Biofilm Engineering Approaches for Improving the Performance of Microbial Fuel Cells and Bioelectrochemical Systems

Angelaalincy, Maria Joseph; Navanietha Krishnaraj, Rathinam; Shakambari, Ganeshan; Ashokkumar, Balasubramaniem; Kathiresan, Shanmugam; Varalakshmi, Perumal

doi:10.3389/fenrg.2018.00063

REVIEW article

Front. Energy Res., 05 July 2018

Sec. Bioenergy and Biofuels

Volume 6 - 2018 | https://doi.org/10.3389/fenrg.2018.00063

Biofilm Engineering Approaches for Improving the Performance of Microbial Fuel Cells and Bioelectrochemical Systems

$\r\nMaria Joseph Angelaalincy$ Maria Joseph Angelaalincy¹

Rathinam Navanietha Krishnaraj²

Ganeshan Shakambari¹

Balasubramaniem Ashokkumar³

Shanmugam Kathiresan⁴

Perumal Varalakshmi¹^*

¹Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai, India
²Department of Chemical and Biological Engineering, Composite and Nanocomposite Advanced Manufacturing – Biomaterials Center, Rapid City, SD, United States
³Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai, India
⁴Department of Molecular Biology, School of Biological Sciences, Madurai Kamaraj University, Madurai, India

Microbial fuel cells (MFCs) are emerging as a promising future technology for a wide range of applications in addition to sustainable electricity generation. Electroactive (EA) biofilms produced by microorganisms are the key players in the bioelectrochemical systems involving microorganism mediated electrocatalytic reactions. Therefore, genetically modifying the organism for increased production of EA biofilms and improving the extra electron transfer (EET) mechanisms may attribute to increase in current density of a MFC and an increased COD removal in wastewater treatment plant coupled MFC systems. Extracellular polysaccharides (EPS) produced by the organisms attribute to both biofilm formation and electron transfer. Although cell surface modification, media optimization and operation parameters validation are established as enhancement strategies for a fuel cell performance, engineering the vital genes involved in electroactive biofilm formation is the future hope. Therefore, in this review we critically address the biofilm formation mechanisms in electro active microorganisms, strategies for improving the biofilm formation leading to improved electrocatalytic rates for applications in bioelectrochemical systems.

Introduction

The inadequate supply of fossil fuels (Demirbas, 2005; Panwar et al., 2011), ever growing population and the escalating energy demand, in the recent years, has become one of the biggest bottlenecks to human survival and economy. Other than this, the associated problems like global warming and pollution (Davis and Higson, 2007) are major impetus for researchers to explore alternative energy sources which are renewable, sustainable and economical. Though wind power and solar cells have already been harnessed and brought into commercial use, fuel cells, which are equally promising, are still the least explored (Angelaalincy et al., 2016).

Another prime concern other than energy demand, in developing countries, is the increasing levels of wastewater (Liu and Ramnarayanan, 2004; Gude, 2015). Incidentally both these concerns can be alleviated by harnessing the microbes for remediating the wastes while colonizing on electrodes with a biofilm and serving as live or microbial fuel cells (MFC) (Aelterman et al., 2006; Li et al., 2013; Chaturvedi and Verma, 2016). The microbes employed in MFCs convert the chemical energy present in organic compounds to electrical energy through catalysts (Chaudhuri and Lovley, 2003). Most commonly, MFCs may at times employ bacteria on the anode to carry out oxidation of organic matters and bacteria or microalgae on the cathode to undergo reduction. Compared to other bioenergy conversion processes like anaerobic digestion, gasification and fermentation, MFCs have an added advantage of reduced amounts of secondary pollutants production (Chouler et al., 2016) and cost-effective operation, as they operate under ambient environmental conditions (Park and Zeikus, 2003). Irrespective of their role in wastewater treatment and electricity generation, MFC based biosensors are of great interest in the recent years pertaining to their advantages such as high sensitivity, stability and remote site applicability without electricity supply. MFC-based biosensor devices have been to test microorganism load, BOD, presence of corrosive biofilms, cytotoxic elements and microbial activity monitoring (Yang et al., 2015a).

Therefore, identifying the loop-holes that hinder MFC performance and its multi facetted applications, has become essential in setting up an effective MFC system. While discussing the role of microorganisms in MFC, it is essential to understand the mechanism of electron transfer contributing to electricity generation. Microorganisms mostly grow on the electrodes to form a biofilm, which is an extracellular polysaccharide (EPS) enthralled surface harboring the microbial community (Rollefson et al., 2011). The EPS physically immobilizes the bacteria, however paves way for cell to cell contact and communication. This cell to cell communication, involves in the electron transfer and electron- electrode interaction in MFCs (Patil et al., 2012; Sarjit et al., 2015). Moreover, the efficiency in electron transfer is inversely proportional to the distance over which the electrons travel to reach the electron acceptor (Breuer et al., 2013).

Therefore, a clear view on the role of biofilms on electron transfer will provide an insight on development of new approaches for improving the performances of microbial fuel cells apart from increased wastewater treatment efficiency. Irrespective of the several operational parameters that influence a fuel cell performance, the extracellular electron transfer (EET) mechanism (Schröder, 2007) and biofilm production (Zhang et al., 2011) in the employed microorganisms always have a positive influence toward power production. Therefore, approaches for biofilm engineering, which involves genetic and surface modification techniques are considered as new avenues in fuel cell research. This review aims to provide a deep insight on the role of extracellular polysaccharides (EPS) in biofilm formation and the role of biofilm in current generation in microbial fuel cells.

Elements Constituting Microbial Fuel Cells

An overview of the basic elements that comprise a MFC is mandatory to understand the major role played by biofilm producing microbes in a fuel cell. Microbial fuel cells are generally made of a cathode, an anode, a PEM (proton exchange membrane) and a resistor, through which the electrons travel to the anode. In most cases, the anode is entrapped within the bacterial consortium (Gouveia et al., 2014). Sometimes, it may contain the organic material to be oxidized or the fuel source (Zhao et al., 2005), however, oxidation occurs at the anode. The cathode is provided with the desired source microbe. After oxidation at the anode protons pass through the PEM to the cathode, where they get reduced into water (He et al., 2014; Figure 1). This is a double—chambered MFC that exists more commonly. A single chambered MFC, on the other hand, contains a single chamber harboring both the anode and the cathode together (Singh et al., 2016) or only the anodic compartment, coupled with an air—cathodic chamber (Tharali et al., 2016). The single chamber MFC lacks a PEM. The various proposed designs for devising a single chamber MFCs have been mentioned in Tharali et al. (2016).

FIGURE 1

Figure 1. Schematic representation of simultaneous waste reduction and electricity generation in a Microbial fuel cell, employing photosynthetic organisms at the anode.

The types of MFCs that operate with a trielectrode system are commonly employed in electrochemical studies (Angelaalincy et al., 2017). The setup consists of three electrodes: the working electrode which acts as a cathode, mostly made up of glassy carbon or platinum electrode that accommodates the microbial consortium; the counter electrode, that functions as the electricity conductor and the reference electrode which is the standard electrode made up of silver in potassium chloride or silver. The current produced is recorded with the help of a cyclic voltammeter.

MBFC (Microbial biofuel cells) contain two chambers made up of polycarbonate, acrylic glass or glass, plexiglass (Du et al., 2007), or plastic bottles (Parkash et al., 2015), or cans (Obasi et al., 2012), holding the two electrodes. In MBFCs a large surface area and a robust structure are mandatory for supporting the biofilm, withstanding water current. Therefore, an assortment of electrode materials such as carbon paper, carbon cloth, graphite plates, granules, rods and RVCs (Reticulated vitreous carbon) are employed in the construction of a fuel cell. The range of cathodic materials is quiet stringent and is limited to the usage of precious metals like platinum (Pt). At times, Pt is replaced with transient materials such as iron and cobalt mixture catalysts, which are yet to be studied in detail (Cheng et al., 2006; Zhao et al., 2006). However, the platform for ambient microbial growth and biofilm formation is mandatory while constructing a MBFC.

EPS in Biofilm Formation and Regulation

The biofilm that forms under natural circumstances increases in density with the age of the culture, however is not electrochemically active throughout, due to the formation of an inner lining of dead or inactive cells in the biofilm matrix (Sun et al., 2016). Therefore, a strategy that induces live or active microbes to produce more electro active biofilms is considered to be a promising approach for improving the power performance of MFCs. In order to achieve this, the major constituents of a biofilm and their role in biofilm formation needs to be understood.

Microbial biofilms have encountered an evolutionary increase in their complexity with due course of time (Kreft and Wimpenny, 2001). A metaphorical representation of biofilms states them as the “city of microbes,” where the EPS are mentioned as the “house of the biofilm cells.” The water content, charge, porosity, hydrophobicity, density, sorption properties and mechanical stability of the biofilm cells are affected by the EPS thus determining the immediate conditions of life (Flemming et al., 2007). In fact, the organisms are found embedded in these biopolymers. The higher the secretion of EPS by the organism, the greater is the density of the biofilm. A denser biofilm harbors more number of organisms than a lighter one. Therefore, the density of the biofilm increases with respect to the age of the organisms. As mentioned early, biofilm formation on the anode, has a pivotal role in MFCs. However, the electrochemical performance is not determined by the density but by the temporal and spatial locations of live and dead cells within a biofilm (Sun et al., 2015). Studies in electro active bacteria Geobacter sulferreducens have evidenced the rapid drop of charge transfer resistance in the presence of rapidly multiplying live cells. However, with time, as the dead cells start to accumulate in the inner layer of the biofilms, there has been a high diffusion resistance observed in the electrochemical system. In such cases, it is inferred that, not the density of the biofilms, but the active electron transferring live organisms present in the outer layer of the biofilms, contribute to the high current generation of the system (Sun et al., 2016). In composition, apart from EPS, the biofim is also comprised of a major portion of polysaccharides and minor portions of glycoproteins, proteins, glycolipids, and negligible amount of nucleotides and in rare cases, some metals (Angelaalincy et al., 2017) that contribute to the structural and functional outlook of the biofilm. However, EPS comprises the major component of the biofilm matrix. Among the known bacterial EPS, at least three polysaccharides have been found to be active in biofilm formation.

They are the Psl, Pel, and alginate polysaccharides. Psl polysaccharides are EPS produced from Psl genes. Reports since 2004, evidently states the pivotal role of Psl polysaccharides in biofilm formation in Pseudomonas aeroginosa (Colvin et al., 2012; Wei and Ma, 2013). The Psl loci consist of 15 co-transcribed genes, out of which 11 contribute for the synthesis of Psl dependent biofilm (Friedman and Kolter, 2004; Jackson et al., 2004; Matsukawa and Greenberg, 2004). The formation of biofilm comprises of five sequential steps including initial attachment, irreversible attachment, microcolony formation, biofilm maturation and biofilm dispersion. Therefore, overexpression of these genes might attribute to increased biofilm formation by live or active microbes in a MFC.

Zhang et al. (2017) reported the role of these polysaccharides in biofilm initiation, which influenzes the surface motility of subsequent cells (Zhao et al., 2013). It has been observed that overproduction of Psl polysaccharides intensifies cell to cell interaction and intercellular adhesion, promoting the first and crucial step in biofilm formation (Ma et al., 2006; Byrd et al., 2009). The Psl polysaccharide attaches firmly in a helical shape on the bacterial cell wall promoting strong intercellular interactions (Ma et al., 2009) thus serving as a scaffold during biofilm matrix formation. During biofilm maturation, they are found attached on the periphery of the three dimensional structured colonies thus providing structure support and enabling biofilm dispersion at the end (Ma et al., 2009). Thus, EPS have a pivotal role in the formation, structural integrity, adhesion property, stability and life of biofilms and thus could be employed as crucial elements in biofilm engineering processes by increasing the copy number of these EPS producing genes.

Factors Influencing Biofilm Formation

Although EPS plays an irresistible role in biofilm formation, the process also depends on the external environmental factors and the gene expression mechanisms that contribute to the biofilms development in individual cells (Toyofuku et al., 2016). The physical and environmental factors to which the cells are subjected influence the biofilms formation along with the surface and extracellular components of the organisms. To be more specific, the lipopolysaccharides (LPS) and exopolysaccharides (EPS) alongs with quorum sensing (QS) signaling molecules prescribe the fate of biofilms formation (Nocelli et al., 2016). Henceforth, it is clear that the factors that externally influence EPS production, quorum sensing signaling molecules production and other stress factors such as heavy metal stress (Chen et al., 2015), salinity (Hong et al., 2016), pH (Christenson, 2011), nutrient starvation (Angelaalincy et al., 2017), nutrient depletion, pathogen invasion, growth substrate, water current in moving water bodies etc. (Angelaalincy et al., 2016) also contribute in influencing biofilm formation. However, the major factors involved in biofilms formation are QS signaling molecules and EPS and genetically engineering these molecules can overcome the other environmental impacts involved in biofilm formation. However, this needs substantial research.

Influence of Quorum Sensing Signaling Molecules

It is well known that the bacterial community communicates with each other through cell to cell interaction thus coordinating their collective behavior. This requires release of autoinducers from the cells resulting in the phenomenon called quorum sensing. Monzon et al. (2016) reported a 95% increase in biofilm mass of Halanaerobium praevalence thereby contributing to a 30% increase in power density upon addition of 100 nM quinolone type signaling molecules. Quinolone are signaling molecules belonging to the LuxR family proteins coded by hmqF genes (Agarwal et al., 2012) in Halanaerobium species. On one hand the entire promotion of biofilms formation in Pseudomonas aeroginosa has been demonstrated (Diggle et al., 2002), where on the other hand, little is known about the effect of autoinducers from other bacteria in stimulating QS of Halanaerobium sp. (Monzon et al., 2016).

Influence of Extracellular Polysaccharides and Other Physical Factors

It has already been registered that apart from the QS signaling molecules, EPS and LPS are also found to play pivotal role in biofilms formation. Bacterial generation of c-di-GMP by diguanylate cyclases (DGC) at high levels enhance matrix exopolysaccharides such as Pel and Alginate synthesis attributing to biofilms formation. Among the three major polysaccharides, Psl, Pel, and alginate, enhanced production of Psl contributes for initiation and maintenance of biofilms structure (Ma et al., 2009), where Pel, a glucose rich extracellular matrix which is specific to gram negative bacteria, aids in the formation of solid surface associated biofilms for non-pilated organisms (Vasseur et al., 2005). Moreover, a bifunctional enzyme produced from algC gene in P. aeroginosa has been identified to be crucial for the biosynthesis of four polysaccharides viz. LPS, Psl, Pel, and alginate which influenze biofilms formation (Wei and Ma, 2013). Almost all the genes that produce any of the above said polysaccharides are invariably contributing biofilms formation in the organism (Wei and Ma, 2013).

Role of EPS in Electron Transfer

Though the extracellular polysaccharides (EPS) play variety of roles in different cells, its function in electron transfer is largely intriguing. Polysaccharides have long since been known to be associated with cell to cell interaction and surface attachments and variations in composition of the EPS can have pleiotropic effects where the surfacecharge is modified and alters surface attachment, which provide an anchor for holding the peripheral proteins that involve cell-cell recognition events as revealed in studies with Shewanella (Korenevsky and Beveridge, 2007). Studies with Geobacter sulfurreducens have been a matter of intense research in this context where a report showed a role for EPS as attachment sites for peripheral redox proteins that allow multicellular communities to transfer electrons to distant acceptors, where the mutant that lacked the gene encoding exopolysaccharidematrix production failed to develop electrogenic biofilms onelectrodes. It was thus observed that G. sulfurreducenspossessed genes (e.g., xapA or xapK) that encode extracellular anchoring polysaccharides that contain binding sites for c-type cytochromes, are essential for the electron transfer to the electrode (Rollefson et al., 2011). Thus the EPS is evidenced to be a crucial factor not only in formation of biofilm but also in electron transfer in a fuel cell.

As mentioned earlier, a widely proposed use of MFCs, one of the several bioelectrochemical systems (BESs), is to focus on simultaneous wastewater treatment and electricity generation (Pant et al., 2012; He et al., 2016a; Santoro et al., 2017). The bottleneck of BESs is the ability of the electroactive microorganisms to participate in extracellular electron transfer (EET) (Yang et al., 2012). In this process of EET, microorganisms serve as electron transfer systems, using direct or mediated mechanisms (Franks, 2015). Direct EET occurs via electron transfer through outer membrane proteins (Shi et al., 2016) or through electrically active bacterial surfaces (Wrighton et al., 2011) which physically contacts the electrode, most probably the anode or the bacteria in the vicinity. In mediated EET, endogenously (e.g., phenazines) or exogenously soluble (eg. humics) mediator molecules, also called as redox shuttles (Qiao et al., 2008) that shuttles electrons from the cells to the anode through the extracellular aqueous matrix (Lovley, 2011) act as mediators. EET where metabolites or mediators are produced by one species and are consumed by another species in a consortia are called mediated interspecies electron transfer or MIET (Cheng and Call, 2016).

A detailed characterization of the various microbes present in the consortia of exoelectrogenic biofilms provides insight into the processes to convert complex organic matter in wastewater streams into electrical current in bioelectrochemical systems (BESs) (Kiely et al., 2011). The past decade has also provided evidence of yet another method, direct interspecies electron transfer (DIET), that happen between organisms or in association with electrically conductive materials and it has been reviewed that they can be stimulated in engineered systems to improvise on required waste treatment goals and also for energy recovery in microbial electrochemical technologies (Cheng and Call, 2016). Thus the role of biofilm in BESs can hardly be understated and hence it allows the focus on extracellular polysaccharides production by the microorganisms that facilitate biofilm formation.

Mechanism of Electron Transfer

The mechanism of electricity generation in MFC can be classified is of two types: the direct electron transfer and indirect electron transfer.

Direct Transfer (DET)

In the first type of MFC, the bacteria transfer the electrons from its membrane to the electrode directly without the intervention of an intermediate fermentation product (Angelaalincy et al., 2016). This is called as the direct transfer. These microbial fuel cells impose the selection of highly active microbial consortium which is either mixed or pure, as these microbes are the catalysts functioning in electrons transfer. The transfer is aided through proteins (cytochrome) that are immobilized on the bacterial cell wall. Rhodoferax ferrireducens (Liu et al., 2007) and Geobacter sulfurreducens (Bond and Lovley, 2003) can be stated as examples of this type of bacteria (Roller et al., 2008). Physical contact of the bacterial membrane possessing EPS or membrane organelle with the fuel cell electrode, an anode in most cases, is crucial for direct electron transfer (Read et al., 2010). No diffusional redox species are involved in this electron transfer process. This type of electron transfer between the organism and the electrode is possible only with an electrochemically active (EA) microorganism or bacteria (Chang et al., 2006). Exoelectrogens possesses the ability to transfer electrons directly to an electrode without employing artificial electron shuttles, by three mechanisms (Figure 2): (i)short-range electron transfer with the aid of redox-active proteins like cytochromes found on the outer surface of bacterialcell membrane and (iii) long-range electron transfer through conductive pili also known as nanowires.

FIGURE 2

Figure 2. Mechanism of short range, electron shuttle mediated and long range electron transfers in a bacterium.

Living cells, in general are believed to be electrically inactive because of their non-conducting nature (Di Domenico et al., 2015). However, studies revealed that an organism possessing a membrane bound electron transport protein entrapped inside an EA biofilm can be efficiently used for the mechanism (Pinto, 2016). But the transfer of electrons from inside the cell to its outside it governed by transport proteins, whereas the transfer of the electrons to an external, solid terminal electron acceptor, here it is an electrode, is mediated by outermembrane (OM) redox proteins (Wrighton et al., 2011). Some sediment inhabiting metal reducing microorganisms like Geobacter (Seeliger et al., 1998), Rhodoferax (Hochkoeppler et al., 1997) and Shewanella (Myers and Myers, 1997) are found to contain c- type cytochromes, which are multi-heme proteins, found along with EA biofilms in these organisms.

As mentioned, the DET requires physical contact of the EA bacteria and the bacterial cytochrome with the fuel cell anode. However, only the first monolayer existing bacteria in the biofilm will be electrochemically active, at the anode (Babauta et al., 2012). Therefore the performance of the MFC is dependent on the cell density in the first monolayer. As stated previously, the density of the biofilm increases with the age of the culture and with the secretion of more EPS. Therefore, the presence of EA biofilms with dense monolayer have been reported to play a significant role in the MFC performance (Eaktasang et al., 2013).

Other than the cytochromes, the bacteria also possess adherence fimbriae—the pili, made up of proteins and sortase enzyme (Proft and Baker, 2009). Reguera et al. (2006) have reported that some Geobacter and Shewanella strains produce pili that possess electroconductivity. These pili enable organisms to reach a distant anode or solid electron acceptors and utilize the electron transfer potency. The organisms also utilize an electrode, which is not in direct contact with the cell, as its sole electron acceptor, with the aid of their pili. These pili, also called as nanowires, also attach to the membrane bound cytochromes of the cells through which electron transfer to the cell's periphery is adepted (Yang et al., 2012). The nanowires also entangle the development of dense EA biofilms thus enhancing the anode performance.

Indirect Transfer

In the indirect way, a mediator is employed for electron transfer. In indirect transfer, the secondary metabolites (endogenous redox mediators) are especially of great interest, as their synthesis makes the electron transfer independent of the presence of exogenous redox shuttles (Schröder, 2007). This is called as Mediated electron transfer (MET) (Zhou et al., 2013). This can also be attributed by microbially secreted soluble electron shuttles, for example, pyocyanin and flavins (Figure 2). The mediator serves as a reversible terminal electron acceptor, transferring electrons from the bacterial cell either to a solid oxidant (the MFC anode) or into aerobic layers of the biofilm, where it becomes re-oxidized and is again available for subsequent redox processes. One molecule can thus serve for thousands of redox cycles (Santoro et al., 2017).

Consequently, the production of small amounts of these compounds (directly in the anodic biofilm) enables the organism to dispose of electrons at sufficiently high rates. For example, the pigment pyocianine produced by Pseudomonas aeruginosa has been found responsible for its electrochemical activity (Rabaey et al., 2004). Quinone-mediator (2-amino-3-dicarboxyl-1,4 naphthoquinone) produced by Shewanella oneidensis increases the power density of MFC by a factor of 2 when compared with the one without the mediator (Schröder, 2007). The bacteria Pseudomonas alcaliphila is also capable of producing its own redox mediators. Other than the redox mediators, the by-products produced as a result of bacterial metabolism also contribute for indirect electron transfer, through the oxidation of the produced by-products. Oxidation of the fermentative hydrogen produced by bacteria (Chen, 2006) at the anode is an example.

In both the cases, electron transfer is through bacterial contact with the electrode either directly or through soluble shuttles that act as mediator molecules such as ubiquinones, pigments, dyes and metal complexes forming reversible redox couples that are readily soluble and non-toxic to the microbial consortium, biologically non-degradable and are highly stable in both the oxidized and reduced forms (Aghababaie et al., 2015).

Microorganisms Engaged in Microbial Fuel Cells

Attributing to the above said potencies, a broad range of photosynthetic and anerobic microrganisms have been employed as electron donors and acceptors in MFCs. They include Chlorella vulgaris (Jeon et al., 2012), Phormidium sp. (Bradley et al., 2012), Saccharomyces cerevisiae (Permana et al., 2015), Leptothrix discophora (Rhoads et al., 2005), Scenedesmus armatus (Angelaalincy et al., 2017), Rhodispirullum rubrum (Bensaid et al., 2015), Thiobacillus ferrooxidance (Ter Heijne et al., 2007), Desulfovibrio desulfuricans (Kang et al., 2014), Klebsiellapneumoniae (Deng et al., 2010), Pseudomonas fluroscens (Friman et al., 2013), Geobacter metallireducens (Poddar and Khurana, 2011), and some anaerobic bacteria. Some of these organisms are genetically engineered to provide exponential results in terms of current production and sustainable biomass generation than the wild type strains. Reports on the usage of algae in fuel cells are limited when compared to those on bacteria.

Parameters Influencing Fuel Cell Performance

Apart from the microorganisms employed, electro active nature of the biofilm produced and the mode of electron transfer used by the organisms, parameters such as temperature, pH, applied potential, flow conditions etc. influence the performance of a microbial fuel cell during field application (Jadhav and Ghangrekar, 2009). Ringeisen et al. (2007) has explored parameters such as electrodic materials, surface area of the electrode and special aerobic cultures for enhanced fuel cell performances. Inspite of the several reviews on the performance of MFCs under controlled conditions, there are still researches going on to determine the influence of various operational parameters on the fuel cell performance. Jadhav and Ghangrekar (2009) have reported a varying MFC performance with variations in the operationg parameters such as pH, temperature and external resistance. The study has reported that a reduction in temperature range (8–22°C) resulted in an increased current upto 1.4 mA from 0.7 mA and increased coulombic efficiency of 5% from 1.5%. However, the COD removal efficiency decreased to 59% from 90%. On the contrary, certain wastewater derived organisms showed increased bioelectrocatalytic performance and increased COD removal (Chan and Li, 2014). Its presumed that increasing temperature increases the oxygen reduction kinetics, thus decreasing the internal resistance of the cell. This results in increased current density and increased Coulombic efficiency. The COD removal rate has also been observed to increase with increase in temperature, which may be the result of an increased biomass due to increased biochemical reaction rate. Therefore, the substrate utility rate increases, resulting in an efficient COD removal (Scott and Yu, 2015). However, there are also reports that a decreased temperature increases current density, power density and cell voltage (Chan and Li, 2014). Therefore, the optimal operation temperature of a MFC can only be determined based on the anodic consortium employed for current production.

Similiarly, a variation in the anodic pH between 5.5 and 7.5 inferred that, a steady pH maintanence at 6.5 resulted in increased current and coulombic efficiency of 4% where a pH more than 7 and less than 6 resulted in decreased current (Jadhav and Ghangrekar, 2009). In general, MFCs are operated at a neutral pH to attain higher power output, because the anodic microbial consortium performs well in a neutral pH rather than in an increased or decreased pH. However, an increased pH at the anodic chamber shall attribute to increased COD removal, whereas an increased pH at the cathodic chamber results in an increased power output (Scott and Yu, 2015). Further, a carbon source, which is soluble has been reported to significantly change the MFC power output than a particulate carbon source (Borole and Hamilton, 2010). The employed microbial consortium greatly feeds on the dissolved carbon source for growth and metabolism, thus resulting in an improved biomass that contributes to increased power output (Angelaalincy et al., 2017). The effect of ionic strength of the anodic chamber has also been found to influence the performance of MFC. An increase in the ionic strength to 400 mM from 100 mM has resulted in 1,330 mW/m² power density from 720 mW/m² thereby reducing the internal resistance to 79–161 Ω (Liu et al., 2005). In addition, the flow rate in a MFC has also been found to influence the anodic and cathodic impedence in fuel cells. Aaron et al. (2010) reported that increasing the anodic flow rate decreased the cathodic impedence by 65% however, the anodic impedence remained significantly unaltered. Similarly, with increasing flow conditions, the anode modules produced a power density of 6.0 ± 0.4 Wm⁻³ which is 1.9 times higher than the control conditions (He et al., 2016b; Table 1). Although a number of factors influence the performance of fuel cells, a genetic approach, which would enhance biofilms production in microorganisms is still considered a promising approach for enhanced fuel cell performance.

TABLE 1

Table 1. Studies on the role of microbial biofilm in MFCs over the past decade.

Biofilm Engineering for Enhanced Fuel Cell Performance

Given the role of EPS in EET of microorganisms and its relavence in biofilm formation on electrodes, the prospect of engineering the biofilm for its enhanced adhesion and EET is just the future of the MFCs. S. oneidensis MR-1, a facultative anaerobe is capable of reducing Mn(IV) and Fe(III) oxides and can produce current in microbial fuel cells. The mechanisms employed by S. oneidensis MR-1 for this process have not been fully elucidated. However, several different S. oneidensis MR-1 deletion mutants were made and tested for current production and metal oxide reduction. The results suggested involvement of certain key cytochromes in all of the processes though with varying degrees in each process thus showing a very complex picture of electron transfer to solid and soluble substrates by S. oneidensis MR-1 (Bretschger et al., 2007). The mechanism involved in EET in S. oneidensis MR-1 involves OmcA and MtrC (outer membrane -OM), decaheme c-cyts in direct electron transfer to solid metal oxides and anodes of MFCs, however another member of the genus Shewanella, S. loihica PV-4 showed different mechanism for current generation (mediated electron transfer) (Newton et al., 2009). Another study involving cell surface polysaccharides of Shewanella oneidensis MR-1 demonstrated that the effect of these polysaccharides on not only the cell adhesion to graphite anodes but also the current generation in MFCs, as the electrically non-conductive capsular polysaccharides can interfere with the contact of OM cytochromes to anodes and direct EET via them. Thus, cel surface engineering was prospected as a valuable scheme to generate higher current in bacterial MFC system (Kouzuma et al., 2010). Genetic engineering approaches have been made in a model organism Shewanella oneidensis MR-1where flavin biosynthesis gene cluster ribD-ribC-ribBA-ribE and metal-reducing conduit biosynthesis gene cluster mtrC-mtrA-mtrB were coexpressed in the bacteria and an improved EET capacity in microbial fuel cells with an increase in maximum current density by approximate 110% was seen (Min et al., 2017).

In yet another approach, a synthetic fermenter-exoelectrogen containing a microbial consortium (Escherichia coli-S. oneidensis) was tested to establish a highly electroactive anodic biofilm. Briefly, a synthetic riboflavin pathway from Bacillus subtilis was expressed into E. coli to overproduce flavins in order to facilitate flavin-mediated electron transfer, and a hydrophobic S. oneidensis strain CP2-1-S1 was employed as the exoelectrogen to increase its adhesion to the carbon electrode. The extremely hydrophobic interactions between S. oneidensis and the anode along with the overproduced flavins produced by the recombinant E. coli added an advantage for S. oneidensis over E. coli in the attachment to the anode surface. This rationally engineered anodic biofilm with the modified microbial community profile showed a higher catalytic current (from 0.19 to 1.84 A/m2 at 0 V vs. SHE). The xylose-fed MFC inoculated with this engineered microbial consortium generated a greater power density which was 6.8 times higher than that inoculated with wild type coculture (Yang et al., 2015b).

Similarly, simple surface modifications for enhanced biofilm formation, increased electron transfer rate and higher current density generation from microbial fuel cell (MFC) have also been demonstrated using partial oxidation of carbon felt material by UV/O₃ treatment, where the electrochemical studies performed suggested that Shewanella oneidensis MR-1 biofilm formation was improved on UV/O₃ treated carbon felt electrodes at an applied potential of −0.3 V vs. Ag/AgCl, where the carbon electrodes exposed to 45 min of UV/O₃ treatment provided the best electrochemical results and enhanced bacterial cell attachment (Cornejo et al., 2015). Further, the experimental evidence of a stimulated voltage production of up to 0.3 V in MFC in amendment with 100 nM quinolone signal compared to the control debates the scope of genetic engineering of quorum sensing signaling molecules, thus attributing increased biofilms formation, for enhanced power production in MFCs.

Further, the effect of different operational conditions on biofilm development and nitrification in three moving-bed biofilm reactors (MBBRs) was investigated (Bassin et al., 2012). Organisms such as Shewanella oneidensis and Geobacter sulfurreducans have been studied on the role of the Cytochrome C and pili production in MFCs (Alfonta, 2010). Genetic engineering and gene silencing strategies to explore the role of these appendages have provided clear-cut information on the pathway of EET from bacteria to the anode (Rosenbaum and Angenent, 2009). News reports about research in in United States have reported 50% more fuel production while employing genetically engineered bacteria in a MFC (Hudson, 2013). However, genetic modification or metabolic engineering of microalgae and its putative efficiency in power production has not been reported so far. Still, enhancing the EPS production of the organism through media optimization strategies have been reported to improve the current (mA) generation in microalgae (Angelaalincy et al., 2017). Compared to bacterial system, the algal system is quite complex depicting a large number of genes attributing to various functions. The red alga Porphyridium purpureum has been reported to possess an unusually simple enzyme network containing 19 genes that involve in many critical biosynthetic steps represented by single enzymes. Starch synthase, a glycosyltransferases 5 (GT5) enzyme, has been explored to be involved in priming polysaccharide synthesis apart from its role in chain elongation of amylopectin and production of novel granules (Bhattacharya et al., 2013). The presence of this gene has also been reported in a green microalga, Coelastrella sp. M60 (Karpagam et al., 2018) indicating the presence and importance of the gene in the algae family. Over expression of such genes contributing to polysaccharide production in microalgae has not been reported so far. Hence, cloning and expression of genes contributing to exopolyssacharide production in microalgae needs substantial research and is thereby supposed to be a promising approach for enhanced power production in photosynthetic algal microbial fuel cells (PAMFCs), as the durability of PAMFCs is longer compared to bacterial biofuel cells. Thus, biofilm engineering is a large avenue open for research.

Conclusion

The contribution of microorganisms toward sustainable energy generation, bioremediation and other industrial applications are incredible though a large part of it remains untapped and un-explored. Their ability to coordinate their metabolism upon achieved cellular density is surprising, which can be attributed to different microbial mechanisms among which the EPS bound biofilms of the organisms have a huge share. The composition, morphology, physical properties and thickness of biofilms show a remarkable impact on bioelectricity production. Although, there are numerous applications of biofilms and EPS in specific, their role in MFCs and bioelectricity generation is note- worthy. Many different strategies to engineer biofilm have been explored to harness this metabolism to sustainable energy production have been attempted but, the rate limiting step to this progress is our limited information of the complete metabolism and genetic regulation and the fact that our knowledge about the EET mechanism is limited to the dissimilatory metal- reducing bacteria mainly in the Geobacter spp. and Shewanella spp.; however, some properties of other proteins involved in in the EET need to be explored. Although these two species have contributed much to the MFCs, other exoelectrogens need also to be discovered employed and tapped for future enhancement of MFC supported technologies. Hence, a deeper insight into the biofilm properties and genetic modification of organisms may open new avenues in improving the performance of a MFC.

Author Contributions

MA and PV have written the manuscript. RN, MA, GS, BA, and SK actively contributed in writing, editing and improving the manuscript. PV provided support, guidelines and manuscript correction.

Funding

The authors thank Department of Science and Technology, Ministry of Science and Technology, New Delhi, India (DST- INSPIRE Fellowship/2014) for funding PV and MA to support this work.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

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