-3-ylamine, 3-hydroxy palmitic acid methyl ester

Bradyrhizobium japonicum, Rhizobium spp., Alphaproteobacteria spp., Pseudomonas aeruginosa, Ralstonia solanacearum

Polysaccharide biosynthesis

Flavier et al. (1997), Bogino et al., 2015

Unidentified compounds

Cys-11-methyl-2dodecenoic acid

Xanthomonas campestris

Virulence

Zhang and Dong (2004)

3Hydroxypalmiticacid-methyl-ester (3OH PAME) and (R)-methyl-3hydroxymyristate (R)-3-OH MAME

3OH palmitic acid methyl ester, PhcB

Ralstonia solanacearum, Ralstonia spp.

Plant pathogenesis

Flavier et al. (1997)

DSF

RpfF

Xanthomonas campestris

Biofilm, virulence, motility, toxin, exopolysaccharides, and extracellular enzymes

Papenfort and Bassler (2016)

CqsA

CAI-1

V. harveyi, Vibrio cholerae

Pathogenesis

Papenfort and Bassler (2016)

AmbBCDE

2(2-Hydroxyphenyl) thiazole-4-carbaldehyde IQS

Pseudomonas aeruginosa

DarABC

Dialkylresorcinols (DARs)

Photorhabdus asymbiotica

Cell-to-cell communication

Papenfort and Bassler (2016)

PpyS

Photopyrones

P. luminescens

Cell-to-cell communication

Papenfort and Bassler (2016)

Papenfort and Bassler (2016)

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TABLE 14.2 Major characteristics of biofilms and their importance for bioremediation. Properties

Characteristics

Quorum sensing (communication)

Biofilm formation at a threshold density

Tolerance towards environmental stresses (complex and toxic waste, dehydration, and pH change)

Extensive genetic diversification of biofilm bacteria, chelation, enzymatic degradation, reaction with EPS, precipitation, volatilization as alkylated metal compounds and precipitation

Metabolic diversity and symbiosis

Waste products utilization, accumulation, and horizontal gene transfer

Heterogeneity and social interactions of biofilms

Inducing biofilm resistance due to different metabolic states in the biofilm, communication, cooperation, and competition

Redox and electron acceptor diversity

Different metabolic functions with respect to electron-acceptor reduction

Tolerance against stress

Persisters and viable-but-nonculturable cells (VBNC)

Porous physical structure with water channels

Allow for transport of nutrients, waste products and electron acceptors

Surfactants

Solubilizing hydrophobic and recalcitrant substrates

Microcolony and gradient formation

Redox potential and nutrient cycling because of aerobic and anaerobic processes

stressful conditions. The activation and expression of different genes within a biofilm are different from the free-floating planktonic cells. These expressions of different genes are dependent on oxygen, nutrient concentrations, and division of labor among microbes, which leads to degradation of various complex pollutants by various metabolic pathways. Chemotaxis and flagellar-dependent motility is important feature of microbes for biofilm formation (Pratt and Kolter, 1999). The QS in the presence of complex pollutants, soil, and water assists microbe in swimming, swarming, chemotaxis, twitching motility, and coordinating movement towards pollutants, improving biodegradation. Under natural environmental conditions, most bacteria persist in biofilm mode covered in an EPS matrix which is a beneficial structure of biofilm-forming microbes in bioremediation. The structure of bacterial biofilms and the content of the EPS production is dependent on the environmental conditions in which the microbes are found (Table 14.2) (Kreft and Wimpenny, 2001; Jung et al., 2013; Miqueleto et al., 2010). Biofilms appear filamentous and in mushroom-like shapes in fast moving and static water respectively as shown in Fig. 14.10. The biofilm matrix offers greater resistance than planktonic cells to microbes from environmental stresses, acid stress, antimicrobial agents, biocides, UV damage, drought conditions, predation, solvents, and toxic pollutants (Davey and O’Toole, 2000; Mah and O’Toole, 2001). In contrast, free-floating planktonic cells degrade pollutants by metabolic activity but these cells are not stationary and are not adapted to persist under environmental stress condition. In contrast, biofilmforming microbes are particularly adept in bioremediation by immobilizing pollutants during degradation in an EPS. The three-dimensional structure of EPS with reduced oxygen concentrations towards the center brings together aerobes and anaerobes, heterotrophs with nitrifiers and sulfate reducers with sulfate oxidizers, which promotes faster degradation of varied pollutants in natural and engineered systems. The presence of the EPS in biofilm facilitates the nutrient exchange and removal of byproducts. An EPS has the capacity to bind with various metals including lead, copper, manganese, magnesium, zinc, cadmium, iron, and nickel (Ferris et al., 1989). Nutrient limitations can also increase EPS production which can enhance the absorption of metals and pollutants from the environment. Biofilms consist of phosphorus-accumulating microorganisms which can remove and recover phosphorus from wastewater. The coexistence of multiple microbial species within biofilms in close proximity promotes interaction among its members which may lead to complete metabolization of complex pollutants due to result of cometabolization. Common places of natural environmental biofilms are soil, aquatic plants, sediments, covering rocks in streams and plants, lakes and rivers, and in wetlands. Natural biofilms in the environment can degrade and remove complex and recalcitrant pollutants from the environment. Biofilms formed on the surface of water consist of bacteria, protozoa, fungi, and algae. Naturally developed biofilms by algae such as Nitzschia in organic sediments can utilize the wastes and produce oxygen which in turn facilitates the aerobic bacteria of the biofilm that can further degrade another complex. Natural biofilms are also an important part of the food chain where grazers or protozoa may feed on biofilm material. Small fish may engulf the protozoa and subsequently small fish are engulfed by larger fish and terrestrial animals. Microbial aggregates in aquatic systems such as microbial mats consisting of aerobic, anaerobic bacteria, protozoans, diatoms, nitrifying bacteria,

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FIG. 14.10 Mechanisms of bioremediation of complex polymers and heavy metals by biofilm.

phototrophic bacteria, and algae are formed on a sediment surface under extreme conditions to survive predation from grazers and stress conditions. Municipal wastewater treatment is also based on the floc, which are fragile structures in activated sludge plants. Floc is a type of biofilm formed on the surface of sand to remove organic compounds and metals from treatment systems or natural reservoirs (Logsdon et al., 2002). Biosorption (microbial or plant cell) and biotransformation (enzymes or metabolites) are probably the most widely explored biological metal and organic hazardous pollutants removal strategies. Bacteria have two types of heavy metals uptake system: one is a fast and unspecific mechanism in which a chemiosmotic gradient is developed across the cytoplasmic membrane. Due to the gradient, heavy metals are removed from the site. The second type of mechanism is slower, specific, and needs energy from ATP hydrolysis. The mechanisms of bioremediation of complex polymers and heavy metals by biofilm are shown in Fig. 14.10. Biosorption is physiochemical process that can be carried out via surface complexation, electrostatic interaction, precipitation, redox processes, and ion exchange, using the cellular structure of microbes. Biosorption is a passive process for metals uptake and it is independent from the metabolic process. The amount of metal sorbed depends on the composition of the metal at the cellular surface and kinetic equilibrium. The biosorption process is a very fast reaction and can reach equilibrium within a few minutes. The mechanism of metal removal from biofilms involves biosorption which is the total passive interactions of the cell wall and metal ions. Biosorption may be a metabolismdependent or metabolism-independent process. At metal sorption sites, sorption may be extracellular accumulation, precipitation, cell surface adsorption/precipitation, and intracellular accumulation. The transfer of metal from outside to inside the cell membrane, i.e., intracellular accumulation, is dependent upon the metabolism of cell as an active defense system of microorganisms.

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Intracellular accumulation is only performed by living cells. Biosorption by dead biomasses is a metabolismindependent process which is the result of complexation of functional groups with metals on the microbial cell surface. Ion exchange, and physical and chemical adsorption are the types of biosorption that are independent from cell metabolism. Microbial cell walls are made up of polysaccharides, proteins, and lipids which have abundant metal-binding groups, such as hydroxyl, carboxyl, alcohol, ester, amine, phosphoryl, sulfhydryl, sulfonate thiol and thioether groups. These ligands are also known to be involved in metal chelation. However, intracellular accumulation of pollutants, mainly heavy metals, in microbes may be governed by the same mechanism as that used for the uptake of metabolically-important ions such as potassium, magnesium, and sodium. Cation transport systems of cell for metabolically-important ions may become confused by heavy metal ions of the same charge and ionic radius. Hence, they can be transported inside the cells then they follow the metabolic-dependent removal mechanism. Macrophytes can be used for the sorption and accumulation mechanisms to remove heavy metals from contaminated water bodies (Mane et al., 2011). Overall, metal mobilization takes place through acidification, protonation, and chelation, while immobilization takes place through precipitation, complexation, and alkalinization. In an ion exchange mechanism, metal removal by cells including divalent metal ions, are exchanged with counter ions of polysaccharides of the microbial cell wall. Another mechanism of detoxification of metals by biofilm-forming microorganisms is the production of several organic acids, i.e., gluonic, fumaric acid, malic acid, and oxalic acid, which chelates toxic metals through metallo-organic molecule formation. The abovementioned organic acids solubilize the metals and enhance their leaching from their surface to facilitate metal removal. An EPS containing biosurfactants may also aid the solubilization of hydrophobic or other refractory substrates which would otherwise be inaccessible to microorganisms. This makes compounds accessible for metabolization or transformation. Acetobacter xylinum, Agrobacterium spp., Alcaligenes faecalis, Bacillus spp., Leuconostoc, Pseudomonas spp., Xanthomonas campestris, and Zymomonas mobilis have been identified as EPS-producing microorganisms. The EPS works as a suitable biosorbent for heavy metals which is a common strategy of biofilm-mediated bioremediation of heavy metals. Some commercial bacterial EPS with anionicity are gellan (Sphingomonas paucimobilis), alginate (Azotobacter vinelandii, P. aeruginosa), xanthan (X. campestris), hyaluronan (Pasteurella multocida, P. aeruginosa), fucopol (Enterobacter A47), and galactopol (Pseudomonas oleovorans) which are involved in the biosorption and biomineralization mechanisms for metal removal. The EPS from cyanobacteria is also involved in removing heavy metals by the biosorbent mechanism. In general, chelation and acidification processes mobilize the metals, while alkalinization, complexation, and precipitation immobilize the metals. Moreover, chemical transformations stimulate the mobilization and immobilization of metals. The other mechanism of metal removal is a process in which the heavy metals enter into the cytoplasm through the cell membrane. This is known as bioaccumulation which is an active uptake process. In bioaccumulation, living cells accumulate the pollutants and metals through physical, chemical, and biological mechanisms. Bioprecipitation of metals is also a type of metal removal strategy adopted by biofilm-forming bacteria in which they produce a substance that reacts with metals or pollutants to form insoluble metal compounds. Soluble metal species are also converted into insoluble compounds of carbonates, hydroxides, phosphates and sulfides during the bioprecipitation process (Fig. 14.10). The peptidoglycan layer of Gram-positive bacteria contains alanine, glutamic acid, meso-di-aminopimelic acid, polymers of glycerol, and teichoic acid, while glycoproteins, lipopolysaccharides, enzymes, phospholipids, and lipoproteins of Gram-negative bacteria are the active sites involved in the metal removal process. Once the metals and similar types of pollutants are bound on cell surfaces, microbial cells reduce their toxicity by converting them from one oxidation state to another. Bioassimilation is the mechanism of microbes to enhance the uptake of metals and complex pollutants by secretion of siderophores. Bacteria, fungi, and plants secrete small molecules which have the capacity to chelate the iron and other metals, and these are known as siderophores. Siderophores are mainly named on the basis of types of ligands used to chelate the iron and other metals. The wide varieties of structurally different siderophores are produced due to adaptation of microbes under stressful conditions. Catecholates, hydroxamates, carboxylates (e.g., derivatives of citric acid), and mixed types of siderophores are present. Sorption of metals and other pollutants in algae involves proteins and many polysaccharides such as alginic acid, glycan, xylan, and mannan. The physical and chemical properties of the metal affect the algae’s sorption capacity. The rigid cell wall of fungi is composed of chitin, proteins, lipids, N2-containing polysaccharide, polyphosphates, and inorganic ions. Fungi detoxify and tolerate metals by transformation, accumulation, and intracellular and extracellular precipitation of metals. The cell wall of fungi has a ligand for binding of metals leading to the metal removal. The first mechanism adopted by fungi is excretion of substances such as proteins and organic acids to mobilize or immobilize metals. Another mechanism is nonspecific binding of cell wall and melanins to metals. Bioremediation or the transformation of complex pollutants involves all known categories of enzyme oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases (synthetases). Oxidoreductases are a group of enzymes that transfer

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electrons from donor atoms to acceptor atoms. The transferases group of enzymes removes a functional group from one compound and transfers it to another compound. Hydrolases cleave the bonding between carbon-carbon, carbon-nitrogen, carbon-oxygen, and other bonds with the help of water. Lyase enzymes cleave the chemical bonds in such a way that a new double bond or ring is formed. Cleavage takes place by means other than oxidation and hydrolysis. Isomerases are involved in a substrate’s isomerization. Ligases catalyze the joining of two molecules. Studies have shown that phosphatase activity, sulfate solubilization, and cometabolism in the matrix of biofilms are key factors in remediation of complex wastewater (Plosz et al., 2010; Rodriguez and Bishop, 2008). On the other hand, organic and inorganic stress in plants triggers a plant’s interlinked molecular and physiological processes to cope. Mechanisms used by plants for inorganic pollutants (metals) tolerance include transport of metals into vacuoles, plant cell wall binding, intracellular complexation with peptide ligands, i.e., metallothioneins (MTs), and phytochelatins (PCs), and sequestration of a siderophore-metal complex in apoplast and soils. The tolerance strategy in hyperaccumulator plants is the secretion of different compounds such as low molecular weight organic acids (LMWOAs) that are able to stimulate microbial growth, solubilization of insoluble nutrients (phosphate, zinc, and iron) and detoxification of some metals. Secretion of LMWOAs such as citric acid, malic acid, oxalic acid, and succinic acid are one of the most important strategies adopted by plants to cope with higher concentrations of metals, by excluding metals through chelation in the rhizosphere and apoplast. Bioaccumulation is another molecular mechanism of phytodegradation which involves intracellular accumulation of metals in the biomass of living cells. Bioaccumulation can be passive biosorption, which is metabolism-independent (physical and chemical adsorption, chelation, metal-ion exchange, surface complexation, and precipitation). or active bioaccumulation, which is metabolism-dependent (entry of metal ions into microbial cells through complex permeation, ion pumps, and endocytosis). Bioaccumulation is a more complex process than biosorption. Bioaccumulation involves the metabolic activities of living cells such as extracellular precipitation and intracellular sequestration. Biosorption involves surface molecules (S-layer proteins) to trap metals either by living or dead cell, while bioaccumulation involves the incorporation of essential nutrients and metal reduction through helper proteins. Recent research on metal removal has focused on biosorption, metal binding with waste materials, and renewable biosorbents (living and dead cells of bacteria, fungi, and plants) which will be more economical. In addition, bioleaching and bioexclusion are molecular mechanisms of metal removal. Root exudates released by plants play a very important role in biofilm-mediated phytoremediation. Root exudates are a mixture of compounds that are secreted by the whole root at certain stages of plant growth. Root exudate is good source of nutrients and energy for microorganisms and in return, the microorganism enhances the secretion of exudates from the plant roots. Furthermore, organic acids of root exudates are bound with metals and influence the solubility, bioavailability, and mobility of metals. The EPS, AHLs, proteins, glycoproteins, glycopeptidolipids and other macromolecular metabolites play a significant role in biofilm formation. Bacterial major OMPs and an array of glycan-binding proteins, e.g., lectin, are involved in host recognition and root colonization along with adhesion and root adsorption (Compant et al., 2010). Moreover, the motility and type IV pili of bacterial cells are also involved in root colonization. Root exudates induce the potentiality of plants against the metal and other organic and inorganic stresses by either a detoxification process (adsorption, transformation, chelation, and in-activation) or allelopathic functions. Additionally, root architecture can be changed due to pH, salinity, moisture, nutrient, and organic and metals content which influences the microbes (PGPR, fungi, rhizobia). Furthermore, microbes are colonized for biofilm formation due to chemotaxis movement. Biofilms are playing a very important role in bioprotectants (induced systemic resistance), phyto-stimulators (triggering hormonal signaling networks), biofertilizers (phytoavailability of minerals, i.e., N, P, K, Ca, and Fe), bioalleviators (reducing ethylene stress), biopesticides (via antibiotic functions), biomodifiers (modifying root biomass and morphology), and bio-control (control adverse effects of pathogenic bacteria via secretion of antibiotic and antifungal) which lead to phytoremediation of organic and metals. These functions enhance plant growth which is a successful phytoremediation mechanism (see Fig. 14.11). Microbes can alter the chemical composition of exudates that lead to changes in physiology through the release of various signaling molecules, i.e., Myc factors, Nod factors, volatile organic chemicals, and EPS. Root exudates and biofilm-forming bacteria play a very important role in changing the bioavailability of mineral nutrients and metals. Metals and nutrients availability may be enhanced either through acidification or through amino acid/organic-metal complexes; enzymatic activity in rhizosphere; intracellular binding compounds (e.g., amino acids, phyto-chelatins, and organic acids) and by stimulation of rhizosphere microbial activity including plant growth-promoting rhizospheric bacteria (PGPR). In PGPR, there are multiple characteristics such as enhanced metal availability, alleviation of metal toxicity (metal-resistant bacteria), indole-3-acetic acid (IAA) as a phytohormone-generating bacteria, siderophore forming and biochelator

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Phyto-stimulators Microbes

Biofertilizers

Breaks the QS

Quorum sensing (QS)

Regulate signaling (VOCs Nod factors, Myc factors and organic volatiles) and physical interaction (adhesion, fimbri flagella interaction)

Chemical signal (flavonoids, QS mimic signal)

Bioalleviators Bio-pesticides Exudate

Bio-modifier Bio-control

Changes root architecture and activity Directly involve in adsorption, chelation, transformation, inactivation of metals

Biofilm formation Chemotaxis and colonization

Influence properties of rhizosphere soils, pH, salinity, moisture, nutrient, metals

Influence microbes (PGPR, fungi, rhizobia)

FIG. 14.11 Involvement of QS for biofilm formation and phytoremediation.

formation (biosurfactants or organic acid generating bacteria), nitrogen fixation and solubilization of minerals (phosphate, sulfate or potassium solubilizing bacteria), production of ethylene synthesis inhibitors 1-aminocyclopropane-1-carboxylate (ACC) and rhizobitoxine antimicrobial enzymes (phytoalexins, chitinases, b-1,3-glucanase, and lysozyme; and polysaccharides (exopolysaccharides and EPSs) for microbe-assisted phytoremediation. The rhizosphere is very thin portion of soil that adheres to the root. It is directly affected by root exudates and associated soil microorganisms. Some microorganisms of rhizospheric soil form biofilms due to the secretion of chemicals or signals by plants as well as bacteria which is very useful for phytoremediation. Comprehensive interaction between plant and microorganisms through signaling plays an important role in adaptation under stress conditions. Flavonoids of root exudates are the main signaling components in various plant-microbe interactions such as mycorrhizae formation and legumerhizobia interaction. Flavonoids also play an important role in arbuscular mycorrhizal fungus (AMF), hyphal growth, and spore germination. When plants are colonized with AMF, the pattern of flavonoid secretion is suddenly altered which affects the plant-AMF interaction. Apart from this, flavonoids also enhance the growth of rhizobacteria and act as chemo-attractants and inducers of Nod genes involved in nodulation. Bacterial VOCs (2,3-butanediol and acetoin) can stabilize the interaction between the plant and microbes leading to the expression of plant defense and growth promoting mechanisms under stress conditions. Furthermore, root architecture can be changed due to synthesis of signaling molecules: Myc factor for AMF and Nod factors for rhizobia. Biofilm formation can be detected by tagging certain molecular markers such as b-glucosidase or tagging with fluorescent protein observed under microscope (Reinhold-Hurek and Hurek, 2011). Moreover, phosphate compounds present in contaminated soil are not easily soluble, hence plants cannot absorb them. Some microorganisms have ability to bind with phosphate and form a metal-phosphate complex, either through the phosphate cycle or the accumulation of high concentrations of phosphate. Insoluble phosphate at a contaminated site can be solubilized by organic acids, enzymes, and some chelating agents secreted by microbes and plants. In contrast, some bacteria in a biofilm can form insoluble metal sulfides which precipitate out, leading to detoxification of toxic metals from the solution. Moreover, siderophores are low molecular weight and complex ferric chelating agents. Siderophores are often produced by bacteria and fungi under low iron availability. Siderophore association is constant for Fe3+ that ranges between 1012 and 1052. Iron is an essential element for plants and microbial growth in numerous metabolic activities such as electron transport systems, acting as cofactors for enzymes, the formation of heme, and the synthesis of chlorophyll. Iron can be converted to a less soluble complex oxyhydroxide in the presence of oxygen. Hence, there is need for specific molecules in the environment which can release the iron from the immobilized complex. A siderophore is the molecule that can release the iron and turn it into an available form. Siderophores are synthesized by both plants and microbes. Siderophores produced by plants are known as phytosiderophores. Actinomycetes and certain algae have the capacity to produce siderophores under iron stress condition. Interestingly, Saccharomyces cerevisiae is unable to produce siderophores but has the capacity to utilize siderophores produced by other microorganisms. Siderophores are useful for phytoremediation of heavy metals and xenobiotic compounds, and during the last two decades many research studies have focused on this aspect. In addition, rhizospheric microorganisms play a significant role in many plants’ physiological activities which are

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also useful for the remediation of environmental pollution. These microbes may form biofilms with the help of QS molecules. Rhizospheric microorganisms can also produce phytohormones such as cytokinins, indole acetic acid, abscisic acid, and gibberellic acid even under stress conditions. Plant-associated microorganisms have the capacity to enhance plant growth by adopting any one or more of the abovementioned mechanisms. Therefore, PGPR can be applied not only in agriculture for food production, but it can be also use for phytoremediation of heavy metals and other organic pollutants.

14.7

Types of pollutants remediated by biofilms

Biofilm-mediated bioremediation is being increasingly used in the removal of different types of pollutants including POPs, oil spills, pesticides, xenobiotics, and heavy metals. POPs are the most persistent pollutants with long half-lives due to their hydrophobicity and can be found in air, water, and sediments. All can be toxic when found in the food chain. Examples of POPs include polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, polychlorinated ethenes, pentachlorophenol (PCP); all of which are listed as toxic or harmful pollutants by the United States Environmental Protection Agency (Shukla et al., 2014; Chen et al., 2015). Many biofilm-forming bacteria have been isolated from the environments that degrade POPs which may be further modified and used for bioremediation (Chen et al., 2015). Bacterial biofilms have been engineered for detoxification of POPs ( Johnsen and Karlson, 2004; Rodriguez and Bishop, 2008; Plosz et al., 2010). The increasing use of petroleum in industry is a major cause of toxicity to marine life leading to an adverse effect on the whole ecosystem (Atlas and Hazen, 2011; Ron and Rosenberg, 2014). In this context, new microbial biofilm-forming strains including Pseudomonas, Rhodococcus, Arthrobacter, Alcanivorax, Bacillus, and Cycloclasticus spp. of gamma proteobacteria have been employed (Dasgupta et al., 2013; Harayama et al., 2004). A microbial consortium of B. subtilis and Acinetobacter radioresistens with a surfactant-producing strain has been shown to be a better degrader of petroleum waste (Mnif et al., 2015). Li et al. (2008) suggest that certain bacterial strains are capable of forming electrochemically active biofilms (EAB) which directly exchange electrons with a conductive solid surface. An EAB can be utilized for heavy metal removal from contaminated soil and groundwater (Cong et al., 2013). Biofilm-forming bacteria have been shown to transform or degrade phenol, hexadecane, phenanthrene, pyrene, nitrogen, and total organic carbon (Kang and Park, 2010; Huang et al., 2013; Zhang et al., 2015). The expression of enzyme production during biofilm-mediated degradation is regulated by QS systems. The RhlI/RQS system of P. aeruginosa is involved in the organic pollutant degradation from municipal and industrial wastewater, and it is also involved in denitrification along with biodegradation. Mutants of DrhlI and DrhlR showed increased denitrification processes and decreased phenol degradation (Yong and Zhong, 2013b). The addition of AHL extracts in the media showed induced denitrification activity in QS mutant strains similar to the wild-type strain. Acinetobacter spp. showed biofilmmediated hexadecane biodegradation due to autoinducer synthases encoded by abaI (Anbazhagan et al., 2012). Degradation of anthranilate and phenol by P. aeruginosa expression of enzyme catechol-1,2-dioxygenase and catechol 2,3-dioxygenase was significantly enhanced by the addition of N-decanoyl-L-homoserine lactone and N-octanoyl-L-homoserine lactone through the rhlI/RQS system, respectively (Chugani and Greenberg, 2010; Yong and Zhong, 2013a). The utilization of bacterial communities has been applied for degradation and detoxification of organic and inorganic contaminants. Heavy metals such as copper, cadmium, nickel, zinc, and cobalt have been remediated by using various biofilm reactors. Smith and Gadd (2000) showed that biofilm-forming sulfate-reducing bacteria (SRB) are useful in mines for scavenging metals from metal-contaminated water into metal sulfide precipitates (Smith and Gadd, 2000; Muyzer and Stams, 2008). Passive oxidation of As and Fe by biofilm was significantly observed at coal mine drainage contaminated sites and gold-quartz mining sites (Guezennec et al., 2012). Biofilm enzymes have been employed as biomarkers for stream water quality and selenium has been reduced and concentrated in biofilms on tubes containing nutrients ((Pool et al., 2013; Williams et al., 2013). In addition, a phosphatase enzyme present in the matrix of biofilm facilitates metal precipitation by both aerobic bacteria and anaerobic conditions. Interestingly, in some cases, biofilm formation is induced by addition of carbon sources in contaminated groundwater to reduce the flow of pollutants away from the site of contamination or create a barrier to minimize its spread.

14.8

Conclusions

Bioremediation will become a necessity of today’s modern life due to excess discharge and accumulation of recalcitrant, xenobiotic compounds into the environment. The great adaptability of microbes will provide simple, economical and ecofriendly techniques to degrade environmentally-recalcitrant pollutants. Recently, comprehensive attention has been focused on the management of environmental pollution caused by toxic, recalcitrant, and hazardous compounds including heavy metals for sustainable development. Various bioremediation processes have been adequately evolved and utilized.

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While, each process has its own advantages and disadvantages, biofilm-induced bioremediation remains an attractive choice in mitigating environmental pollutions due to the wide range of adaptability of biofilm-forming bacteria. With the involvement of QS, bioremediation kinetics can be enhanced. A biofilm provides a protective environment to cells by cell-to-cell communication through signaling molecules, the exchange of genetic material and metabolites. Thus, biofilm-mediated bioremediation will be a better technique for treatment of industrial wastewater. Xenobiotic, recalcitrant stress might induce QS responses in a biofilm community that can be applied for effective bioremediation. Besides the biofilm formation, QS regulates EPS production, biosurfactant synthesis, siderophore formation and genetic competence, which are additionally important for environmental cleaning of organic and inorganic pollutants. Moreover, the development of bacterial consortia containing QS between various species along with other specificities can also act as advanced bioremediation practices. One of the essential constituents of biofilms is the release of EPS which provides a self-defense against stress conditions of pollutants. The net negative charge of biofilm effectively sequesters positively-charged pollutants, including heavy metals. Most of the functional groups of a biomass that have binding capacity with metals are present on cell walls. A biosorbent contains a variety of functional groups such as amino, amide, carbonyl, carboxyl, hydroxyl, imidazole, phosphate, sulfate, sulfydryl, and thioether moieties as metal binding sites. It has been also reported that the sorption through EPS is generally nonspecific. A novel approach for improving bioremediation uses natural transformation processes within biofilms, utilizing the uptake of e-DNA with catabolic genes that facilitate biodegradation of selected pollutants. Cell-to-cell interaction, i.e., QS, among members of biofilm community can be explored further to improve the bioremediation process. Mechanisms involved in biofilm-mediated degradation are the biosorption process including transport across cell membrane, ion exchange, complexation, and physical and adsorption precipitation. In addition, extracellular enzymes, i.e., ligninolytic enzymes, phosphatase, dehydrogenase, or oxidoreductase within the EPS of biofilms, decontaminate pollutants such as heavy metals and organic compounds. Genetically modified microorganisms (GEM) have been also constructed with the capacity to degrade diverse recalcitrant pollutants. Moreover, horizontal gene transfer of genes capable of degradation of hazardous pollutants from GEM to members of biofilm populations can further enhance the biodegradation process. Cloning of genes for chemotactic ability and biosurfactant production of GEM can further enhance the biodegrading capability of modified microbes. Nevertheless, the release and use of GEM in nature and its transmission is under much debate and viewed as controversial. During the biofilm-mediated remediation of pollutants from contaminated sites, bioavailability of the pollutants, aerobic, anaerobic conditions, the presence of toxic substances, low temperature and potentiality of microorganisms should be considered prior to the application of the process. Moreover, in certain cases of recalcitrant pollutants, biofilm-mediated bioremediation can be used in combination with phytoremediation or with chemical treatments. The involvement of QS in biofilm growth cycle has been extensively studied. However, successful application of biofilm-mediated bioremediation is limited at this point. Thus, there is a need to understand more about QS in biofilm-mediated bioremediation of recalcitrant pollutants. Commercializing biofilmmediated bioremediation for recalcitrant pollutants is still a challenge for researchers.

Acknowledgments Project funding from DST (SERB) grant number YSS/2015/000768 and DBT, New Delhi grant number BT/PR13922/BCE/8/1129/2015 are gratefully acknowledged.

References Anbazhagan, D., Mansor, M., Yan, G.O., Yusof, M.Y., Hassan, H., Sekaran, S.D., 2012. Detection of quorum sensing signal molecules and identification of an autoinducer synthase gene among biofilm forming clinical isolates of Acinetobacter spp. PLoS One 7, e36696. Atlas, R.M., Hazen, T.C., 2011. Oil biodegradation and bioremediation: a tale of the two worst spills in U.S. history. Environ. Sci. Technol. 45, 6709–6715. Bassler, B.L., Wright, M., Showalter, R.E., Silverman, M.R., 1993. Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol. Microbiol. 9, 773–786. Becker, P., Hufnagle, W., Peters, G., Herrmann, M., 2001. Detection of different gene expression in biofilm-forming versus planktonic populations of Staphylococcus aureus using micro representational-difference analysis. Appl. Environ. Microbiol. 67, 2958–2965. Bogino, P.C., Nievas, F.L., Giordano, W., 2015. A review: quorum sensing in Bradyrhizobium. Appl. Soil Ecol. 94, 49–58. Burton, E.O., Read, H.W., Pellitteri, M.C., Hickey, W.J., 2005. Identification of acyl-homoserine lactone signal molecules produced by Nitrosomonas europaea strain Schmidt. Appl. Environ. Microbiol. 4906–4909. Chen, M., Xu, P., Zeng, G., Yang, C., Huang, D., Zhang, J., 2015. Bioremediation of soils contaminated with polycyclic aromatic hydrocarbons, petroleum, pesticides, chlorophenols and heavy metals by composting: applications, microbes and future research needs. Biotechnol. Adv. 33, 745–755. Chen, M.Y., Lee, D.J., Tay, J.H., 2007. Distribution of extracellular polymeric substances in aerobic granules. Appl. Microbiol. Biotechnol. 73, 1463–1469.

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14

201

Chen, X., Schauder, S., Potier, N., Van Dorsselaer, A., Pelczer, I., Bassler, B.L., Hughson, F.M., 2002. Structural identification of a bacterial quorum sensing signal containing boron. Nature 415, 545–549. Chong, G., Kimyon, O., Rice, S.A., Kjelleberg, S., Manefield, M., 2012. The presence and role of bacterial quorum sensing in activated sludge. Microb. Biotechnol. 5, 621–633. Chugani, S., Greenberg, E.P., 2010. Lux Rhomolog-independent gene regulation by acyl-homoserine lactones in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 107, 10673–10678. Combaret, P.C., Prensier, G., Le Thi, T.T., Vidal, O., Lejeune, P., Dorel, C., 2000. Developmental pathway for biofilm formation in curli-producing Escherichia coli strains: role of flagella, curli and colanic acid. Environ. Microbiol. 2 (4), 450–464. Compant, S., Clement, C., Sessitsch, A., 2010. Plant growth-promoting bacteria in the rhizo- and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biol. Biochem. 42, 669–678. Cong, Y., Xu, Q., Feng, H., Shen, D., 2013. Efficient electrochemically active biofilm denitrification and bacteria consortium analysis. Bioresour. Technol. 132, 24–27. Daniels, R., Reynaert, S., Hoekstra, H., Verreth, C., et al., 2006. Quorum signal molecules as biosurfactants affecting swarming in Rhizobium etli. Proc. Natl. Acad. Sci. U. S. A. Dasgupta, D., Ghosh, R., Sengupta, T.K., 2013. Biofilm-mediated enhanced crude oil degradation by newly isolated Pseudomonas species. ISRN Biotechnol. 1–13. Davey, M.E., O’Toole, G.A., 2000. Microbial biofilms: from ecology to molecular genetics. Microbiol. Mol. Biol. Rev. 64, 847–867. Decho, A.W., Visscher, P.T., Ferry, J., Kawaguchi, T., He, L.J., Przekop, K.M., Norman, R.S., Reid, R.P., 2009. Autoinducers extracted from microbial mats reveal a surprising diversity of N-acylhomoserine lactones (AHLs) and abundance changes that may relate to diel pH. Environ. Microbiol. 11, 409–420. Decho, A.W., Frey, R.L., Ferry, J.L., 2011. Chemical challenges to bacterial AHL signaling in the environment. Chem. Rev. 111, 86–99. Defoirdt, T., Boon, N., Bossier, P., Verstraete, W., 2004. Disruption of bacterial quorum sensing: an unexplored strategy to fight infections in aquaculture. Aquaculture 240, 69–88. Degrassi, G., Aguilar, C., Bosco, M., Zahariev, S., Pongor, S., Venturi, V., 2002. Plant growth-promoting Pseudomonas putida WCS358 produces and secretes four cyclic dipeptides: cross-talk with quorum sensing bacterial sensors. Curr. Microbiol. 45 (4), 250–254. Deng, Y., Wu, J., Eberl, L., Zhang, L.H., 2010. Structural and functional characterization of diffusible signal factor family quorum-sensing signals produced by members of the Burkholderia cepacia complex. Appl. Environ. Microbiol. 76, 4675–4683. Dobretsov, S., Dahms, H.U., Huang, Y., Wahl, M., Qian, P.Y., 2007. The effect of quorum-sensing blockers on the formation of marine microbial communities and larval attachment. FEMS Microbiol. Ecol. 60, 177–188. Dobretsov, S., Teplitski, M., Bayer, M., Gunasekera, S., Proksch, P., Paul, V.J., 2011. Inhibition of marine biofouling by bacterial quorum sensing inhibitors. Biofouling 27, 893–905. Drews, A., 2010. Membrane fouling in membrane bioreactors: characterisation, contradictions, cause and cures. J. Membr. Sci. 363, 1–28. Esparza-Soto, M., Westerhoff, P., 2003. Biosorption of humic and fulvic acids to live activated sludge biomass. Water Res. 37, 2301–2310. Ferris, F.G., Schultze, S., Witten, T.C., Fyfe, W.S., Beveridge, T.J., 1989. Metal interactions with microbial biofilms in acidic and neutral pH environments. Appl. Environ. Microbiol. 55, 1249–1257. Flavier, A.B., Clough, S.J., Schell, M.A., Denny, T.P., 1997. Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum. Mol. Microbiol. 26, 251–259. Gieg, L.M., Fowler, S.J., Berdugo-Clavijo, C., 2014. Syntrophic biodegradation of hydrocarbon contaminants. Curr. Opin. Biotechnol. 27, 21–29. Guezennec, A.G., Michel, C., Joulian, C., Dictor, M.C., Battaglia-Brunet, F., 2012. Treatment of arsenic contaminated mining water using biofilms. In: Interfaces Against Pollution. Nancy, France. Harayama, S., Kasai, Y., Hara, A., 2004. Microbial communities in oil-contaminated seawater. Curr. Opin. Biotechnol. 15, 205–214. Henke, J.M., Bassler, B.L., 2004. Three parallel quorum-sensing systems regulate gene expression in Vibrio harveyi. J. Bacteriol. 186, 6902–6914. Horemans, B., Breugelmans, P., Hofkens, J., Smolders, E., Springael, D., 2013. Environmental dissolved organic matter governs biofilm formation and subsequent linuron degradation activity of a linuron-degrading bacterial consortium. Appl. Environ. Microbiol. 79, 4534–4542. Houdt, R.V., Aertsen, A., Moons, P., Vanoirbeek, K., Michiels, C.W., 2006. N-acyl-homoserine lactone signal interception by Escherichia coli. FEMS Microbiol. Lett. 256, 83–89. Huang, Y.L., Dobretsov, S., Ki, J.S., Yang, L.H., Qian, P.Y., 2007. Presence of acyl-homoserine lactone in subtidal biofilm and the implication in larval behavioural response in the polychaete Hydroides elegans. Microb. Ecol. 54, 384–392. Huang, Y., Zeng, Y., Yu, Z., Zhang, J., Feng, H., Lin, X., 2013. In silico and experimental methods revealed highly diverse bacteria with quorum sensing and aromatics biodegradation systems—a potential broad application on bioremediation. Bioresour. Technol. 148, 311–316. Jiang, B., Liu, Y., 2012. Roles of ATP-dependent N-acylhomoserine lactones (AHLs) and extracellular polymeric substances (EPSs) in aerobic granulation. Chemosphere 88, 1058–1064. Johnsen, A.R., Karlson, U., 2004. Evaluation of bacterial strategies to promote the bioavailability of polycyclic aromatic hydrocarbons. Appl. Microbiol. Biotechnol. 63, 452–459. Jung, J.H., Choi, N.Y., Lee, S.Y., 2013. Biofilm formation and exopolysaccharide (EPS) production by Cronobacter sakazakii depending on environmental conditions. Food Microbiol. 34, 70–80. Kang, Y.S., Park, W., 2010. Contribution of quorum-sensing system to hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1. J. Appl. Microbiol. 109, 1650–1659.

202

New and future developments in microbial biotechnology and bioengineering

Kim, M., Lee, S., Park, H.D., Choi, S.I., Hong, S., 2012. Biofouling control by quorum sensing inhibition and its dependence on membrane surface. Water Sci. Technol. 66, 1424–1430. Kim, S.R., Oh, H.S., Jo, S.J., Yeon, K.M., Lee, C.H., Lim, D.J., Lee, C.H., Lee, J.K., 2013. Biofouling control with bead-entrapped quorum quenching bacteria in membrane bioreactors: physical and biological effects. Environ. Sci. Technol. 47, 836–842. Kreft, J.U., Wimpenny, J.W., 2001. Effect of EPS on biofilm structure and function as revealed by an individual-based model of biofilm growth. Water Sci. Technol. 43, 135–141. Lee, J., Wu, J., Deng, Y., Wang, J., Wang, C., Wang, J., Chang, C., Dong, Y., Williams, P., Zhang, L.H., 2013. A cell-cell communication signal integrates quorum sensing and stress response. Nat. Chem. Biol. 9 (5), 339–343. Li, Y., Hao, W., Lv, J., Wang, Y., Zhong, C., Zhu, J., 2014a. The role of N-acyl homoserine lactones in maintaining the stability of aerobic granules. Bioresour. Technol. 159, 305–310. Li, Y., Lv, J., Zhong, C., Hao, W., Wang, Y., Zhu, J., 2014b. Performance and role of N-acyl-homoserine lactone (AHL)-based quorum sensing (QS) in aerobic granules. J. Environ. Sci. 26, 615–1621. Li, Z., Zhang, X., Lei, L., 2008. Electricity production during the treatment of real electroplating wastewater containing Cr6+ using microbial fuel cell. Process Biochem. 43, 1352–1358. Liu, A.B., Ahn, I.S., Mansfield, C., Lion, L.W., Shuler, M.L., Ghiorse, W.C., 2000. Phenanthrene desorption from soil in the presence of bacterial extracellular polymer: observations and model predictions of dynamic behavior. Water Res. 35, 835–843. Logsdon, G.S., Kohne, R., Abel, S., LaBonde, S., 2002. Slow sand filtration for small water systems. J. Environ. Eng. Sci. 1, 339–348. Lv, J., Wang, Y., Zhong, C., Li, Y., Hao, W., Zhu, J., 2014. The effect of quorum sensing and extracellular proteins on the microbial attachment of aerobic granular activated sludge. Bioresour. Technol. 152, 53–58. Mah, T.F., O’Toole, G.A., 2001. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 9, 34–39. Mane, P.C., Bhosle, A.B., Kulkarni, P.A., 2011. Biosorption and biochemical study on water hyacinth (Eichhornia crassipes) with reference to selenium. Arch. Appl. Sci. Res. 3, 222–229. McSwain, B.S., Irvine, R.L., Hausner, M., Wilderer, P.A., 2005. Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge. Appl. Environ. Microbiol. 71, 1051–1057. Miller, M.B., Bassler, B.L., 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55, 165–199. Miqueleto, A.P., Dolosic, C.C., Pozzi, E., et al., 2010. Influence of carbon sources and C/N ratio on EPS production in anaerobic sequencing batch biofilm reactors for wastewater treatment. Bioresour. Technol. 101, 1324–1330. Mnif, I., Mnif, S., Sahnoun, R., Sameh, M., Younes, A., Ellouze-Chaabouni, S., Dhouha, G., 2015. Biodegradation of diesel oil by a novel microbial consortium: comparison between co-inoculation with biosurfactant-producing strain and exogenously added biosurfactants. Environ. Sci. Pollut. Res. Int. 22, 14852–14861. Morgan-Sagastume, F., Boon, N., Dobbelaere, S., Defoirdt, T., Verstraete, W., 2005. Production of acylated homoserine lactones by Aeromonas and Pseudomonas strains isolated from municipal activated sludge. Can. J. Microbiol. 51, 924–933. Mulcahy, H., Charron-Mazenod, L., Lewenza, S., 2010. Pseudomonas aeruginosa produces an extracellular deoxyribonuclease that is required for utilization of DNA as a nutrient source. Environ. Microbiol. 12, 1621–1629. Muyzer, G., Stams, A.J., 2008. The ecology and biotechnology of sulphate-reducing bacteria. Nat. Rev. Microbiol. 6, 441–454. Nielsen, P.H., Jahn, A., 1999. Extraction of EPS. In: Wingender, J., Neu, T.R., Flemming, H.C. (Eds.), Microbial Extracellular Polymeric Substances: Characterization, Structure and Function. Springer-Verlag, Berlin Heidelberg, pp. 49–72. Oh, H.S., Kim, S.R., Cheong, W.S., Lee, C.H., Lee, J.K., 2013. Biofouling inhibition in MBR by Rhodococcus sp. BH4 isolated from real MBR plant. Appl. Microbiol. Biotechnol. 97, 10223–10231. Pan, X.L., Liu, J., Zhang, D.Y., Chen, X., Song, W.J., Wu, F.C., 2010. Binding of dicamba to soluble and bound extracellular polymeric substances (EPS) from aerobic activated sludge: a fluorescence quenching study. J. Colloid Interface Sci. 345, 442–447. Papenfort, K., Bassler, B.L., 2016. Quorum sensing signal–response systems in Gram-negative bacteria. Nat. Rev. Microbiol. 14, 576–588. Plosz, B.G., Vogelsang, C., Macrae, K., Harald, H.H., Antonio Lopez, H.L., Katherine, H.L., 2010. The BIOZO process—a biofilm system combined with ozonation: occurrence of xenobiotic organic micro-pollutants in and removal of polycyclic aromatic hydrocarbons and nitrogen from landfill leachate. Water Sci. Technol. 61, 3188–3197. Ponnusamy, K., Kappachery, S., Thekeettle, M., Song, J., Kweon, J., 2013. Anti-biofouling property of vanillin on Aeromonas hydrophila initial biofilm on various membrane surfaces. World J. Microbiol. Biotechnol. 29, 1695–1703. Ponnusamy, K., Paul, D., Kweon, J.H., 2009. Inhibition of quorum sensing mechanism and Aeromonas hydrophila biofilm formation by vanillin. Environ. Eng. Sci. 26, 1359–1363. Ponnusamy, K., Paul, D., Kim, Y.S., Kweon, J.H., 2010. 2(5H)-Furanone: a prospective strategy for biofouling-control in membrane biofilm bacteria by quorum sensing inhibition. Braz. J. Microbiol. 41, 227–234. Pool, J.R., Kruse, N.A., Vis, M.L., 2013. Assessment of mine drainage remediated streams using diatom assemblages and biofilm enzyme activities. Hydrobiologia 709 (1), 101–116. Poonguzhali, S., Madhaiyan, M., Sa, T., 2007. Quorum-sensing signals produced by plant-growth promoting Burkolderia strains under in vitro and in planta conditions. Res. Microbial. 158, 287–294. Prakash, B., Veeregowda, B.M., Krishnappa, G., 2003. Biofilms: a survival strategy of bacteria. Curr. Sci. 85 (9), 1299–1307. Pratt, L.A., Kolter, R., 1999. Genetic analyses of bacterial biofilm formation. Curr. Opin. Microbiol. 2, 598–603. Reinhold-Hurek, B., Hurek, T., 2011. Living in side plants: bacterial endophytes. Curr. Opin. Plant Biol. 14, 435–443.

Biofilm-mediated bioremediation of pollutants Chapter

14

203

Ren, T., Yu, H., Li, X., 2010. The quorum-sensing effect of aerobic granules on bacterial adhesion, biofilm formation, and sludge granulation. Appl. Microbiol. Biotechnol. 88, 789–797. Ren, T.T., Li, X.Y., Yu, H.Q., 2013. Effect of N-acy-L-homoserine lactones-like molecules from aerobic granules on biofilm formation by Escherichia coli K12. Bioresour. Technol. 129, 655–658. Rodriguez, S., Bishop, P., 2008. Enhancing the biodegradation of polycyclic aromatic hydrocarbons: effects of nonionic surfactant addition on biofilm function and structure. J. Environ. Eng. 134, 505–512. Ron, E.Z., Rosenberg, E., 2014. Enhanced bioremediation of oil spills in the sea. Curr. Opin. Biotechnol. 27, 191–194. Schaefer, A.L., et al., 2008. A new class of homoserine lactone quorum-sensing signals. Nature 454, 595–599. Sheng, G.P., Zhang, M.L., Yu, H.Q., 2008. Characterization of adsorption properties of extracellular polymeric substances (EPS) extracted from sludge. Colloids Surf. B Biointerfaces 62, 83–90. Sheng, G.-P., Yu, H.-Q., Li, X.-Y., 2010. Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review. Biotechnol. Adv. 28, 882–894. Shukla, S.K., Mangwani, N., Rao, T.S., et al., 2014. Biofilm-mediated bioremediation of polycyclic aromatic hydrocarbons. In: Das, S. (Ed.), Microbial Biodegradation and Bioremediation. Elsevier, Oxford, pp. 203–232. Singh, R., Paul, D., Jain, R.K., 2006. Biofilms: implications in bioremediation. Trends Microbiol. 14, 389–397. Smith, W.L., Gadd, G.M., 2000. Reduction and precipitation of chromate by mixed culture sulphate-reducing bacterial biofilms. J. Appl. Microbiol. 88, 983–991. Song, X.N., Cheng, Y.Y., Li, W.W., Li, B.B., Sheng, G.P., Fang, C.Y., Wang, Y.K., Li, X.Y., Yu, H.Q., 2014. Quorum quenching is responsible for the underestimated quorum sensing effects in biological wastewater treatment reactors. Bioresour. Technol. 171, 472–476. Sperandio, V., Torres, A.G., Giron, J.A., Kaper, J.B., 2001. Quorum sensing is a global regulatory mechanism in entero hemorrhagic Escherichia coli O157:H7. J. Bacteriol. 183, 5187–5197. Steyn, B., Oosthuizen, M.C., MacDonald, R., Theron, J., Brozel, V.S., 2001. The use of glass wool as an attachment surface for studying phenotypic changes in Pseudomonas aeruginosa biofilms by two-dimensional gel electrophoresis. Proteomics 1 (7), 871–879. Tan, C.H., Kai, S.K., Chao, X., Martin, T., Yan, Z., Rohan, W., Wun, J.N., Scott, A.R., Staffan, K., 2014. The role of quorum sensing signalling in EPS production and the assembly of a sludge community into aerobic granules. ISME J. 8, 1186–1197. Vu, B., Miao, C., Russell, J., Crawford, E.P.I., 2009. Bacterial extracellular polysaccharides involved in biofilm formation. Molecules 14, 2535–2554. Wang, Z.W., Liu, Y., Tay, J.H., 2005. Distribution of EPS and cell surface hydrophobicity in aerobic granules. Appl. Microbiol. Biotechnol. 69, 469–473. Williams, K.H., Wilkins, M.J., N’Guessan, A.L., Arey, B., Dodova, E., Dohnalkova, A., Holmes, D., Lovley, D.R., Long, P.E., 2013. Field evidence of selenium bioreduction in a uranium-contaminated aquifer. Environ. Microbiol. Rep. 5, 444–452. Xu, H., Liu, Y., 2011. Reduced microbial attachment by D-amino acid-inhibited AI-2 and EPS production. Water Res. 45, 5796–5804. Yang, S.F., Liu, Q.S., Tay, J.H., Liu, Y., 2004. Growth kinetics of aerobic granules developed in sequencing batch reactors. Lett. Appl. Microbiol. 38, 106–112. Yeon, K.M., Lee, C.H., Kim, J., 2009. Magnetic enzyme carrier for effective biofouling control in the membrane bioreactor based on enzymatic quorum quenching. Environ. Sci. Technol. 43, 7403–7409. Yong, Y.C., Zhong, J.J., 2013a. Impacts of quorum sensing on microbial metabolism and human health. In: Zhong, J.J. (Ed.), Future Trends in Biotechnology.pp. 25–61. Yong, Y.C., Zhong, J.J., 2013b. Regulation of aromatics biodegradation by rhl quorum sensing system through induction of catechol meta-cleavage pathway. Bioresour. Technol. 136, 761–765. Yuncu, B., Sanin, F.D., Yetis, U., 2006. An investigation of heavy metal biosorption in relation to C/N ratio of activated sludge. J. Hazard. Mater. 137, 990–997. Zhang, L.H., Dong, Y.H., 2004. Quorum sensing and signal interference: diverse implications. Mol. Microbiol. 53, 1563–1571. Zhang, S.H., Yu, X., Guo, F., Wu, Z.Y., 2011. Effect of interspecies quorum sensing on the formation of aerobic granular sludge. Water Sci. Technol. 64, 1284–1290. Zhang, K., Zheng, X., Shen, D.S., Wang, M.Z., Feng, H.J., He, H.Z., et al., 2015. Evidence for existence of quorum sensing in a bioaugmented system by acylated homoserine lactone-dependent quorum quenching. Environ. Sci. Pollut. Res. Int. 22, 6050–6056.

Further reading Chan, D.I., Vogel, H.J., 2010. Current understanding of fatty acid biosynthesis and the acyl carrier protein. Biochem. J. 430, 1–19. Oh, H.S., Yeon, K.M., Yang, C.S., Kim, S.R., Lee, C.H., Park, S.Y., Han, J.Y., Lee, J.K., 2012. Control of membrane biofouling in MBR for wastewater treatment by quorum quenching bacteria encapsulated in microporous membrane. Environ. Sci. Technol. 46, 4877–4884. Waters, C.M., Bassler, B.L., 2005. Quorum sensing: cell-to-cell communication in bacteria. Annu. Rev. Cell Dev. Biol. 21, 319–346.