Mechanisms by which bacteria generate power in MFCs have been intensively investigated in recent years. How might we make "super-bug" electrogens using the power of genetic manipulation? There are many factors that we may ponder. The MFC literature is rife with bio­logical, biochemical and biophysical aspects of highly electrogenic bacteria including members of the genera Geobacter, Shewanella, and Rhodoferax species, and many other Gram-positive and — negative electrogenic bacteria (Huang et al., 2012; Guo et al., 2012). Many of these organ­isms can exist and/or thrive in a myriad of different niches, including those involving significant variations in temperature, pH, osmolarity, pollutants, biocides, and metabolizable/nonmetabolizable carbon sources. As such, many studies involving MFCs house single spe­cies bacteria, bacterial "consortia," media or feedstock, anode/cathode materials, MFC design and design mate­rial, flow rates, Coulombic efficiency and other MFC pa­rameters that can impact power density measurements.

Bacterial Metabolism: How to Power MFCs through Respiratory/Anaerobic Fluxes

Many bacteria are metabolically versatile organisms, and can utilize nearly every carbon-containing com­pound produced in nature. As stated earlier, they are capable of aerobic and/or anaerobic respiration as well as fermentation. Aerobic respiration requires molecular oxygen (O2) while the latter can use alternative electron acceptors including but are not limited to NOT, SO4_, Fe3+, dimethyl sulfoxide (DMSO), trimethylamine N-oxide (TMAO), and CO2. A member of the tricarbox­ylic acid cycle (TCA) cycle, fumarate, can also be used. Interestingly, forms of electron acceptors including oxides of iron and manganese (Shi et al., 2007), vana­dium, selenium, tellurium and toxic metals including chromium, arsenic and cobalt can also be used by some organisms. Thus, depending on the organism(s), the goal of genetically unmodified bacteria is to couple oxidation of organic matter and reduction of terminal electron acceptor (in most cases, the anode of the MFC). However, in MFCs, the electron acceptor in most cases, with the exception of biocathodes, and bioanode/bio — cathode MFCs, is the anodic surface. This can occur in what are commonly termed mediator-dependent or mediator-less MFCs (discussed below). Many facultative and obligatory anaerobic bacteria can undergo an anaer­obic process known as fermentation that does not require functional cytochromes, respiratory chains and produces far less energy in the form of adenosine triphosphate (ATP) than, for example, glucose respiration in E. coli.

Mediator-Less Factors Affecting MFC Performance

Many studies have been conducted in the past ~ 7 years to either isolate superior unknown electrogens or improve the electrogenic properties of existing organ­isms possessing such capacity. The power of various mo­lecular genetics tools (mutations, deletions, gene transfer, overexpression, etc.) is the central force under­lying the discovery of such strains. However, surface — localized factors such as bacterial type IV pili (TFP) represent a major mediator-less protein that contributes significantly to some of the more extensively studied electrogenic bacteria.

TFP (or "Nanowires"): Geobacter and Shewanella Species as Model Organisms

TFP have been found to be critical for the transfer of electrons generated metabolically to metal oxides (e. g. iron oxides; Reguera et al., 2005) that represent just one component of an MFC anode. These are the extendable (fully extended outside the cell, followed by retraction, degradation, and new pilus synthesis) proteinaceous appendages (for fundamental structures of three TFP of P. aeruginosa, N. gonorrhoeae and V. chol — erae, see Figure 9.5(a—c) from Craig et al., 2004). Pili are often essential for optimal biofilm formation in many bacterial genera (Zechner et al., 2012), a require­ment for mediator-less current on the MFC electrode(s) surface. Geobacter members are capable of reducing oxide of either insoluble iron (Fe3+) or manganese (Mn4+) that are directly coupled with organic carbon oxidation. The pilus extends from many bacteria to bind to and retract from surfaces for biofilm formation and disper­sion in some bacteria (O’Toole and Kolter, 1998) and is capable of a "grappling hook" retraction mechanism, followed by degradation, new pilus synthesis and extension, followed again by the complete retraction— degradation—synthesis—extension loop. The pili of elec­trogenic G. sulfurreducens have been termed "nanowires" due to their highly conductive properties (Reguera et al.,

2005) that appear to differ markedly from similar mem­bers of the same genus (e. g. G. metallireducens). These "nanowires" have also been described in S. oneidensis (Gorby et al., 2006) and are likely in many other bacteria.

The electrogenic importance of the pilus was proven in a deletion mutant strain of G. sulfurreducens pilA that generated nearly 10-fold less the power density than that of wild-type, pilus+ bacteria (Reguera et al., 2006).

These results were independent of anode composition, whether it is inexpensive, highly reproducible conduc­tive graphite, or gold, an expensive yet sometimes prob­lematic (reproducibility issues) anodic material (Richter et al., 2008). A longer isoform of PilA is critical for optimal power generation than a shorter PilA (Richter et al., 2012). Many genes, including those involved in flagella and pilus biosynthesis in G. sulfurreducens (and even bacterial human opportunistic pathogens, Proteus mirabilis and P. aeruginosa (Totten et al., 1990; Zhao et al., 1999)) are controlled by the nitrogen sigma factor, RpoN (Leang et al., 2009), as identified by microarray analyses. Thus, predictably strains lacking RpoN are not electrogenic when compared to wild-type bacteria. Yi et al. (2009) demonstrated indirect evidence that T4P in KN400 strain of G. sulfurreducens not only formed more robust biofilms but also provided superior power generation (KN400 current (7.6 A/m2) and power (3.9 W/m2); wild-type DL1-(1.4 A/m2 and 0.5 W/m2)). An excellent review by Lovley et al. (2011) has shed light on the unique processes involved in G. sulfurreducens metabolism and how such unique metabolic proper­ties lends to its reputation as a highly electrogenic organism.

Gorby et al. (2006) have shown that S. oneidensis MR-1 produce conductive pili in response to a reduction in or a lack of a terminal electron acceptor. Those researchers linked electron carrier proteins (c-type decaheme cyto­chromes MtrC and OmcA, see below) as well as muta­tions in the type II secretion pathway, where there are often periplasm protein modifications (e. g. disulfide bond formation) within Gram-negative bacteria. Thus, despite possessing pili, bacteria lacking specific cyto­chromes possessed reduced electrogenic properties.

Yi et al. (2009) isolated a mutant of G. sulfurreducens DL1 (KN400 strain) that was more effective in current production than wild-type bacteria. The paradoxical results were manifested with KN400 forming thinner biofilms, increased current production, great nanowire production, flagellum production, far less outer — surface c-type cytochromes and, above all, lower MFC internal resistance. Recently, however, an artificial matrix termed a conductive artificial biofilm (CAB) was developed that allows for adherence and nearly

11- fold increased conductive properties of Shewanella biofilm bacteria (Yu et al., 2011).

Cytochromes (Cell-Bound)

Redox properties of some bacterial cytochromes (either membrane-bound or soluble cytochromes (e. g. cytochrome c) electron carriers) have been connected with the conductive properties of pili (described above). Typically, these are critical for normal respiratory functions in both prokaryotic and eukaryotic cells.

In recent years, electrogenesis by metal-oxidizing Shewanella and Geobacter species as described above are facilitated by the production of pili and flagella, yet cy­tochromes have also emerged as one of the major drivers of the electrogenic process. This is due, in part, to the or­ganisms harboring such compounds transport and cellular localization of these redox-active cytochromes to the surface or near-surface of the aforementioned or­ganisms (Figure 9.6). Thus, the surface (e. g. an iron oxide (Fe3+) anode) has to be readily accessible to compo — nent(s) of the respiratory pathway of such organisms for optimal electrogenesis to occur. Using S. oneidensis as a model organism for examining the role of cyto­chromes in the electrogenic process, there are at least

10 gene products involved in iron reduction that are crit­ical for some features of electrogenesis in this organism, an event that has been studied by many research groups for more than two decades (Arnold et al., 1990). Conve­niently, most of the genes (especially mtr genes) involved in the process of iron oxide reduction and elec­trogenesis in MFCs are located in close proximity on the S. oneidensis genome (Figure 9.6). Figure 9.6 lists the or­ganisms that are also iron-oxidizing bacteria for

comparative purposes. Of the loci involved in metal reduction, these include mtrDEF, outer membrane cytochrome (omcA), followed by the mtrCAB genes. Figure 9.7 is a simplified recent schematic diagram of the mechanism of precisely how this process functions in S. oneidensis, elegantly described by Shi et al. (2012b). Prior to this exhaustive process of mechanistic functionality, the first genes found to be required for iron and manganese oxide reduction were performed

in S. putrefaciens using transposon mutagenesis in 1998 (Beliaev and Saffarini, 1998). MtrB was found to be an outer membrane cytochrome while the upstream locus, mtrA, encodes a periplasmic decaheme cytochrome. MtrC of both S. putrefaciens and S. oneidensis is also an outer membrane cytochrome with apparent terminal iron reductase activity (Beliaev et al., 2001; Hartshorne et al., 2007). Shi et al. (2006) demonstrated that OcmA, yet another decaheme cytochrome, binds under acidic conditions to MtrC and, in fact, these form a high — affinity protein complex with one another. MtrF, MtrD and MtrE appear to be homologs of MtrCAB, yet one set cannot replace the other functionally, although some components can coaggregate. The outlier is that the mtrFDE loci appear to be highly expressed in bio­films and the Mtr system, in general, is required for optimal biofilm formation (Coursolle et al., 2010), similar to aggregation of bacteria on a conductive sur­face such as an iron oxide anode in MFCs.

In summary, the order of electron flow for optimal electrogenesis of S. oneidensis is the following: cyto­plasmic membrane-bound menaquinone, periplasmic tetraheme CymA with electron flowing through the b-barrel of MtrB to the decaheme cytochromes MtrA/ F and finally to two other decaheme proteins, MtrC and OmcA (Figure 9.7). The final destination for elec­trons prior to reduction of iron oxides is MtrC. Thus, again, it is intuitive that the genes encoding those proteins involved in iron reduction are localized in the following order on the S. oneidensis genome, mtrDEF—omcA—mtrCAB, respectively. Similar to the discovered mechanism of proton pumping in the F1F0-ATP synthase using bacteriorhodopsin in mem­brane vesicles, scientists have proven that the protein complex of MtrCAB conducts electron when embedded within membrane vesicles (Hartshorne et al., 2009). In 2010, Tai et al. (2010) assessed potential networks of transcriptional regulation between chemotactic and electron transport properties and found that previously unknown roles of genes including cheA (a chemotaxis gene), mgtE-1 (an Mg2+ transport gene) and SO4572 (a triheme cytochrome gene). More recently, Leang et al. (2010) showed that the redox-cytochrome OmcS of G. sulfurreducens actually binds to the conductive pili, thereby contributing to their electrogenic proper­ties. However, using a whole-cell cyclic voltammetric analysis of various mutant strains including (DmtrC/ DomcA), transmembrane pili (DpilM-Q, DmshH-Q, and DpilM-Q/DmshH-Q) and flagella (Dflg), Carmona et al. (2011) demonstrated that even with such mutations in place, often there are "by-pass" mechanisms of electron transfer, still allowing for some level of electro­genesis using cyclic voltammetric techniques. A synop­sis of these results is shown in schematic form in Figure 9.8.

Brief Synopsis of the S. oneidensis MR-1 Bioelectrochemical Machinery in Reverse:

Potential Role in the Biosynthesis of Biofuels in MFCs

The multiple proteins and other factors involved in bacterial electrogenesis in MFCs are complex. A pro­cess termed electron flow reversal, or, better put, electron diversion, is critical for a nonelectrogenic process for the purpose of generating single or multiple com­pounds of value. Ross et al. (2011) have helped simpli­fying many features of this process in their 2011 publication. Obviously, the goal of scientists working with electrogenic bacteria is to maximize their power density while wasting the energy harness in the carbon skeletons they consume for sustenance. From the above information collectively, it appears that TFP and cyto­chromes involved in the Mtr respiratory pathway facil­itate the transfer of direct current in the form of electrons to one or more electrodes. Figure 9.9 helps simplifying what is currently understood of these sys­tems and other drivers that will be discussed below. This process is also clearly dependent upon the carbon sources (or feedstock in more complex, multisubstrate systems). In that study, multiple isogenic mutants were created that (1) lacked the periplasmic fumarate reductase (FccA) and thus could not reduce fumarate using electrons derived from electrodes, a process adversely affected by nearly 90% by (2) deletion of mtrB, or worse, (3) the periplasmic cytochrome, MtrA, and prevention of menaquinone biosynthesis.