Microbial Electroactive Biofilms - American Chemical Society

Chapter 8

Microbial Electroactive Biofilms

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Rashmi Kiran and Sunil A. Patil* Department of Earth and Environmental Sciences, Indian Institute of Science Education and Research Mohali, Sector 81, S. A. S. Nagar, Manauli PO 140306, Punjab, India *E-mail: [email protected].

Microbial metabolism coupled with extracellular electron transfer (EET) plays a crucial role in redox cycling of major elements in natural environments. The EET capabilities of microorganisms are exploited in bioelectrochemical systems to drive transfer of electrons to and from electrodes for electricity generation, bioremediation, biosensing, and biocatalysis applications. The microorganisms that can use the electrodes to achieve their respiratory or metabolic processes via EET are commonly referred to as electrochemically active or electroactive microorganisms. Several microbes have evolved to perform EET via direct and indirect mechanisms. In the case of the direct electron transfer mechanism, physical contact between microorganisms and electrodes is necessary. The irreversible attachment of electroactive microorganisms to the electrode surface eventually leads to the growth and development of biofilms commonly referred to as “electroactive biofilms” (EAB). The formation and functioning of EAB are critical to the performance of different types of microbial electrochemical technologies such as microbial fuel cells and microbial electrolysis cells. This chapter first describes the electroactive microorganisms that have been reported to form biofilms on the anode and cathode surfaces. The electron transfer mechanisms between the EAB and electrode are then discussed. It is followed by a brief overview of the major tools and techniques that are used to study the formation and functioning of EAB as well as the electron transfer mechanisms at the biofilm–electrode interface. Finally, the main application areas and future research prospects of EAB are presented.

© 2019 American Chemical Society

Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

Introduction Microbial metabolism involves a network of a series of oxidative and reductive reactions, which are coupled to intracellular and extracellular electron transfer (EET) from electron donors to electron acceptors to sustain the energy requirement of cells (1). Cellular respiration comprises a cascade of reactions that are linked through a system of electron carrier proteins with different reduction potentials leading to electron transfer to the terminal electron acceptor. Microorganisms can use soluble compounds (e.g., O2, nitrate, and sulfate) as the terminal electron acceptors. Some microorganisms can also respire on solid-state electron acceptors through EET (2). Such reactions play a key role in natural mineral redox cycles and can link the microbial cellular respiration to the environment. The microbial EET is important in a variety of biogeochemical cycles and can be exploited for the bioremediation of organic and inorganic contaminants. In anaerobic environments where soluble electron acceptors are absent, Fe(III) and Mn(IV) are the most important non-soluble electron acceptors for microorganisms (3, 4). A diversity of microorganisms can perform electron transfer to iron minerals via dissimilatory metal reduction in different types of environments (5). In dissimilatory metal reduction, Fe(III) and Mn(IV) act as external electron acceptors (3). For example, Geobacter is a strict anaerobe and predominates iron-reducing populations in a wide range of subsurface environments (5, 6). Shewanella is a facultative anaerobe and thrives at redox interfaces (1). These microorganisms can couple cellular respiration and growth to the reduction of metals such as Fe(III) and Mn(IV) (3, 7). Desulfuromonas acetoxidans, a marine microorganism, is known to grow anaerobically by oxidizing acetate with concomitant reduction of elemental sulfur and Fe(III) (3). A few other microbes such as Thiobacillus thiooxidans, Thiobacillus ferrooxidans, and Sulfolobus acidocaldarius can use elemental sulfur (S°) as the electron donor to reduce Fe(III) to Fe(II) (8). Organisms belonging to the γ subclass of proteobacteria such as Shewanella putrefaciens, Shewanella alga, and Ferrimonas balearica can generate energy for growth from the reduction of Fe(III) with H2 or organic acids serving as the electron donor (3). Those microorganisms that can transfer electrons to the extracellular insoluble or solid-state electron acceptors or accept electrons from the extracellular electron donors can be defined as electrochemically active or electroactive microorganisms (9). These microorganisms are of particular interest to the field of microbial electrochemistry and technology (MET). METs are emerging technologies that use microorganism–electrode interactions for electricity production, wastewater treatment, bioremediation, and production of high-value- reduced products (9, 10). Bioelectrochemical systems (BES) are unique multidimensional systems that are used to accomplish different MET applications (Figure 1). A BES system commonly consists of a dual chamber setup in which an ion exchange membrane separates the cathode and anode electrodes and thus individual electrode reactions. Electrodes are surrounded by an electrolyte, which can be an aqueous solution (9, 11). The underlying driver of anodic and cathodic reactions in BES is the EET performed by microorganisms. Microbial fuel cell (MFC) is the most extensively studied BES. In MFC, the microbial oxidation of organic substrate occurs in the anode chamber. It leads to the production of electrons, protons, and carbon dioxide. Protons migrate to the cathode side through an ion exchange membrane, which is placed between the cathode and anode electrodes. The electrons are transferred to the anode, which acts as a terminal electron acceptor for microbes. These electrons then flow to the cathode through the external circuit, and electricity can be harvested. At the cathode, oxygen is the most commonly used terminal electron acceptor that is reduced to the water. Microbial electrolysis 160 Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

cell (MEC) is the second-most studied BES and is used to produce or synthesize reduced products such as hydrogen or organic chemicals at the cathode with an additional input of electrical energy (12, 13). Another type of BES is microbial desalination cell (, in which oxygen acts as the electron acceptor at the cathode to achieve wastewater treatment coupled with water desalination at low energy consumption (14). Microbial electrosynthesis (MES), an emerging MET, offers the unique possibility of effective metabolic reduction of CO2 to synthesize multicarbon organic compounds (11, 15). All these processes are based on the EET capabilities of electroactive microorganisms.

Figure 1. Schematic of a typical bioelectrochemical system showing the EAB at the anode and cathode surfaces.

Electroactive Microorganisms and Biofilms Microorganisms have evolved different strategies to transfer electrons in and out of their cells (16). The first and foremost reason for EET is cellular respiration, which is achieved with solid-state metal oxides such as Fe oxides. During respiration, electrons are released from a terminal oxidase in the respiratory chain to Fe(III) outside the cell to produce Fe(II) (1). The second reason is the syntropy in which one microbe can transfer electrons directly to another microbe without the need for intermediates such as H2. For example, fermentative bacterium Pelotomaculum thermopropionicum has been observed to be linked to the methanogen Methanothermobacter thermautotrophicus through electrically conductive appendages. It was the first direct evidence of interspecies electron transfer (17). A third possible reason for EET can be electron transfer for cell–cell communication. Opportunistic pathogens such as Pseudomonas aeruginosa produce pyocyanins that promote upregulation of quorum-sensing controlled genes. They can also act as electron shuttles to allow electrical current generation in MFCs (8). 161 Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

In natural environments, there is a great diversity of electroactive microorganisms. Such bacteria can grow in suspension as planktonic cells, and they can also form a complex community structure by attaching to a solid electrode surface, forming electroactive biofilms (EAB) (Figure 1) (18). In general, biofilm formation is an effective strategy that microbes adapt for their survival in different environmental stress conditions such as lack of substrate, the presence of antimicrobial substances, and changes in optimal pH and temperature conditions (19). The key reason for the formation of EAB on electrode surfaces is the use of electrodes either as electron acceptors or donors by microbes for sustaining their respiratory or metabolic processes. A wide variety of extracellular polymeric substances (EPS) are secreted by microbial consortia embedded in biofilms. EPS form the threedimensional structure of biofilms and are responsible for cell adhesion to solid electrode surfaces. A wide range of molecular interactions occurs in the EPS matrix. Microbial communities in biofilms exhibit novel properties as compared with planktonic cells. Biofilm cells are immobilized and remain intact due to the action of EPS. They allow intense interactions and efficient electron transfer between the microbial cells, leading to the formation of synergistic microbial consortia. Many microorganisms have been reported to be electroactive but not characterized in detail for their electroactivity. Those microbes that use the anode electrode as the terminal electron acceptor are commonly referred to as exoelectrogens. The term “electrotrophs” is increasingly being used for those microbes that can take up electrons from solid-state donors such as the cathode electrode. The best-characterized electroactive microorganisms belong to Geobacter and Shewanella genera (Tables 1 and 2). These microbial groups have been studied by various research groups to understand the EET mechanisms. Geobacter sulfurreducens strain PCA and KN400 and Shewanella oneidensis strain MR-1 are used as the model organisms to study microbial electrochemistry (20). These model organisms differ in their electron transfer mechanisms and have efficient anodic and cathodic electron transfer capabilities. Table 1. Most Notable or Well-Studied Anodic Electroactive Microorganisms S. No.

Name

Inoculum source

Reaction catalyzed

Reference

1

Geobacter sulfurreducens

Pure culture

Acetate oxidation

(21, 22)

2

Geobacter sulfurreducens PCA

Pure culture

Lactate oxidation

(23)

3

Geobacter metallireducens

Pure culture

Acetate oxidation

(24)

4

Geobacter anodireducens

Pure culture

Acetate oxidation

(25)

5

Geoalkalibacter subterraneus

Pure culture

Acetate oxidation

(26)

6

Shewanella oneidensis MR-1

Pure culture

Lactate oxidation

(28)

7

Shewanella putrifaciens

Pure culture

Lactate oxidation

(29)

8

Pseudomonas aeruginosa

Pure culture

Glucose oxidation

(30)

9

Thermincola potens

Pure culture

Acetate oxidation

(31)

10

Thermincola ferriacetica

Pure culture

Acetate oxidation

(32)

11

Shewanella loihica PV-4

Pure culture

Lactate oxidation

(33)

12

Rhodopseudomonas palustris DX-1

Strain isolated from MFC

Acetate oxidation

(34)

162 Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

Table 2. Most Notable or Well-Studied Cathodic Electroactive Microorganisms S. No.

Name

Inoculum source

Reaction catalyzed

Reference

1

Geobacter sulfurreducens

Pure culture

Fumarate to succinate reduction, U(VI) to U(IV) reduction

(24, 35)

2

Geobacter metallireducens

Pure culture

Nitrate to nitrite reduction

(24)

3

Geobacter lovleyi

Pure culture

Dechlorination (tetrachloroethene (PCE) (36) to cis-dichloroethane)

4

Acidithiobacillus ferrooxidans

Pure culture

Oxygen reduction

(37)

5

Pseudomonas alcaliphila

Pure culture

Nitrate reduction

(38)

6

Methanobacterium palustre Pure culture

Hydrogenotrophic methanogenesis

(39)

7

Desulfovibrio desulfuricans Pure culture

Sulphate reduction

(40)

8

Methanococcus maripaludis

Pure culture

Electromethanogensis

(41)

9

Sporomusa ovata

Pure culture

CO2 reduction

(42)

10

Sporomusa sphaeroides

Pure culture

CO2 reduction

(42)

11

Shewanella putrefaciens IR-1

Pure culture

Oxygen reduction

(43)

12

Shewanella oneidensis MR-1

Pure culture

Oxygen & fumarate reduction

(44)

13

Shewanella oneidensis DSP10

Pure culture

Oxygen reduction

(27)

14

Moorella thermoacetica

Pure culture

CO2 reduction

(42)

15

Thiobacillus denitrificans

Pure culture

O2, nitrate reduction

(45)

16

Clostridium ljungdahlii

Pure culture

CO2 reduction

(42)

17

Clostridium pasteurianum Pure culture

Glucose fermentation

(46)

Anodic Electroactive Biofilms Anodic EABs are the most well-studied, as they are common to most BES. The basic requirement is the establishment of direct electron transfer (DET) to the anode from the microbial cell membrane. The use of anode as the electron acceptor plays a major role in the selection of electroactive microorganisms community structure in EAB (47, 48). The procedure of anodic EAB formation is very straightforward. An electrode material (e.g., carbon-based) is placed into an anoxic substrate or growth medium. It is inoculated with a sewage or sludge or sediment sample. The electrode is polarized at a slightly positive potential (e.g., 0.2 vs Ag/AgCl reference electrode when acetate is used as the substrate) to facilitate the electrochemical selection and enrichment of electroactive microorganisms or operated without any applied potential (49). An EAB consisting of layers of microbial cells that can perform EET using the anode as the terminal electron acceptor is then developed. 163 Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

Two commonly found and well-studied exoelectrogens that form anodic EAB belong to Geobacter and Shewanella spp. Members of Geobacteriaceae family are ubiquitous Gram-negative, strictly anaerobic bacteria that are found mainly in iron-rich sedimentary environments. The majority of microbes on the anode of the MFCs harvesting electricity from aqueous sediments and acetate or complex waste substrates belong to the Geobacteriaceae family (6, 21–25). Geobacter sulfurreducens is a well-known exoelectrogen that forms anodic EAB. This bacterium grows in freshwater environments and can oxidize acetate when cultured at the anode side. It forms uniform thick biofilms on the carbon anodes (21). Geobacter metallireducens has also been reported in MFCs (24). This bacterium can perform oxidation of a variety of aromatic contaminants along with the reduction of Fe(III). Most of the microorganisms in anodic EAB that were grown in marine sediments belonged to the family of Geobacteriaceae and showed 95% similarity to D. acetoxidans (50). Bacteria from Geobacteriaceae family can perform anaerobic degradation of sedimentary organic carbon by directly transferring electrons to graphite anodes without any requirement of electron transfer mediators (50). A variety of outer membrane multiheme c-type cytochromes (OMCs) and conductive filamentous appendages referred to as nanowires are involved in EET in Geobacter-based EABs (51). Electrochemical techniques are routinely used to study the electrocatalytic activity of Geobacter biofilms (51, 52). Members of Shewanellaceae family are facultative anaerobes, and several species of this family such as S. oneidensis MR-1, S. oneidensis MR-4, Shewanella putrefacians, S. putrefacians IR-1, and Shewanella loihica PV-4 have been reported to form anodic EABs. These bacteria have the unique property of performing both direct and mediated or indirect electron transfer (IET). IET is performed using redox shuttles, which include melanin, humic substances, menaquinone, riboflavin, flavin mononucleotides, and their derivatives. Particularly, riboflavin molecules secreted by S. oneidensis MR-1 have demonstrated electrochemical activity at the biofilm-electrode surface. They can also act as a cofactor for the outer membrane cytochrome omcA (53). It has also been reported that nanowires produced by S. oneidensis MR-1 exhibit nonlinear electrical transport property along their length (20). Compared with the pure cultures, mixed microbial cultures obtained from wastewater or sludge have been reported to produce greater power densities and higher coulombic efficiency in MFCs (21). That can be attributed to the flexibility of biofilm toward changes in external factors such as medium composition and operational conditions. Symbiotic interactions in a mixed culture allow microbes to utilize a wide range of substrates and metabolic pathways, allowing complete oxidation of organic components (16). The mixed microbial communities colonizing the anode are complex. Microbes such as P. aeruginosa and Bacillus tequilensis were found to exhibit low electrochemical performance when used as a pure culture in MFCs. However, when they were grown as a coculture, a substantial increase in the power output was observed (39, 54). The majority of research groups focusing on applied aspects use mixed microbial culture enriched from natural sediments, soil, primary wastewater, or sewage sludge in MFCs. Such cultures can efficiently metabolize a wide range of substrates from simple organic acids to complex carbohydrates like starch and cellulose. Although the electrochemical selection procedure can be applied to enrich electroactive microorganisms (55), nonelectrogenic microorganisms may attach to the anode surface and drastically diminish biofilm electrical conductivity (56) with the mixed culture inoculum sources. Furthermore, although the presence of different bacteria has been found to increase the metabolic capacity of biofilm, that does not necessarily mean that more diverse biofilms will be more conductive and will have more electrical output. For instance, a defined co-culture of Escherichia coli and G. sulfurreducens produced less power as compared with the system in which the pure culture 164 Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

of G. sulfurreducens was used (57). Defined co-cultures have also been demonstrated to enhance electrical output. A co-culture of G. sulfurreducens, P. aeruginosa, and S. oneidensis were found to produce more power output when grown in co-culture with Gram-positive Enterococcus faecium as compared with the pure culture biofilms of these bacteria. However, when these three exoelectrogens were grown in paired co-cultures with Clostridium acetobutylicum, the resulting power produced was less than that of each pure culture (58). Within MFC reactors, a complex metabolic interplay occurs between fermentative and non-fermentative microbes. A pure culture of S. oneidensis when fed with lactate performed nearly identically in terms of coulombic efficiency and current production as compared with the co-culture of S. oneidensis and Lactococcus lactis fed with glucose. S. oneidensis cannot utilize glucose on its own but can utilize lactate. It was shown that S. oneidensis consumed the lactate that was formed by L. lactis by fermenting glucose (59). Such defined co-culture experiments have made it clear that some co-cultures can enhance current production as compared with pure cultures. However, mutualistic syntrophic interactions can even have a negative impact due to competition or inhibitory effects. Such unique characteristics brought on by each defined co-culture interaction still need to be studied in detail to be able to predict unidentified interspecies interactions in a better way. Selection of a suitable bacterial consortium along with the type of electrode material determines the rate of enrichment of EAB. The porous and fibrous nature along with conductivity and biocompatibility of the electrode materials play a critical role in EAB formation (60). Improvement of anode materials by physical surface modification and chemical catalysts can allow thicker and more efficient EAB formation. In general, the electrode material should have good conductivity, excellent longevity, and good biocompatibility, and it should be cost-effective. Carbon materials that are commonly used include carbon paper, glassy carbon, carbon cloth, graphite plate, granule graphite, carbon mesh, and graphite brush (61). Effective surface modification methods include treatment with chemical or physical methods, immobilization of electrocatalytically active and conductive polymers, and deposition of various metals (62). Varying the anode size and anode material can greatly affect biofilm thickness and conductivity (21). Also, electrode potential, medium conditions such as pH, and operational conditions such as temperature can affect the biofilm growth and development (63, 64). The potential difference between the electron donor and acceptor determines the amount of energy that will be obtained by bacteria and also determines the efficiency of the process. In an MFC system, the anode potential can be varied over a wide range to enable efficient electron transfer from bacteria to electrodes. Bacteria in mixed culture community utilize several metabolic pathways, which is an advantage for the entire community to be able to maximize the energy efficiency of the MFC system by adapting to the applied anode potential (22). The applied electrode potential has been reported to determine the bacterial composition and activity of the anodic biofilms (65). The overall efficiency and current production depend on the type of microbes dominating the biofilm layer on the anode. The thickness of anodic EABs varies in a wide range from a few micrometers to over 100 micrometers because of various factors (66). Cathodic Electroactive Biofilms The cathode can provide an endless supply of electrons to the electrotrophic microbes. These microorganisms can accept electrons from the cathode for the reduction of terminal electron acceptors such as CO2, nitrate, sulfate, metal ions, chlorinated compounds, organic acids, protons, and O2. Only a few pure cultures have been found to use Fe(II) as an electron donor, and the 165 Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

mechanisms of electron transfer have not been intensively studied. Several microorganisms can catalyze various reactions at different redox potentials. This ability of electrotrophic microorganisms can be used for a wide range of applications. These include, for example, O2 reduction in MFCs, and hydrogen production, metal reduction and recovery, nitrate and sulfate reduction, and synthesis of reduced multicarbon compounds in MECs. The promising application is in the field of microbial electrosynthesis (MES), in which CO2 and H2O are converted to multicarbon compounds that are released extracellularly (67). Although documented in several research papers, cathodic EABs are not well-studied as compared with their counterpart anodic EABs. Biological cathodes are an inexpensive and sustainable alternative for chemical cathodes. For instance, cathodic EABs have been reported to be used as catalysts for O2 reduction in MFCs (68). Many electrotrophs have been reported to perform O2 reduction to water at the cathodes of MFCs. A few examples are P. aeruginosa, P. fluorescens, S. putrefaciens, Shigella flexneri, E. coli, Kingella denitrificans, Enterobacter cloacae, Micrococcus luteus, Moraxella catarrhalis, Bacillus subtilis, Burkholderia cepacia, Brevundimonas diminuta, and various species of Acinetobacter and Sphingobacterium (69). Nitrate and sulfate-reducing bacteria (SRB) are anaerobic bacteria that use nitrate and sulfate or sulfur, respectively, as the terminal electron acceptor and can also form cathodic EABs. In nature, they are found in diverse anaerobic environments such as soils and marine sediments (70). SRB have been reported to remediate sulfate compounds. Anaerobic hyperthermophile sulfate-respiring bacteria such as Archeoglobus fulgidus have also been reported to possess respiratory flexibility (71). A pure culture of Desulfovibrio desulfuricans ATCC27774 has been found to be capable of forming EAB at the cathode. Biofilm electroactivity is correlated with the enzymatic activity within the matrix that is immobilized. Peroxidases and catalase and superoxide dismutase enzymes can also catalyze redox reactions that involve oxygenated species to allow SRB bacteria to form natural biofilms even in oxygenated environments. The proposed mechanism is the involvement of H2 intermediates and hydrogenase enzymes. D. desulfuricans can directly perform the exchange of electrons with different electrode materials such as stainless steel and graphite (40). In soils, nitrate reduction has not been found to occur at a high rate, because of the limited supply of electron donors (72). With the cathode electrodes embedded into the soil, the growth of nitratereducing bacteria can be enhanced (73). Metagenomic analysis of nitrate-reducing biofilms revealed that these biofilms were comprised of Thiobacillus sp. in one study and Clostridium and Nitrosomonas spp. in another study (74–76). Thiobacillus has been previously implicated as a potential electrotroph due to its ability to reduce nitrate by using Fe(II) as an electron donor. It can perform interspecies electron transfer with G. sulfurreducens through conductive magnetite nanoparticles (77). Nitrate reduction through electrolytic reduction has been extensively studied. G. metallireducens has been found to reduce nitrite to nitrate with electrons derived from the cathode (73). Other than nitrate, Geobacter species at the cathode are known to uptake electrons to reduce a wide range of electron acceptors such as fumarate, U(VI), and chlorinated compounds as well (78). Shewanella spp. are facultative anaerobes, which have a highly adaptive metabolism that allows them to survive in diverse environments. Because of diverse respiratory capabilities, they can reduce different organic and inorganic compounds. They are of interest for researchers because of their possible use in bioremediation (79). For example, S. putrefacians has been reported to perform bioremediation of uranium-VI to uranium-IV (80). Methanogens are the methane-producing bacteria that can perform electron transfer directly or indirectly using H2-mediated channels (39). The same is also applicable to the acetogenic 166 Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

microorganisms that produce acetate. Respiratory process in methanogens is coupled with the generation of an H+ or Na+ electrochemical gradient. They utilize CO2 as electron acceptors. H2 acts as an electron donor in the methanogenesis process, which occurs under strictly anaerobic conditions. Methanococcus janaschii and Methanobacterium thermoacetotrophicum (71) have been reported to produce CH4 at the cathode via the electromethanogenesis process. Mixed cultures that are dominated with Methanobacterium palustre and pure cultures of Methanococcus maripaludis have also been shown to perform electromethanogenesis (39). Acetogenic bacteria such as Sporomusa ovata, S. silvacetica, Sporomusa sphaeroides, Clostridium ljungdahlii, and Morella thermoacetica have been reported to accept electrons directly from the cathode to reduce CO2 to form acetate in an MES process (42). These acetogens and others such as Acetobacterium spp. in mixed culture cathodic EABs have been reported to use electrochemically produced H2 as an energy source to fix CO2 (81, 82). Mixed culture biofilms that are established at the anode can also draw electrons from the cathode (13, 39). For example, Gram-positive bacteria such as Clostridium, some species of Archaea, and microalgae have been reported to be able to transfer electrons to the anode or take electrons from the cathode. Clostridium spp. can be used at the anode in MFCs to produce current (39) as well as to derive electrons from the cathode to produce acetate from CO2 in MES systems. Under anaerobic conditions, cathodic biofilms are more difficult to grow as compared with the anodic biofilms. That means the formation of thinner biofilms than that of the anodic EABs (83). For example, a cathodic biofilm of Geobacter lovleyi visualized by confocal laser microscopy was found to be thinly distributed on the electrode surface (84). Cathodic EABs require more input of energy (i.e., application of more negative potential at the cathode electrode) (85).

Electron Transfer Mechanisms in Electroactive Biofilms As elaborated in the following sections, the electron transfer between EAB and electrodes can occur via outer membrane proteins or enzymes, mediators, and other conductive structures. Electron transfer between microorganisms within the cathodic and anodic biofilms also can occur through intraspecies or interspecies interactions. Mechanisms of Electron Transfer in Anodic EABs Even though many microorganisms have been reported to be capable of transferring electrons to an anode, in most of these bacteria, the electron transfer mechanisms are still unknown. A wide range of mixed microbial cultures enriched from marine sediments, wastewater, and anaerobic sludge have been reported to be capable of transferring electrons to the anode (21). Enrichment conditions determine the type of microorganisms that will grow on the anode surface. Geobacter and Shewanella sp. have been widely studied to understand EET mechanisms (86). Microbial communities at anodes are frequently dominated by Geobacter species, highly related to G. sulfurreducens (42). G. sulfurreducens can form a thick biofilm on the anode, and throughout the biofilm, cells can be metabolically active (87). The architecture of the biofilm matrix not only comprises bacterial cells but also has pockets and channels that are filled with nutrients, exopolysaccharides, and waste products from the metabolism of cells. These systems are heterogeneous and comprise various niches with varying proton gradients, metabolic activities, and cell viability (88). Microorganisms can transfer electrons to the anode through direct electron transfer (DET) and indirect electron transfer (IET) mechanisms. 167 Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

For DET, Geobacter and Shewanella are the most extensively studied microorganisms (Figure 2). These are metal (Fe-III)-respiring bacteria and are best-known to perform DET. For DET, a physical contact between the bacterial cell and electrode is required. The anode can act as an inexhaustible electron acceptor on which Geobacter can form persistent biofilms. In MFCs, current production has been shown to be linear with the increase in biomass and biofilm thickness to a certain extent at the anode. It suggests that not only the cells that are near the anode contribute to current production but also the cells that are at distance from the anode surface through long-distance electron transport (89). DET is not limited just to the electrode surface. Microorganisms can thus perform transfer of electrons to the anode either via long-range EET or via short-range EET. DET in combination with high cell surface density enables efficient electron transfer and also allows Geobacter to achieve higher anodic current densities than any other microbes (22). Microorganisms require electron transport proteins that can transfer electrons from the inside to the outside of the cell. C-type cytochromes are the key components that require adherence of the bacterial cell to the anode surface. C-type cytochromes are multiheme proteins that are part of a bacterial membrane, which mediates electron transport in many organisms and are present in metal-reducing bacteria (90).

Figure 2. The proposed Mtr and Pcc EET pathways. In the metal-reducing (Mtr) pathway of Shewanella oneidensis MR-1 (a) and the porin–cytochrome (Pcc) pathways of Geobacter sulfurreducens (b), electrons are transferred from quinol (QH2) in the cytoplasmic membrane, through the periplasm, and across the outer membrane to the bacterial surface, where MtrC transfers electrons to surface iron atoms directly through its solvent-exposed heme iron atom (inset of a; brown sphere). For simplicity, OmcA on the bacterial surface and flavins are not shown in a. Reproduced with permission from ref (86)). Copyright Macmillan Publishers Limited, part of Springer Nature 2016 Nature Reviews Microbiology. The genome of G. sulfurreducens can encode for more than 100 c-type cytochromes, including OmcB, OmcE, OmcS, and OmcZ. Among them, a crucial role is played by OmcZ, which is located in the outer membrane of the bacteria (20). A series of periplasmic and outer membrane cytochromes are responsible for EET (Figure 2b) (16, 91). Another model organism, S. oneidensis MR-1, can perform both DET as well as IET (91). It was among the first to be identified as a microorganism that can use minerals such as Fe(III), Mn(III), or Mn(IV) as terminal electron acceptors (92). S. oneidensis mutants that are deficient in genes for c-type deca heme cytochromes, MtrC, OmcA, and also for type II secretion pathway have been reported to display poorly conductive 168 Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

nanowires (93). S. oneidensis can form thin biofilms on anodes and also can grow as planktonic cells in MFCs (28). In the case of Shewanella, MtrA, MtrB, and MtrC encode periplasmic deca heme ctype cytochromes, which can be involved in Fe(III) and Mn(IV) as well as in electrode reduction (Figure 2a) (94). In similar conditions, G. sulfurreducens biofilms have been found to have 3- to 70fold higher amounts of attached cells as compared with S. oneidensis biofilms (95). In co-culture of G. sulfurreducens and S. oneidensis, Geobacter was found to dominate the biofilm (95). The exact mechanism of electron transport through various cytochromes is not clear. The following general steps have been proposed: (1) oxidation of a substrate electron donor and transfer of electrons to inner membrane or periplasmic cytochromes, (2) electron transfer from periplasmic to outer membrane cytochromes, and (3) electron transfer from the outer membrane cytochromes to the electrode surface (51, 96). The rate of electron transfer of each step can differ.

Figure 3. Schematic of an EAB formed on the anode, showing the distribution of outer membrane cytochromes, pili, and EPS. A schematic of the electron transfer process that can occur within Geobacter EAB is shown in Figure 3. In the biofilm matrix, microbes form a physical connection among themselves and also at the solid electrode surface (89). Efficient DET depends on the architecture of the biofilm on the anode surface (18). Cytochromes and pili or nanowires can form a dense network within the biofilm matrix. The biofilm thickness is correlated with the amount of current generated (89). However, biofilms >50 µm have been found to no longer contribute to current production (97). Geobacter mutants without nanowires have been shown to produce less current and form thin biofilms (20, 51). Redox shuttle molecules have also been shown to be critical in electron transport within biofilms. They are self-secreted by microbes and enable transfer of electrons between the external electron acceptor or donor and the microorganism even at long distances. Shewanella has been wellcharacterized for the production of redox shuttles. It secretes flavins as electron transport mediators (98). P. aeruginosa secretes phenazines to perform high-rate electron transfer to the anode in the MFC systems. Mutant P. aeruginosa strains that could not produce phenazines were not able to perform efficient electron transfer (17). Another possible way to perform long-range EET can be through long cellular appendages called nanowires or conductive pili. These are responsible for electron shuttling across the biofilm layers (93). Both Shewanella and Geobacter spp. produce nanometer-scale diameter, micrometer-scale long proteinaceous filaments that are referred to as pili 169 Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

or microbial nanowires that extend from their outer surface to the extracellular matrix as shown in Figure 4 (99, 100). The conductive pili of Geobacter comprise flexible, surface motifs that can stimulate electrostatic interactions with the extracellular electron acceptors (101). On Fe(III) oxide coatings and poised electrodes at metabolizing electrode potentials, pili have also been shown to promote bacterial cells aggregation to form a biofilm (20).

Figure 4. Tapping atomic force microscopy phase images of S. oneidensis MR-1 cells after producing bacterial nanowires in the perfusion flow system. The sample is fixed and air-dried before atomic force microscopy imaging. (Scale bar: 2 μm). (Insets) In vivo fluorescence images of the same cells/nanowires at the surface/solution interface in the perfusion platform. The cells and the nanowires are stained by the membrane stain FM 4-64FX. (Scale bar: 1 μm). The morphologies observed range from vesicle chains (A) to partially smooth filaments incorporating vesicles (B). (C) Transmission electron micrographs of G. metallireducens strain Aro-5. A and B reproduced with permission from ref. (99). Copyright 2010 National Academy of Sciences, U.S.A. C reproduced with permission from ref. (100). Copyright 2018 American Society for Microbiology. Mechanisms of Electron Transfer in Cathodic EABs Different mixed, as well as pure, microbial communities can use cathodes as the electron donor (16, 69). This can be coupled to the reduction of various electron acceptors ranging from O2 to protons and CO2 (102). Microorganisms can utilize different pathways for the uptake of electrons from the cathode. It can occur directly from the electrode surface and indirectly via soluble redox mediators and energy carriers such as H2. These electron transfer mechanisms seem similar to the anodic electron transfer, but the components that are involved function differently at different potentials (103). Unlike anodic electron transfer, cathodic electron uptake is essentially not an 170 Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

energy-conserving mechanism for microorganisms (69). H2 gas can be readily produced at the cathode. It can serve as an electron shuttle to transfer electrons from the cathode to the microbial cell. A versatile range of products can be formed as a result of microbial metabolism that is driven by H2 (104). The ability to generate H2 depends on the expression of hydrogenases. For example, Desulfovivrio paquaseii harbor hydrogenase enzyme that catalyzes the production of H2 via direct transfer of electrons from the cathode (105). However, the exact mechanism is not clear. Electron transfer via hydrogenases is still not completely understood. Cathodic electron transfer can also occur through electron shuttles, which offer a major advantage because they can dissolve at a higher concentration than can H2. The most efficient mechanism of cathodic electron transfer is through direct biocatalysis. G. Metallireducens has been reported to achieve cathode-driven nitrate reduction. Geobacter spp. can accept electrons directly from the surface of an electrode (24). Sporomusa ovata has also been reported to produce acetate and oxo-butyrate from CO2 through direct electron uptake from the polarized cathode (106). Organisms such as C. jungdahlii have a unique proton-dependent electronshuttling complex that can generate energy and may play a role in EET (107). In S. oneidensis MR1, which form biofilm at the cathode, electron transfer can occur through structural proteins and cytochromes. Reverse Mtr respiratory pathway has been suggested to be involved in electron flow from the cathodes to the Shewanella cells (108). Electromethanogenesis occurs when methanogens accept electrons from the cathode and use them for the reduction of CO2 to methane. A recent study of M. maripaludis has provided genetic evidence for a hydrogenase-independent mechanism of direct (without diffusible electron mediator) electron transport from cathodes to microbes (109). G. sulfurreducens can directly take electrons from the cathodes. For instance, even after the replacement of the medium, biofilm formation could be observed on the surface of the electrode along with current consumption (24). C-type cytochromes are involved in uptake of electrons from the cathode. A microarray transcriptional analysis of G. sulfurreducens biofilms on cathode was performed to study the difference in anode biofilm versus cathode biofilm EET mechanisms (110). Interestingly, as compared with cathodic biofilms, it was found that pili and outer membrane cytochromes were highly expressed in anodic biofilms. Moreover, a distinct gene, GSU3247, was expressed at the electron-consuming biofilms at the cathode. This gene encodes a mono heme c-type cytochrome, PccH, which was not highly expressed in the anodic biofilm (111). In acidic environments such as mine drainage, where Fe(II) and sulfur oxidation are the predominant microbial activity, cytochrome-mediated uptake of electrons from solid electron donors is a common process. It has been shown that Acidithiobacillus ferrooxidans can directly accept electrons from Fe(II) minerals (Pyrite) (112, 113).

Tools and Techniques Used To Study Electroactive Biofilms A combination of electrochemical and spectroscopic techniques along with microscopic imaging can be used to gain insights into the microbial EAB–electrode interactions (Table 3). Some of these techniques are discussed here. The thickness of anode EABs varies from monolayers to multiple layers with different niches. These biofilms can be several microns thick depending on electrode material, BES reactor design, and microbial consortia. Morphological characterization of EABs on the electrode surfaces can be done using scanning electron microscopy (SEM), fluorescent microscopy, and confocal laser microscopy (CLSM). SEM is the most commonly used microscopic 171 Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

technique for the observation of EABs attached to the electrode surface (Figure 5a). SEM analysis can be used to examine the spatial distribution and morphology of the biofilm (114–116). CLSM allows in situ imaging of EABs to observe several characteristic parameters such as surface coverage, thickness, and morphological changes in the biofilm (Figure 5b). CLSM of EABs stained with a pHsensitive fluorescence dye can unveil proton gradients, with higher concentrations of protons found in proximity (51, 117). Spectroscopic and chromatographic methods can be used to detect substrates that are utilized in the system, composition of the products that are synthesized, and extracellular mediators. Spectroscopic methods such as UV-visible spectroscopy and chromatographic methods such as high-performance liquid chromatography and liquid chromatography–mass spectrometry are useful to identify and quantitatively measure the concentration of redox mediators. Resonance Raman spectroscopy is suitable to study the role of cytochromes in electron transfer mechanisms. The enhancement of the signal is achieved when the signal frequency of the laser line is in the proximity of the electron transitions that occur in the heme group of cytochromes (118). Electrochemical techniques such as cyclic voltammetry (CV), linear sweep voltammetry, and differential pulse voltammetry can be used to identify the components that are responsible for shuttling of electrons in the form of redox current peaks, based on their reversible electrochemical activities. These tests are conducted with the potentiostat/galvanostat device. CV and linear sweep voltammetry are electroanalytical techniques that have been used in several BES studies. The current flowing through the working electrode is recorded while the electrode potential is swept linearly (back and forward) between two potentials at a specific scan rate (119). A linear polarization potential is applied and scanned from the initial potential to the final potential and the current is measured, which helps to determine a potential range at which EET can take place in EABs (53, 51). CV can be used to identify the redox potential of the redox moieties of the EAB that are involved in electron transfer processes (Figure 5c). It also helps to understand the redox mediators secreted by bacteria and electrochemical characterization of mixed and pure cultures. Current (i) is monitored as a function of voltage (E), and the rate of change of E (voltammetric scan rate v) yields i-E-v dependencies, which can provide information about the electron transfer mechanisms. CV can also be used to study the effect of pH change in the biofilm formation (64). The CV technique has helped identify flavins and their role in EET (53) Differential pulse voltammetry is a voltammetric technique in which potential is scanned with a series of pulses. This technique is capable of detection of nanomolar levels of substrates by minimizing nonfaradaic current and is, therefore, one of the most suitable voltammetric techniques that can be used to monitor trace amounts of redox species present in biological systems (120). Electrochemical impedance spectroscopy (EIS) is another powerful technique used to study the dynamics of the mobile or bound charges in the interfacial layer of the biofilm (121). It is a semiquantitative, nonintrusive, and efficient technique to study and analyze the performance of BES. It is conducted using a potentiostat. A potentiostat is connected with the BES in a two or three electrode mode. The BES is perturbed with an alternating current of small magnitude and then the response of the system is studied at a steady state condition. EIS can be used to study the electrochemical properties of biofilms. Quantification of charge transfer, biofilm capacitance, and biofilm and diffusion resistance are some parameters that can be studied by EIS and are essential to improve the performance of the BES. For instance, high internal resistance limits the maximum power produced by MFCs. Measurement of impedance using EIS gives an understanding of the contribution of different types of resistances to the overall resistance of the system. This technique can be used to optimize the electrochemical behavior of the biological system (121). Bode and 172 Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

Nyquist plots are commonly used for the representation of impedance measurements. Both CV and EIS have been used in numerous studies to study the biofilms developed by G. sulfurreducens or mixed cultures on electrodes (Figure 5 c & d) (122, 123). Table 3. An Overview of the Tools and Techniques Commonly Used To Study Microbial Electroactive Biofilms S. no.

Technique

Use

Reference

1

Confocal microscopy

Biofilm architecture, thickness, roughness

(84, 124)

3

Fluorescence microscopy

To visualize electrode biofilm

(44, 125)

4

Scanning electron microscopy

Biofilm morphologies, putative nanowire structures

(22, 53, 134)

5

Transmission electron microscopy

Quantitative analysis at high resolution across

(93)

the sections of the biofilms and spatial arrangement and cellular ultrastructure 6

Atomic force microscopy

Uses a sharp probe or tip to map the contours of

(126)

a sample and allows the precise measurement of dimensions of individual bacteria 7

(88)

16S rRNA gene sequencing and

Characterization of the mixed microbial

phylogenetic analysis

community composition

8

Fluorescent in situ hybridization

Microbial species or strain-specific functionality

9

Cyclic voltammetry

Electron transfer mechanism studies, midpoint (53, 119, 122) potentials of the catalytic moieties, prediction of rate-limiting steps

10

Linear sweep voltammetry

Polarization curves, electron transfer processes

(127, 128)

11

Chronoamperometry

Potential of the working electrode is set and the

(22, 53)

(56)

resulting current from faradaic processes occurring at the electrode is monitored as a function of time 12

Differential pulse voltammetry

Detection of nanomolar levels of substrates by

(53, 120)

minimizing nonfaradaic current 13

Electrochemical impedance spectroscopy

12

15

Biofilm capacitance, information on charge transfer, biofilm resistance, and diffusion resistance

(53, 121, 123, 129)

High-performance liquid chromatography

Identify and quantify the concentration of redox (77, 118)

UV/VIS spectroscopy

Measurement of absorption spectra, determination of the spectro-electrochemical properties of cell membrane–associated

mediators (118, 130)

multiheme

c-type cytochromes; quantitatively measure the concentration of redox mediators 16

Surface-enhanced infrared absorption spectroscopy

Intimate contact interface between the bacteria and electrode


(131)

Table 3. (Continued). An Overview of the Tools and Techniques Commonly Used To Study Microbial Electroactive Biofilms S. no. 17

18

Technique Surface-enhanced resonance Raman spectroscopy

Use Characterization of OMCs in a catalytically

Reference (132)

active microbialbiofilm

Confocal resonance Raman microscopy To determine the presence and distribution of c- (133) type chromosomes

Figure 5. (a) SEM image of Shewanella oneidensis MR-1 biofilm on the anode surface. (b) Confocal laser scanning microscopy of a fully-grown biofilm on a gold interdigitated microelectrode array (scale bar: 10 μm.) (c) Cyclic voltammogram of acetate metabolizing G. sulfurreducens biofilm. Scan rate: 5 mV/s, 268 h. Inset: first derivatives of the voltammetric curve showing the midpoint potential detectable in catalytic waves of mature biofilms. (d) Nyquist diagrams corresponding to anodic biofilm (mixed culture) at different times of growth: (a) week 1 and (b) week 20. a: Reproduced with permission from ref. (134). Copyright (2013) The Royal Society of Chemistry. b: Reproduced with permission from ref. (87). Copyright (2012) PNAS: National Academy of Sciences. c: Reproduced with permission from ref. (122). Copyright (2008) The Royal Society of Chemistry. d: Reproduced with permission from ref. (123). Copyright (2014) Biomed Central. 174 Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

Applications of Electroactive Biofilms Microbial EET linked to anodes or cathodes in BES offers a wide range of applications. The major ones include electricity generation, wastewater treatment, resource recovery, biosensing, bioremediation, and chemicals production. In the majority of BES applications, EAB at the anode are used for the oxidation of organic substrates present in different wastewaters. Except for pure cultures, anodic biofilms are generally formed from natural sources like wastewater (49). Anodic EABs can have a remarkable impact on the field of wastewater treatment and renewable energy production via MFCs and MECs. There has been an increasing interest in applying EABs for the treatment and removal of oxidizable matter from industrial and domestic wastewaters. Based on various MFC types, chemical oxygen demand removal mostly in the range of 60 to >90% has been reported in the literature (135). Additionally, EABs can be utilized in electrochemical biosensors, which can be used for monitoring biofilm development in facilities where their presence is not desirable (12). In wastewater treatment plants, EAB that is coated onto an electrode such as a quantitative sensor can help obtain in situ monitoring and useful information about microbial respiration (136). Other promising applications include bioremediation of contaminated or polluted water. For example, microbial reduction of soluble uranium(VI) to insoluble uranium(IV) can be used as a strategy to immobilize uranium in a contaminated environment. G. sulfurreducens has been found to perform a reduction of U(VI) to U(IV) with an electrode serving as an electron donor (35). Wastewater also contains inorganic matter such as sulfide, which is a hazardous substance. MFC systems have been found to be effective for sulfide removal with simultaneous electricity generation (137). Hydrogen peroxide (H2O2), which is a potent oxidant with many industrial applications, is produced in industries using highly energy-intensive processes. Bioelectrochemical oxidation of wastewater-enriched microbial consortia at the anode can be coupled with the cathodic reduction of oxygen to form H2O2 (13). Biocathodes offer several applications for the treatment of wastewater, CO2 fixation, chemical production, and bioremediation of toxic pollutants. For instance, MECs can be used for the transformation of organic matter into hydrogen. This process has the potential to produce hydrogen in a sustainable biorefinery platform (12). Microbial metabolism in biocathodes can also be utilized for the removal of nitrogen during the treatment of wastewater by reduction of nitrate compounds (i.e., denitrification). The cathode can serve as the sole electron donor for the reduction of nitrate to nitrite. G. metallireducens has been found to be involved in nitrate removal (24). CO2 is a greenhouse gas and a low-value feedstock. It has been used in BES to produce multicarbon compounds such as acetate (138). It is a novel process to reduce CO2 emissions coupled with chemical production (139). This electricity-driven bioproduction approach could also have significant implications for the storage of renewable energy (140). Electromethanogensis at biocathode has been demonstrated for methane production (83). Enriched mixed culture originated from brewery wastewater at cathode has been reported to reduce CO2 to a mixture of hydrogen, methane, and formate (82). There are many reports of transformation of syngas (CO, CO2, and H2) to multicarbon compounds, such as formate, acetate, butyrate, butanol, lactate, and ethanol, and 2-oxobutyrate at the cathode. Such reactions have been found to be catalyzed by acetogenic bacteria such as Acetobacterium woodi, Clostridium aceticum, Sp. sphaeroides, M. thermoacetica, and C. ljungdahlii (138, 141).


Conclusions and Future Prospects Various aspects related to the microbial EAB are discussed in this chapter. G. sulfurreducens and S. oneidensis are the most intensively studied microorganisms that can form EAB on electrodes. Genetic knowledge of these bacteria has propelled more research into understanding electron transfer mechanisms at the EAB–electrode interface. Anodic electron transfer mechanisms have been more widely studied than cathodic electron transfer mechanisms. Initial research on biofilms has mainly focused on electroactive microbes that are associated with the anode. The study of mechanisms of EET to the anode surface and their electrochemical, biochemical, and physical dynamics paved the way to understanding cathodic biofilms and the complex microbial metabolisms within the mixed culture biofilms. A better understanding of the electron transport mechanisms and molecular pathways involved in cathodic electron uptake is paramount to the implementation of several BES technologies. There are many key differences between anode and cathode biofilms due to the various components involved in EET, different functionalities, and the wide range of microbial populations that grow on the anode or cathode surfaces. New and interdisciplinary strategies to understand EET are required in order to advance the field of microbial electrochemistry and technology and its implications to electron transfer in natural environments. Pure culture and metagenomic approaches are required to identify more exoelectrogens and electrotrophs and also to elucidate more EET components. It is well-known that mixed culture biofilms produce more power output than pure culture biofilms, with the exception of G. sulfurreducens pure culture biofilms (21). Understanding the inner workings of mixed-species interactions within the biofilm matrices is thus beneficial. The ultimate goal of studying community dynamics in EABs is to gain necessary information on engineering biofilms that can be powerful and efficient in field-scale applications, such as bioremediation, wastewater treatment, corrosion control, and biosensing. Metabolomics studies of the anode and cathode biofilms can enhance our understanding of microbial activity and viability within mixed culture biofilms. Also, there is a need to assess competitive and syntrophic interactions that occur within the mixed-culture biofilms. Electron transfer, whether it is direct or mediated, is usually coupled with proton transfer to maintain electroneutrality in solution. The imbalance can cause pH shifts within the biofilm matrix. Proton transport determines the anodic current density, and it has been reported that current density generated by acclimatized anodic bacterial biofilm is limited by proton transport out of the biofilm. Diffusion kinetics depends on the buffer concentration. Optimization of the buffer concentration opens a new door to mitigate this limitation (142) but poses limitations on the real-world applications. Voltammetric and modeling approaches suggest that the biofilm itself can act as a conductive matrix that facilitates EET from microbes to the electrodes (53). In addition to the conductive pili, a multistep electron hopping through redox cofactors like c-type cytochromes results in biofilm conduction. EET in electrode-attached living bacterial biofilms depends on the conductivity of the biofilms. It has been reported that electrochemical activity decreases with the increasing thickness of the biofilm. The highest electrochemical activity has been achieved with a much thinner biofilm, which suggests that live and active cell mass rather than the biofilm thickness is responsible for the high-current generation (143). With the increase in biofilm thickness, many inactive or dead cells accumulate in the inner layer of the biofilm, which results in high diffusion resistance. Biological techniques, shear forces, or dynamic approaches can be used to control the cell growth rate within biofilms and sustain the desired biofilm activity. Modification of the organisms genetically for developing highly conductive biofilms and improvement of EET mechanisms may also help to improve the performance of microbial EAB (144). 176 Rathinam and Sani; Introduction to Biofilm Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

Abbreviations BES, Bioelectrochemical systems CV, Cyclic voltammetry DET, Direct electron transfer EAB, Electroactive biofilm EET, Extracellular electron transfer EIS, Electrochemical impedance spectroscopy EPS, Extracellular polymeric substances IET, Indirect electron transfer MFC, Microbial fuel cell MEC, Microbial electrolysis cell MES, Microbial electrosynthesis MET, Microbial electrochemical technology

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Microbial Electroactive Biofilms - American Chemical Society

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