Process Biochemistry 47 (2012) 1707–1714
Contents lists available at SciVerse ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Bacterial extracellular electron transfer in bioelectrochemical systems Yonggang Yang a,b,c , Meiying Xu a,b,c,∗ , Jun Guo a,b,c , Guoping Sun a,b,c a
Guangdong Provincial Key Laboratory of Microbial Culture Collection, Guangdong Institute of Microbiology, Guangzhou 510070, China State Key Laboratory of Applied Microbiology (Ministry-Guangdong Province Jointly Breeding Base), South China, Guangzhou 510070, China c Guangdong Open Laboratory of Applied Microbiology, Guangzhou 510070, China b
a r t i c l e
i n f o
Article history: Received 5 June 2012 Received in revised form 22 July 2012 Accepted 27 July 2012 Available online 4 August 2012 Keywords: Bioelectrochemical systems Microbial fuel cell Bioﬁlms Electron transfer Extracellular respiration
a b s t r a c t Bioelectrochemical systems (BES), typically microbial fuel cells (MFCs), have attracted increasing attention in the past decade due to their promising applications in many ﬁelds, such as bioremediation, energy generation and biosynthesis. Current-generating microorganisms play a key role in BES. The process of transferring electrons to electrode has been considered as a novel anaerobic bacteria respiration, and more and more bacteria capable of exchanging electrons with electrodes have been isolated. Among those bacteria, Shewanella and Geobacter genera are the most frequently used model organisms in the studies of BES, as well as the bacteria-electrode electron transfer mechanisms. Many signiﬁcant new ﬁndings in the ﬁeld of the bacterial extracellular electron transfer in BES have been reported recently. A better understanding of the mechanisms of bacterial extracellular electron transfer would provide more efﬁcient strategies to enhance the applicability of BES. This review summarizes the recent advances of extracellular electron transfer mechanisms with foci on Shewanella and Geobacter species in BES. © 2012 Elsevier Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electron transfer by c-type cytochromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bacterial nanowire . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electron shuttles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electron transfer in bioﬁlms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Bioelectrochemical systems (BES), typically microbial fuel cells (MFCs), have emerged as promising technologies for bioremediation, energy generation and many other potential applications. Microorganisms, especially those attached to electrodes as bioﬁlms, play a key role in current generation, biodegradation or biosynthesis in BES. Due to the capacity of extracellular electron transfer (EET) to electrode, directly or indirectly, the currentgenerating bacteria could be deﬁned as exoelectrogens . Electron transfer from exoelectrogens to electrode could couple with energy conservation and support their growth, thus could be considered as
∗ Corresponding author at: Guangdong Institute of Microbiology, Guangzhou 510070, China. Tel.: +86 20 87684471; fax: +86 20 87684587. E-mail address: [email protected]
(M. Xu). 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.07.032
1707 1708 1709 1710 1710 1712 1712 1712 1712
electrode/anode respiration [2–4]. Driven by the increasing interests in BES, bacterial electrode respiration and electromicrobiology has received plenty of attention in recent years [1,5–10]. Despite the reported differences, bacterial dissimilatory metal reduction (BDMR) has been considered as the most similar process to electrode respiration [9,11]. The BDMR model bacteria, Shewanella and Geobacter species, are also the most frequently used bacterial models in BES. Many excellent reviews focusing on the BDMR have been reported previously [12–15]. This review article will focus on the recent advances of bacterial extracellular electron transfer mechanisms in BES with foci on Shewanella and Geobacter species. Shewanella species are gram-negative, facultative bacteria which widely thrive in aquatic and sediment environments . Despite the pyruvate fermentative capacity , Shewanella species are usually considered as obligate respiratory bacteria capable of utilizing a wide range of carbon/energy resources (e.g., lactate,
Y. Yang et al. / Process Biochemistry 47 (2012) 1707–1714
glucose and DNA, but not acetate under anaerobic condition) . Shewanella have the most diverse respiration strategies among all the known bacteria to date, including direct electron transfer via outer membrane c-type cytochromes (OMCs), nanowires and indirect electron transfer via endogenously secreted ﬂavins. To date, 24 Shewanella genomes have been completely sequenced and published (www.genomesonline.org), providing a powerful database for comparative genomic analysis of gene functions, regulations, and biochemical pathways in this genus. As far as we know, 10 Shewanella species, including over 20 strains and over 50 mutants, have been used in BES studies (Table S1). Geobacter species are gram-negative obligatory anaerobic bacteria, which are also ubiquitous in aquatic and sediment environments . In addition to acetate, Geobacter spp. can use H2 , lactate and formate as electron donors in current generation [19,20]. Geobacter species are usually the most dominant members in BES electrode bioﬁlms . In contrast with Shewanella, Geobacter utilize only direct electron transfer manners (via nanowire and/or OMCs) in BDMR and electrode respiration. Nine Geobacter genomes have been completely sequenced and published (www.genomesonline.org) to date.
2. Electron transfer by c-type cytochromes c-Type cytochromes (CTCs), which have been considered as one of the most important electron transfer strategy in current generation by exoelectrogens, are widespread heme-containing proteins in most bacteria and archaea. S. oneidensis MR-1 has 42 putative CTCs and 80% of them locate in the outer membrane, covering 8–34% of the cell surface . CymA is a tetraheme-CTC with the N-terminal anchored in the inner membrane and the C-terminal exposed to periplasm (Fig. 1) [12,23]. CymA participates in many Shewanella anaerobic respiration processes. In the case of electrode reduction, a deletion of cymA gene caused >80% decrease in current generation [24,25]. CymA could directly interact with many terminal reductases in periplasm, such as fumarate reductase (FR) and nitrate reductase. Bacterial two-hybrid showed pairwise interactions among CymA, MtrA and some other periplasmic redox proteins (e.g., FR, iron-induced ﬂavocytochrome), indicating that CymA is the major electron conduit to periplasmic space and could directly interact with periplasmic redox proteins by forming a transient protein complex [26,27]. In support of this hypothesis, a study using electrode as electron donor for fumarate reduction by S. oneidensis MR-1 showed that 85% electrons were transferred to FR via CymA and 15% via MtrA, suggesting a CymA–FR–MtrA complex in periplasm . The MtrABC conduit could be considered as an extended branch of the periplasmic complex. It has been reported that IS-insertion activated SirCD expression could partially replace CymA, forming an alternative electron transfer conduit for reduction of several soluble electron acceptors . However, the role of this substitution in electrode reduction has not been studied. MtrABC complex plays a key role as an electron conduit linking the electrode and the intracellular electron ﬂux, by which the electrons transfer across the ∼40 A˚ span of the outer membrane . The three proteins in this complex are physically associated with and functionally dependent on each other. MtrA is a periplasmic protein delivering electrons from CymA to MtrC. Moreover, MtrA is necessary for the stability of MtrB, which is a ␤-barrel trans-membrane protein containing no heme moiety and essential for the proper localization and interaction of MtrA and MtrC [29,30]. MtrC and OmcA are two lipoproteins which have been considered as the terminal extracellular reductases of S. oneidensis MR-1 . Although OmcA could donate electrons to many solid electron acceptors in vitro, MtrC was considered to be more
important than OmcA in many Shewanella EET processes [15,32]. A deletion of mtrC resulted in >90% current decrease in BES . A similar decrease was also found in the reduction of electron shuttles by mtrC mutant while the omcA mutant showed comparable shuttle-reducing capacity with the wild-type strain . The electron transfer rate constant (k0 ) of S. oneidensis MR-1 MtrABC complex on a graphite electrode was estimated to be 195 s−1 , which is at the same order of magnitude with the constant of each individual protein in the complex  (Table 1). Those values are also comparable to the k0 of S. loihica PV-4 OMCs (150 ± 10 s−1 ) , as well as the enzyme-electrode optimized for biosensors [35,36]. In comparison with other anaerobic respiration processes, the electron transfer rate of electrode reduction by S. oneidensis MR-1 in several orders of magnitude higher than haematite reduction but is signiﬁcantly lower than the reductions of soluble electron acceptors . Single or multiple deletion of the primary gene involved in electron transfer of S. oneidensis MR-1, such as cymA or mtrABC, could not completely eliminate the current generation, indicating the existence of alternative electron transfer conduits. Genome analysis has identiﬁed a serial of mtrABC-homologues encoding MtrDEF in the same operon [16,24]. MtrF, the homologue (identity >30%) of MtrC and OmcA, showed higher reducing capacity of ﬂavins and soluble electron acceptors than OmcA or even MtrC in vitro . In S. oneidensis MR-1 mutants with reconstructed Mtr homologues, MtrE could functionally replace MtrB and the complementation of mtrF/mtrA gene pair showed a comparable Fe(III)-reducing capacity with the complementation of mtrC/mtrA . Mtr homologues form an overlapping and branching respiratory network in Shewanella, which might be crucial for the survival of Shewanella in subsurface environments. Recently, the small angle X-ray scattering properties of OmcA and X-ray crystal structure of MtrF were reported [39,40], providing molecular information to understand the intramolecular electron transfer mechanism of CTCs, such as the functions and interactions of hemes and the structural responses of CTCs to the electron donor/acceptor. Comparative genomic analyses of six Geobacter species showed an average of 79 putative CTCs in each Geobacter genome and only 14% of which are conserved in all genomes . It has been proposed that MacA delivers electrons from inner membrane to PpcA in periplasm, and PpcA subsequently transfers electrons to the OMCs (e.g., OmcB, OmcE, OmcS, OmcZ) . 3D structures and the electronic interaction of MacA and PpcA have been determined . Although inconsistent results on the roles of several OMCs in current generation have been shown in different studies [43,44], the OMCs involved in Geobacter electrode reduction is different from those for dissimilatory metal reduction [15,20]. For instance, OmcB and OmcS are crucial for Fe(III) reduction while the mutants lacking omcB or omcS gene showed no or less impact on current generation [15,45]. OmcZ is the only OMC considered to be essential for current generation by G. sulfurreducens . Comparative genome analyses showed that omcZ is not conserved in all Geobacter species . A deletion of omcZ resulted in >90% current decrease while showed no effect on the reductions of other electron acceptors including Fe(III) oxide . Although no signiﬁcant difference was observed for the transcription level of several OMC genes throughout the G. sulfurreducens bioﬁlm based on microarray data , immunogold labeling showed that OmcZ were mostly accumulated at the bioﬁlm–electrode interface in MFCs for electron transferring , which could be due to the electrostatic responses and spatial redistributions of OmcZ in electrode bioﬁlms. Moreover, more signiﬁcant effects on the electron transfer were observed in thick G. sulfurreducens bioﬁlms (>10 m) than in the thinner bioﬁlm when omcZ gene was deleted, suggesting that OmcZ is particularly important for long-distance electron transfer in bioﬁlms.
Y. Yang et al. / Process Biochemistry 47 (2012) 1707–1714
Fig. 1. Extracellular electron transfer model of Geobacter and Shewanella. (I) The OMC-based direct electron transfer conduit in Geobacter; (II) bacterial nanowire; (III) electron transfer network of Shewanella including ﬂavins and CTCs; (IV) electrode respiration-coupled proton motive force and energy (ATP) generation.
Table 1 Electrode respiration related c-type cytochromes in Shewanella and Geobacter spp. Bacteria
Redox potential (midpoint/window)
S. oneidensis MR-1
CymA MtrA MtrC OmcA
Inner membrane Periplasm Outer membrane Outer membrane
4 10 10 10
140 s−1 126 s−1 276 s−1 Unknown
−200/250 mV  −100/300 mV  −138/275 mV  −175/300 mV 
Outer membrane Outer membrane
−180/360 mV  −212/320 mV 
An counterintuitive phenomenon has been shown and discussed in several in vitro studies that the redox potentials of the upstream of electron transfer components are usually higher than those of the downstream OMCs in Shewanella and Geobacter (Table 1) [29,34,49], which seems to be contrary to the presumed energy-generating electron transfer chain. However, the electrochemical properties of the CTCs in vivo might be different due to the protein–protein interactions and the speciﬁc chemical intercellular environments. To date, most of the reported exoelectrogens are gram-negative (G− ) bacteria. The gram-positive (G+ ) cell wall was considered unfavorable for EET . However, G+ bacteria have been found as the dominant dissimilatory metal reducing bacteria in some environments. Recently, the direct electron transfer mechanism of a G+ exoelectrogen, Thermincola potens, was elucidated. T. potens has 32 putative CTC genes in its genome and several CTCs were proposed to be anchored and closely arranged across the cell wall, forming an electron conduit for electrode reduction [51,52].
3. Bacterial nanowire Bacterial nanowire has been known as a novel electron transfer strategy since it was found as electrically conductive pili during Fe(III) oxide reduction by G. sulfurreducens . Subsequently, bacterial nanowires were observed in S. oneidensis MR-1 and some other bacteria, or between different bacterial species , indicating a widely environmental distribution of those bacterial appendages. S. oneidensis MR-1 nanowires (50–150 nm in diameter) are generally present in the form of bundles of individual conductive pilus-like appendages with a diameter of 3–5 nm [50,56] under the electron acceptor limiting or low agitating condition [56,57]. Atomic force microscopy (AFM) showed that the nanowires divided into the membrane pores and extended to tens of micrometers to contact with solid electron acceptors or bacteria partners.
Electrical conductivity across the diameter or along the length of the sheared nanowire has been demonstrated [56,58]. The measured resistivity of S. oneidensis MR-1 nanowire was 1 /cm, ranged in the scope of semiconductor. The k0 of S. oneidensis MR-1 nanowire was estimated to be 1 × 109 s−1 while that of the whole cell was 2.6 × 106 s−1 , both of which are several orders of magnitude higher than the k0 of OMCs. Those results suggest that nanowires might be the most thermodynamically favorable electron pathway in EET. Deletion of mtrC/omcA gene or interruption of type II secretion system could signiﬁcantly decrease the conductivity of Shewanella nanowires, indicating that CTCs are necessary for the conductivity of Shewanella nanowires . However, the molecular compositions and the electron transfer mechanisms of Shewanella nanowires are still unclear. S. oneidensis MR-1 generates different types of ﬁlamentous appendages, including Msh-pili, type IV pili and ﬂagella. Gene deletion analysis showed that Msh pilin were necessary for optimal current generation while the deletions of several other genes (pilD, pilM-Q or ﬂg) involved in type IV pili or ﬂagella generation could increase current generation [59,60]. G. sulfurreducens nanowires (3–5 nm in diameter and up to 20 m in length) locate on one-side of the cell when the cell grow on the surface of metal oxides or during fumarate reduction at a low temperature (25 ◦ C) like some other bacterial pili [55,57]. There are two PilA isoforms with distinct functions in bioﬁlm formation and electron transfer in G. sulfurreducens nanowires . The deletion of pilA gene could diminish the nanowire production and signiﬁcantly decrease the current generation capacity of thick bioﬁlms (>50 m)  and showed less effect on the current generation by thin bioﬁlms (<10 m) , which is similar to the deletion of omcZ in G. sulfurreducens. It has been hypothesized that OmcS may facilitate the nanowire electron transfer as electron mediators or conductive elements since OmcS always distributes along the nanowires in extracellular space . However, the mean interval (28.6 nm) between two nanowire-binding OmcS exceeds the threshold distance (2 nm) of direct electron transfer between two proteins . It is possible that some other redox proteins such as OMCs
Y. Yang et al. / Process Biochemistry 47 (2012) 1707–1714
participate in and facilitate the electron transfer along the nanowires. The electronic conductivity of G. sulfurreducens nanowires was measured to be 6 mS/cm , which is comparable to the synthetic metallic nanostructures and higher than the proposed threshold (10−3 mS/cm) for efﬁcient current generation in BES . In contrast to the OMC-dependent conductivity of Shewanella nanowires, Geobacter nanowires showed intrinsic, metal-like electronic property, which could not be decreased by the deﬁcient in OMCs . In addition to electron transfer, nanowires have been shown to participate in bioﬁlm formation or as a protective mechanism of G. sulfurreducens in uranium reduction [64,65]. However, it should be noted that many nanowire-like structures might exist in microbial samples, such as dehydrated extracellular polysaccharides or protein-transporting nanotubes between bacterial cells [66,67]. Thus, speciﬁc devices (e.g., AFM) or reliable experiments must be used to identify the conductive nanowires.
4. Electron shuttles A proper electron shuttle in BES should be (1) dissolvable, (2) stable, (3) reusable, (4) environment-friendly, and (5) have a proper redox potential. Many bacteria, mostly G− bacteria, are capable of secreting electron shuttles in BES (Table 2). Moreover, it has been suggested that bacterial shuttle secretion could be stimulated by current generation in MFCs . The endogenously secreted ﬂavins by Shewanella species (Fig. 1), mainly riboﬂavin (RF) and ﬂavin mononucleotide (FMN) [37,69,70], were the mostly documented electron shuttles in BES. RF is produced as the precursor for ﬂavin adenine dinucleotide (FAD) synthesis in cytoplasm. FAD is then transported to periplasmic space wherein UshA hydrolyzes FAD to FMN and AMP. FMN could freely diffuse through the outer membrane and then be converted to RF in extracellular space while RF could not be transported back to Shewanella periplasm [71,72]. Kinetic measurements showed that electron transfer rate of puriﬁed S. oneidensis MR-1 OMCs to insoluble iron mineral was 100–1000 times lower than the whole cell and the addition of ﬂavins could increase the reduction efﬁciency to a level comparable with the whole cell . Secretion of microgram level ﬂavins by S. oneidensis MR-1 could enhance the electron transfer efﬁciency by over 3.7 folds while the ATP cost on ﬂavin secretion was negligible compared with the resulting energy beneﬁt . The estimated k0 of ﬂavins absorbed on an electrode (<0.7 s−1 ) was approximately two orders of magnitude lower than that of the OMCs . Shewanella Mtr complex plays an essential role in ﬂavins reduction. MtrC accounted for 50% of the ﬂavins reduction activity and two putative ﬂavin-binding domains were founded in its homologue (MtrF) [33,40]. In addition to CTCs, some other redox proteins of Shewanella might participate in ﬂavin reduction and contribute to current generation [32,38]. Our recent results indicated signiﬁcant current-generating capacity in S. decolorationis S12 ccmA-mutant which was deﬁcient in CTCs biosynthesis when sufﬁcient ﬂavins were offered (Yang et al., unpublished data). Despite the evidences for endogenous secretion of ﬂavins by Shewanella, the possibility that ﬂavins are by-products of cell lysis were discussed recently . Although ﬂavin secretion is commonly observed in many organisms, the function of ﬂavins could be limited in ﬁeld-applied BES since they are light-degradable and could be utilized as carbon resource by some bacteria or archaea . Phenazines generated by a diverse of bacteria, particularly Pseudomonas species, were also intensively studied as intrinsic electron shuttles in MESs . The presence of phenazines enhanced the current-generating capacity of a gram positive bacterium Brevibacillus sp. PTH1 , indicating that electron shuttles in MESs
provide a synergic strategy for microbial members in currentgeneration. It should be noted that phenazines can act as virulence factors or cell signals impacting microbial community compositions and bioﬁlm formations . In addition to the bacteria-generated organics, H2 generated from microbial fermentation or geochemical reaction in BES could be considered as electron shuttles or donors for Shewanella and Geobacter. However, utility of H2 for current generation could become negligible in the presence of H2 -utilizing methanogens . To successfully start-up and operate of a BES, the prevention of methanogenesis, especially in anode chamber, is essential. Furthermore, some other redox chemicals or compounds widely distributed in environments (e.g., humics, manganese species or polysulﬁde) could also be used as electron shuttles. However, compared with direct electron transfer, this EET strategy in BES has several drawbacks such as (1) higher overpotential, and (2) low diffusion co-efﬁciency in bioﬁlms, especially for the organic shuttles [77,78]. Moreover, for exoelectrogens, shuttle secretion would be a metabolic pressure and have competitive disadvantage, especially in the case that the shuttles were degraded or utilized by other members in BES .
5. Electron transfer in bioﬁlms Bioﬁlms consist of numerous microbial cells which are densely stacked and spatially distributed in the extracellular polymeric substances (EPS). A complex electron network involving various electron transfer components could be presumed in a currentgenerating bioﬁlm. In addition to the electron delivers, other factors, such as the diffusion co-efﬁciency, pH gradient and the arrangement of the electron transfer components in EPS, could signiﬁcantly impact the electron transfer in bioﬁlms (Fig. 2). A prevalence of redox proteins including OMCs has been identiﬁed in the EPS of Shewanella bioﬁlms . It was estimated that an electrode colonized by a single-layer of Shewanella bioﬁlm has 10–30% surface coverage of OMCs , indicating a key role of OMCs in the electron transfer network in Shewanella bioﬁlms. Microarray analysis showed that the transcription of the entire mtr operon was up-regulated in a current-generating bioﬁlm compared with that in soluble Fe(III) reduction and aerobic respiration . Likewise, the expression of RF biosynthesis enzyme (RibB) was also up-regulated when S. oneidensis MR-1 was grown in a bioﬁlm , which is consistent with the reports that ﬂavin concentrations in S. oneidensis MR-1 culture medium were generally lower (0–5.8 M/g of protein) than those in electrode bioﬁlm (7.7 M/g of protein) [37,69,70]. Intriguingly, UshA, which was previously suggested as a FAD hydrolyase in periplasm, was abundantly identiﬁed in bioﬁlm EPS . Voltammetry showed that the redox potential of ﬂavins was −200 mV (vs standard hydrogen electrode, SHE) while the representative potential of OMCs in Shewanella bioﬁlm was ∼0 mV (vs SHE) . Therefore, ﬂavins might be more thermodynamically favorable than OMCs in electron transfer to an electrode with negative potential (<0 mV vs SHE). Considering the importance of electrostatic interactions in Shewanella bioﬁlm formation, the electronegative and hydrophilic cellular surface of Shewanella is favorable for binding and donating electrons to a positively poised electrode [25,81]. Polysaccharide is one of the dominant components in EPS of S. oneidensis MR-1 bioﬁlm. However, the insulate polysaccharide would impede the electron transfer in bioﬁlms. Disruption of the gene (SO 3177) involved in polysaccharide biosynthesis by S. onidensis MR-1 could enhance the cell adhesion to electrode and increase the current generation by 50% compared with the wild-type strain . Mobility and chemotaxis are also important for bioﬁlm formation. Interestingly, deletions of the chemotaxis-related genes (e.g., cheA,
Y. Yang et al. / Process Biochemistry 47 (2012) 1707–1714
Table 2 Bacteria-secreted electron shuttles in BES. Bacteria
Shewanella spp. Lactococus lactis Pseudomonas aeruginosa Pseudomonas sp. CMR12a P. alcaliphila MBR Klebsiella pneumoniae Klebsiella sp. ME1 E. coli K-12 Bacillus subtilis Geothrix fermentans
G− G+ G− G− G− G− G− G− G+ G−
RF, FMN  2-Amino-3-dicarboxy-1,4-naphthoquinone  Pyocyanin  Phenazine-1-carboxamide  Phenazine-1-carboxylic acid  2,6-Di-tertbutyl-p-benzoquinon  Quinone  Hydroquinone  Unknown  Unknown 
−208 mV, −219 mV −71 mV −32 mV −150 mV −275 mV −475 mV −296 mV −45 mV −13 mV Unknown
Fig. 2. The presumed electron transfer network in current-generating bioﬁlms. Exoelectrogens and their bacterial partners are densely embedded in the bioﬁlm EPS. The relative diffusion coefﬁciency decreases from 1.0 at the bioﬁlm–liquid culture interface to a mean value of 0.4 in bioﬁlms , and pH could decrease from 7.0 to 6.0 due to proton accumulation in bioﬁlms . Organic electron shuttles like ﬂavins tend to be absorbed in bioﬁlms or on the surface of electrode. OMCs or other redox proteins are prevalent in bioﬁlms and some of them, such as OmcZ, accumulate at the electrode–bioﬁlm interface as a conductive layer.
pilD or ﬂg) increased the current generation by S. oneidensis MR-1 . Studies showed that the bioﬁlm formation and currentgenerating manners of Shewanella species are species-dependant. Current generation by S. onidensis MR-1 was planktonic celldominated [60,83,84] while the current generation by S. loihica PV-4 and S. decolorationis S12 were proposed to be bioﬁlmdominated [84,85]. Bioﬁlm conductivity has been shown as decisive variable for current generation . The electronic conductivity of G. sulfurreducenus KN400 bioﬁlm was measured to be 5 mS/cm, which is comparable to that of synthetic organic metallic nanostructures , and nanowires were suggested to be the major components accounting for the intrinsic conductivity. Signiﬁcant electronic conductivity was also detected in the co-cultured Geobacter aggregates , methanogenic aggregates  and multispecies currentgenerating bioﬁlms . Geobacter species were abundant in all these conductive samples. In contrast, no signiﬁcant conductivity was detected in the bioﬁlms of E. coli and P. aeruginosa , suggested that the conductivity was speciﬁcally contributed by Geobacter species, even though many biomaterials (e.g., DNA, cell wall) have comparable conductivity . In bioﬁlms with metal-like conductivity, electrons are delocalized and can be transferred without thermal activation . A different bioﬁlm electron transfer mechanism, superexchange, was described and discussed in several studies [57,90,91], in which electrons were transferred via a succession of closely arranged redox
species (e.g., OMCs, shuttles). The prevalence of redox proteins like OMCs in current-generating bioﬁlms were presumed to be essential for superexchange, especially in a shuttle-free bioﬁlms. Moreover, in vivo analysis showed that OMCs in G. sulfurreducenus bioﬁlm could function as supercapacitors for charge storage and release . Extracellular polysaccharide network in bioﬁlm EPS has been shown to be essential for anchoring and arranging of OMCs in G. sulfurreducenus bioﬁlm , which is in contrast with the suggested impeding role of polysaccharide in S. onidensis MR-1 bioﬁlm . Spectral and electrochemical analyses showed that the composition of the Geobacter bioﬁlm OMC network varied with the electrode potential . Compared with the efﬁcient EET process in bioﬁlms, substrate oxidation by exoelectrogens was presumed to be the rate-limiting step in bacterial electrode reduction . Starvation and proton accumulation resulting from the low diffusion coefﬁciency in bioﬁlms can strongly suppress the viability of the bioﬁlm cells, especially for the bottom layers [78,93]. Indeed, a live-top/dead-bottom stacking proﬁle was usually observed in noncurrent-generating bioﬁlms . However, confocal laser microscopy and microarray analyses showed that the bottom layers of the current-generating Shewanella and Geobacter bioﬁlms performed similar or even higher viability than the outer layers [48,85,94]. It has been shown that the electron transfer rate of G. sulfurreducenus bioﬁlm increased proportional with bioﬁlm growth until the bioﬁlm reached a thickness of 20 m . The low electron transfer rate of the bioﬁlm cells with a distance greater than 20 m from electrode would decrease the bioﬁlm growth and
Y. Yang et al. / Process Biochemistry 47 (2012) 1707–1714
conductivity . However, Geobacter bioﬁlm across a 100 m gap showed a comparable conductivity with that of a thinner bioﬁlm (50 m) . For the gram-positive bacterium T. potens, direct electron transfer via OMCs was the only proposed EET strategy and the bioﬁlm cells in bottom layers have been suggested to be primary for current generation . Similar to the Geobacter bioﬁlm lacking nanowires , the outer layer cells in T. potens bioﬁlms showed low viability , which highlighted the importance of nanowires or electron shuttles in long-distance electron transfer and the growth of bioﬁlms.
6. Conclusions and future directions In addition to power supply and bioremediation, many new functions and advantages of BES have been developed, such as microbial electrolysis cells, microbial electrosynthesis cells, microbial solar cells, and microbial electrochemical snorkels [98–101]. Bacterial EET plays key roles in these systems. Therefore, understanding the mechanisms of bacterial EET is not only signiﬁcant for the optimization of BES but also explore bio-geochemical science (e.g., BDMR and the natural electric current ). From microbiological viewpoint, several newly emerged issues or questions in EET should be further investigated.
Acknowledgements We acknowledge that this work has been supported by the Team Project of the Natural Science Foundation of Guangdong, China (9351007002000001), Postdoctoral Foundation of Guangdong Academy of Sciences (20120001), the Natural Science Foundation of Guangdong Province (S2011010004267), the National Basic Research Program of China (973 Program) (2012CB22307), the Chinese National Programs for High Technology Research and Development (863 Program) (2011AA060904), the Outstanding Scholarship Foundation of Guangdong Academy of Sciences (200902), the Guangdong-Hongkong Technology Cooperation Funding (2009A030902003), and the Guangdong Province-Chinese Academy of Sciences strategic cooperative project (2009B091300023, 2010B090301048). We thank Dr. Zhili He (Oklahoma University) and Dr. Weimin Wu (Stanford University) for their helpful suggestions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.procbio. 2012.07.032. References
(1) The mechanism of cell-to-cell electron transfer. In addition to the indirect electron transfer from G. sulfurreducens to a partner bacterium via electron mediators [103,104], direct intercellular electron transfer between cells of Geobacter species was reported recently . Moreover, symbiotic correlation and direct electron transfer between Geobacter and methanogens (e.g., Mechanococcus and Mechanosaeta) have been suggested [88,105]. OMCs and nanowires were demonstrated to be essential for Geobacter cell-to-cell electron transfer. However, most methanogens have no CTCs. How the electrically linked microbes recognize each other and donate/accept electrons remains unknown. (2) Electrons transfer from electrode to bacterial cells. Electrode could be utilized as an electron acceptor or electron donor for Shewanella or Geobacter electron transfer [25,106]. Compared with bacterial electrode reduction, less is known about the mechanism of the bacterial electrode oxidation [100,107]. Microarray and electrochemical analyses have shown that the electrode oxidation by G. sulfurreducens shared almost no common respiratory components with electrode reduction . In contrast, Ross et al. suggested that electron transfer from electrode to S. oneidensis MR-1 utilized the same Mtr pathway but in a reversed direction (i.e. electrode–MtrCBA–CymA) compared with Shewanella electrode reduction . However, in vitro voltammetry suggested that the Shewanella CTCs could not transfer electrons in the reversed direction . How the electrode-oxidizing bacteria accept electrons from electrode and along which pathway the electrons were transferred is needed to be elucidated.
Given the electron transfer components are arranged in a thermodynamically favorable order for electron transfer from inside to outside of a bacterium, the reversed electron transfer using the same set of components seems unfavorable, such as the accepting electrons from an electrode or a bacterial partner. How bacteria address those problems, change the chemical properties of the electron transfer components by modifying their molecular structures or employ a different electron transfer pathway, is yet unknown.
 Logan BE. Exoelectrogenic bacteria that power microbial fuel cells. Nat Rev Microbiol 2009;7:375–81.  Rosenbaum MA, Bar HY, Beg QK, Segre D, Booth J, Cotta MA, et al. Transcriptional analysis of Shewanella oneidensis MR-1 with an electrode compared to Fe(III)citrate or oxygen as terminal electron acceptor. PLoS One 2012;7:e30827.  Torres CI, Marcus AK, Rittmann BE. Kinetics of consumption of fermentation products by anode-respiring bacteria. Appl Microbiol Biotechnol 2007;77:689–97.  Kim BH, Kim HJ, Hyun MS, Park DH. Direct electrode reaction of Fe(III)-reducing bacterium, Shewanella putrefaciens. J Microbiol Biotechnol 1999;9:127–31.  Lovley DR. Reach out and touch someone: potential impact of DIET (direct interspecies energy transfer) on anaerobic biogeochemistry, bioremediation, and bioenergy. Rev Environ Sci Biol 2011;10:101–5.  Lovley DR. Future shock from the microbe electric. Microb Biotechnol 2009;2:139–41.  Lovley DR. The microbe electric: conversion of organic matter to electricity. Curr Opin Biotechnol 2008;19:564–71.  Lovley DR. Bug juice: harvesting electricity with microorganisms. Nat Rev Microbiol 2006;4:497–508.  Lovley D. Taming electricigens. Scientist 2006;20:46.  Lovley D. Electromicrobiology. Annu Rev Microbiol 2012;66:391–409.  Logan BE, Regan JM. Electricity-producing bacterial communities in microbial fuel cells. Trends Microbiol 2006;14:512–8.  Shi L, Squier TC, Zachara JM, Fredrickson JK. Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes. Mol Microbiol 2007;65:12–20.  Gralnick JA, Newman DK. Extracellular respiration. Mol Microbiol 2007;65:1–11.  Shi LA, Richardson DJ, Wang ZM, Kerisit SN, Rosso KM, Zachara JM, et al. The roles of outer membrane cytochromes of Shewanella and Geobacter in extracellular electron transfer. Environ Microbiol Rep 2009;1:220–7.  Richter K, Schicklberger M, Gescher J. Dissimilatory reduction of extracellular electron acceptors in anaerobic respiration. Appl Environ Microbiol 2012;78:913–21.  Fredrickson JK, Romine MF, Beliaev AS, Auchtung JM, Driscoll ME, Gardner TS, et al. Towards environmental systems biology of Shewanella. Nat Rev Microbiol 2008;6:592–603.  Meshulam-Simon G, Behrens S, Choo AD, Spormann AM. Hydrogen metabolism in Shewanella oneidensis MR-1. Appl Environ Microbiol 2007;73:1153–65.  Mahadevan R, Palsson BO, Lovley DR. In situ to in silico and back: elucidating the physiology and ecology of Geobacter spp. using genome-scale modelling. Nat Rev Microbiol 2011;9:39–50.  Speers AM, Reguera G. Electron donors supporting growth and electroactivity of Geobacter sulfurreducens anode bioﬁlms. Appl Environ Microbiol 2012;78:437–44.  Stams AJM, de Bok FAM, Plugge CM, van Eekert MHA, Dolﬁng J, Schraa G. Exocellular electron transfer in anaerobic microbial communities. Environ Microbiol 2006;8:371–82.
Y. Yang et al. / Process Biochemistry 47 (2012) 1707–1714  Lovley DR. Live wires: direct extracellular electron exchange for bioenergy and the bioremediation of energy-related contamination. Energ Environ Sci 2011;4:4896–906.  Lower BH, Hochella MF, Lower SK. Putative mineral-speciﬁc proteins synthesized by a metal reducing bacterium. Am J Sci 2005;305:687–710.  Ross DE, Ruebush SS, Brantley SL, Hartshorne RS, Clarke TA, Richardson DJ, et al. Characterization of protein–protein interactions involved in iron reduction by Shewanella oneidensis MR-1. Appl Environ Microbiol 2007;73:5797–808.  Coursolle D, Gralnick JA. Modularity of the Mtr respiratory pathway of Shewanella oneidensis strain MR-1. Mol Microbiol 2010;77:995–1008.  Ross DE, Flynn JM, Baron DB, Gralnick JA, Bond DR. Towards electrosynthesis in Shewanella: energetics of reversing the mtr pathway for reductive metabolism. PLoS One 2011;6:e16649.  Borloo J, De Smet L, Van Beeumen JJ, Devreese B. Bacterial two-hybrid analysis of the Shewanella oneidensis MR-1 multi-component electron transfer pathway. J Integr OMICS 2011;1:260–7.  Firer-Sherwood MA, Bewley KD, Mock JY, Elliott SJ. Tools for resolving complexity in the electron transfer networks of multiheme cytochromes c. Metallomics 2011;3:344–8.  Cordova CD, Schicklberger MFR, Yu Y, Spormann AM. Partial functional replacement of CymA by SirCD in Shewanella oneidensis MR-1. J Bacteriol 2011;193:2312–21.  Hartshorne RS, Reardon CL, Ross D, Nuester J, Clarke TA, Gates AJ, et al. Characterization of an electron conduit between bacteria and the extracellular environment. Proc Natl Acad Sci U S A 2009;106:22169–74.  Schicklberger M, Bucking C, Schuetz B, Heide H, Gescher J. Involvement of the Shewanella oneidensis decaheme cytochrome MtrA in the periplasmic stability of the beta-barrel protein MtrB. Appl Environ Microbiol 2011;77:1520–3.  Shi L, Chen BW, Wang ZM, Elias DA, Mayer MU, Gorby YA, et al. Isolation of a high-afﬁnity functional protein complex between OmcA and MtrC: two outer membrane decaheme c-type cytochromes of Shewanella oneidensis MR-1. J Bacteriol 2006;188:4705–14.  Baron D, LaBelle E, Coursolle D, Gralnick JA, Bond DR. Electrochemical measurement of electron transfer kinetics by Shewanella oneidensis MR-1. J Biol Chem 2009;284:28865–73.  Coursolle D, Baron DB, Bond DR, Gralnick JA. The Mtr respiratory pathway is essential for reducing ﬂavins and electrodes in Shewanella oneidensis. J Bacteriol 2010;192:467–74.  Firer-Sherwood M, Pulcu GS, Elliott SJ. Electrochemical interrogations of the Mtr cytochromes from Shewanella: opening a potential window. J Biol Inorg Chem 2008;13:849–54.  Honeychurch MJ, Hill AO, Wong LL. The thermodynamics and kinetics of electron transfer in the cytochrome P450cam enzyme system. Febs Lett 1999;451:351–3.  Okamoto A, Nakamura R, Ishii K, Hashimoto K. In vivo electrochemistry of c-type cytochrome-mediated electron-transfer with chemical marking. ChemBioChem 2009;10:2329–32.  Ross DE, Brantley SL, Tien M. Kinetic characterization of OmcA and MtrC, terminal reductases involved in respiratory electron transfer for dissimilatory iron reduction in Shewanella oneidensis MR-1. Appl Environmental Microbiol 2009;75:5218–26.  Coursolle D, Gralnick JA. Reconstruction of extracellular respiratory pathways for iron(III) reduction in Shewanella oneidensis strain MR-1. Front Microbiol 2012;3:56.  Liang L, Johs A, Shi L, Droubay T, Ankner JF. Characterization of the decaheme c-type cytochrome OmcA in solution and on hematite surfaces by small angle X-ray scattering and neutron reﬂectometry. Biophys J 2010;98:3035–43.  Clarke TA, Edwards MJ, Gates AJ, Hall A, White GF, Bradley J, et al. Structure of a bacterial cell surface decaheme electron conduit. Proc Natl Acad Sci U S A 2011;108:9384–9.  Butler JE, Young ND, Lovley DR. Evolution of electron transfer out of the cell: comparative genomics of six Geobacter genomes. BMC Genomics 2010;11:40.  Morgado L, Paixao VB, Schiffer M, Pokkuluri PR, Bruix M, Salgueiro CA. Revealing the structural origin of the redox-Bohr effect: the ﬁrst solution structure of a cytochrome from Geobacter sulfurreducens. Biochem J 2012;441: 179–87.  Holmes DE, Chaudhuri SK, Nevin KP, Mehta T, Methe BA, Liu A, et al. Microarray and genetic analysis of electron transfer to electrodes in Geobacter sulfurreducens. Environ Microbiol 2006;8:1805–15.  Nevin KP, Kim BC, Glaven RH, Johnson JP, Woodard TL, Methe BA, et al. Anode bioﬁlm transcriptomics reveals outer surface components essential for high density current production in Geobacter sulfurreducens fuel cells. PLoS One 2009;4:e5628.  Kim BC, Postier BL, Didonato RJ, Chaudhuri SK, Nevin KR, Lovley DR. Insights into genes involved in electricity generation in Geobacter sulfurreducens via whole genome microarray analysis of the OmcF-deﬁcient mutant. Bioelectrochemistry 2008;73:70–5.  Inoue K, Leang C, Franks AE, Woodard TL, Nevin KP, Lovley DR. Speciﬁc localization of the c-type cytochrome OmcZ at the anode surface in current-producing bioﬁlms of Geobacter sulfurreducens. Environ Microbiol Rep 2011;3:211–7.  Richter H, Nevin KP, Jia HF, Lowy DA, Lovley DR, Tender LM. Cyclic voltammetry of bioﬁlms of wild type and mutant Geobacter sulfurreducens on fuel cell anodes indicates possible roles of OmcB, OmcZ, type IV pili, and protons in extracellular electron transfer. Energ Environ Sci 2009;2:506–16.
 Franks AE, Nevin KP, Glaven RH, Lovley DR. Microtoming coupled to microarray analysis to evaluate the spatial metabolic status of Geobacter sulfurreducens bioﬁlms. ISME J 2010;4:509–19.  Liu Y, Kim H, Franklin RR, Bond DR. Linking spectral and electrochemical analysis to monitor c-type cytochrome redox status in living Geobacter sulfurreducens bioﬁlms. Chemphyschem 2011;12:2235–41.  Rabaey K, Rodriguez J, Blackall LL, Keller J, Gross P, Batstone D, et al. Microbial ecology meets electrochemistry: electricity-driven and driving communities. ISME J 2007;1:9–18.  Carlson HK, Iavarone AT, Gorur A, Yeo BS, Tran R, Melnyk RA, et al. Surface multiheme c-type cytochromes from Thermincola potens and implications for respiratory metal reduction by gram-positive bacteria. Proc Natl Acad Sci U S A 2012;109:1702–7.  Wrighton KC, Thrash JC, Melnyk RA, Bigi JP, Byrne-Bailey KG, Remis JP, et al. Evidence for direct electron transfer by a gram-positive bacterium isolated from a microbial fuel cell. Appl Environ Microbiol 2011;77:7633–9.  Inoue K, Qian X, Morgado L, Kim BC, Mester T, Izallalen M. Puriﬁcation and characterization of an OmcZ, an outer-surface, ocataheme c-type cytochrome essential for optimal current production by Geobacter sulfurreducens. Appl Environ Microbiol 2010;76:8.  Qian XL, Mester T, Morgado L, Arakawa T, Sharma ML, Inoue K, et al. Biochemical characterization of puriﬁed OmcS, a c-type cytochrome required for insoluble Fe(III) reduction in Geobacter sulfurreducens. BBA-Bioenergetics 2011;1807:404–12.  Reguera G, McCarthy KD, Mehta T, Nicoll JS, Tuominen MT, Lovley DR. Extracellular electron transfer via microbial nanowires. Nature 2005;435:1098–101.  Gorby YA, Yanina S, McLean JS, Rosso KM, Moyles D, Dohnalkova A, et al. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proc Natl Acad Sci U S A 2006;103:11358–63.  Strycharz-Glaven SM, Snider RM, Guiseppi-Elie A, Tender LM. On the electrical conductivity of microbial nanowires and bioﬁlms. Energ Environ Sci 2011;4:4366–79.  El-Naggar MY, Wanger G, Leung KM, Yuzvinsky TD, Southam G, Yang J, et al. Electrical transport along bacterial nanowires from Shewanella oneidensis MR1. Proc Natl Acad Sci U S A 2010;107:18127–31.  Bouhenni RA, Vora GJ, Bifﬁnger JC, Shirodkar S, Brockman K, Ray R, et al. The role of Shewanella oneidensis MR-1 outer surface structures in extracellular electron transfer. Electroanalysis 2010;22:856–64.  Bifﬁnger JC, Ray R, Little BJ, Fitzgerald LA, Ribbens M, Finkel SE, et al. Simultaneous analysis of physiological and electrical output changes in an operating microbial fuel cell with Shewanella oneidensis. Biotechnol Bioeng 2009;103:524–31.  Richter LV, Sandler SJ, Weis RM. Two isoforms of Geobacter sulfurreducens PilA have distinct roles in pilus biogenesis, cytochrome localization, extracellular electron transfer, and bioﬁlm formation. J Bacteriol 2012;194:2551–63.  Leang C, Qian X, Mester T, Lovley DR. Alignment of the c-type cytochrome OmcS along pili of Geobacter sulfurreducens. Appl Environ Microbiol 2010;76:4080–4.  Malvankar NS, Vargas M, Nevin KP, Franks AE, Leang C, Kim B-C, et al. Tunable metallic-like conductivity in microbial nanowire networks. Nat Nano 2011;6:573–9.  Cologgi DL, Lampa-Pastirk S, Speers AM, Kelly SD, Reguera G. Extracellular reduction of uranium via Geobacter conductive pili as a protective cellular mechanism. Proc Natl Acad Sci U S A 2011;108:15248–52.  Reguera G, Pollina RB, Nicoll JS, Lovley DR. Possible nonconductive role of Geobacter sulfurreducens pilus nanowires in bioﬁlm formation. J Bacteriol 2007;189:2125–7.  Dohnalkova AC, Marshall MJ, Arey BW, Williams KH, Buck EC, Fredrickson JK. Imaging hydrated microbial extracellular polymers: comparative analysis by electron microscopy. Appl Environ Microbiol 2011;77:1254–62.  Rollefson JB, Stephen CS, Tien M, Bond DR. Identiﬁcation of an extracellular polysaccharide network essential for cytochrome anchoring and bioﬁlm formation in Geobacter sulfurreducens. J Bacteriol 2011;193:1023–33.  Rabaey K, Boon N, Siciliano SD, Verhaege M, Verstraete W. Biofuel cells select for microbial consortia that self-mediate electron transfer. Appl Environ Microbiol 2004;70:5373–82.  von Canstein H, Ogawa J, Shimizu S, Lloyd JR. Secretion of ﬂavins by Shewanella species and their role in extracellular electron transfer. Appl Environ Microbiol 2008;74:615–23.  Marsili E, Baron DB, Shikhare ID, Coursolle D, Gralnick JA, Bond DR. Shewanella secretes ﬂavins that mediate extracellular electron transfer. Proc Natl Acad Sci U S A 2008;105:3968–73.  Covington ED, Gelbmann CB, Kotloski NJ, Gralnick JA. An essential role for UshA in processing of extracellular ﬂavin electron shuttles by Shewanella oneidensis. Mol Microbiol 2010;78:519–32.  Gescher J, Richter K, Bucking C, Schicklberger M. A simple and fast method to analyze the orientation of c-type cytochromes in the outer membrane of Gram-negative bacteria. J Microbiol Methods 2010;82:184–6.  Grininger M, Staudt H, Johansson P, Wachtveitl J, Oesterhelt D. Dodecin is the key player in ﬂavin homeostasis of Archaea. J Biol Chem 2009;284:13068–76.  Pham TH, Boon N, Aelterman P, Clauwaert P, De Schamphelaire L, Vanhaecke L, et al. Metabolites produced by Pseudomonas sp enable a Gram-positive bacterium to achieve extracellular electron transfer. Appl Microbiol Biotechnol 2008;77:1119–29.
Y. Yang et al. / Process Biochemistry 47 (2012) 1707–1714
 Pierson LS, Pierson EA. Metabolism and function of phenazines in bacteria impacts on the behavior of bacteria in the environment and biotechnological processes. Appl Microbiol Biotechnol 2010;86:1659–70.  Parameswaran P, Torres CI, Lee HS, Krajmalnik-Brown R, Rittmann BE. Syntrophic interactions among anode respiring bacteria (ARB) and nonARB in a bioﬁlm anode: electron balances. Biotechnol Bioeng 2009;103: 513–23.  Torres CI, Marcus AK, Lee HS, Parameswaran P, Krajmalnik-Brown R, Rittmann BE. A kinetic perspective on extracellular electron transfer by anode-respiring bacteria. FEMS Microbiol Rev 2010;34:3–17.  Stewart PS. Diffusion in bioﬁlms. J Bacteriol 2003;185:1485–91.  Cao B, Shi L, Brown RN, Xiong Y, Fredrickson JK, Romine MF, et al. Extracellular polymeric substances from Shewanella sp. HRCR-1 bioﬁlms: characterization by infrared spectroscopy and proteomics. Environ Microbiol 2011;13:1018–31.  De Vriendt K, Theunissen S, Carpentier W, De Smet L, Devreese B, Van Beeumen J. Proteomics of Shewanella oneidensis MR-1 bioﬁlm reveals differentially expressed proteins, including AggA and RibB. Proteomics 2005;5: 1308–16.  Korenevsky A, Beveridge TJ. The surface physicochemistry and adhesiveness of Shewanella are affected by their surface polysaccharides. Microbiology-Sgm 2007;153:1872–83.  Kouzuma A, Meng XY, Kimura N, Hashimoto K, Watanabe K. Disruption of the putative cell surface polysaccharide biosynthesis gene SO3177 in Shewanella oneidensis MR-1 enhances adhesion to electrodes and current generation in microbial fuel cells. Appl Environ Microbiol 2010;76:4151–7.  Jiang X, Hu J, Fitzgerald LA, Bifﬁnger JC, Xie P, Ringeisen BR, et al. Probing electron transfer mechanisms in Shewanella oneidensis MR-1 using a nanoelectrode platform and single-cell imaging. Proc Natl Acad Sci U S A 2010;107:16806–10.  Newton GJ, Mori S, Nakamura R, Hashimoto K, Watanabe K. Analyses of current-generating mechanisms of Shewanella loihica PV-4 and Shewanella oneidensis MR-1 in microbial fuel cells. Appl Environ Microbiol 2009;75:7674–81.  Yang Y, Sun G, Guo J, Xu M. Differential bioﬁlms characteristics of Shewanella decolorationis microbial fuel cells under open and closed circuit conditions. Bioresour Technol 2011;102:6.  Malvankar NS, Tuominen MT, Lovley DR. Bioﬁlm conductivity is a decisive variable for high-current-density Geobacter sulfurreducens microbial fuel cells. Energ Environ Sci 2012;5:5790–7.  Summers ZM, Fogarty HE, Leang C, Franks AE, Malvankar NS, Lovley DR. Direct exchange of electrons within aggregates of an evolved syntrophic coculture of anaerobic bacteria. Science 2010;330:1413–5.  Morita M, Malvankar NS, Franks AE, Summers ZM, Giloteaux L, Rotaru AE, et al. Potential for direct interspecies electron transfer in methanogenic wastewater digester aggregates. Mbio 2011;2:e00159–211.  Borole AP, Reguera G, Ringeisen B, Wang ZW, Feng YJ, Kim BH. Electroactive bioﬁlms: current status and future research needs. Energ Environ Sci 2011;4:4813–34.  Polizzi NF, Skourtis SS, Beratan DN. Physical constraints on charge transport through bacterial nanowires. Faraday Discuss 2012;155:43–62.  Bonanni PS, Schrott GD, Robuschi L, Busalmen JP. Charge accumulation and electron transfer kinetics in Geobacter sulfurreducens bioﬁlms. Energ Environ Sci 2012;5:6188–95.  Malvankar NS, Mester T, Tuominen MT, Lovley DR. Supercapacitors based on c-type cytochromes using conductive nanostructured networks of living bacteria. Chemphyschem 2012;13:463–8.  Franks AE, Nevin KP, Jia HF, Izallalen M, Woodard TL, Lovley DR. Novel strategy for three-dimensional real-time imaging of microbial fuel cell communities: monitoring the inhibitory effects of proton accumulation within the anode bioﬁlm. Energ Environ Sci 2009;2:113–9.  Lanthier M, Gregory KB, Lovley DR. Growth with high planktonic biomass in Shewanella oneidensis fuel cells. FEMS Microbiol Lett 2008;278:29–35.
 Liu Y, Kim H, Franklin R, Bond DR. Gold line array electrodes increase substrate afﬁnity and current density of electricity-producing G. sulfurreducens bioﬁlms. Energ Environ Sci 2010;3:1782–8.  Marsili E, Sun J, Bond DR. Voltammetry and growth physiology of Geobacter sulfurreducens bioﬁlms as a function of growth stage and imposed electrode potential. Electroanalysis 2010;22:865–74.  Reguera G, Nevin KP, Nicoll JS, Covalla SF, Woodard TL, Lovley DR. Bioﬁlm and nanowire production leads to increased current in Geobacter sulfurreducens fuel cells. Appl Environ Microbiol 2006;72:7345–8.  Erable B, Etcheverry L, Bergel A. From microbial fuel cell (MFC) to microbial electrochemical snorkel (MES): maximizing chemical oxygen demand (COD) removal from wastewater. Biofouling 2011;27:319–26.  Strik DPBTB, Timmers RA, Helder M, Steinbusch KJJ, Hamelers HVM, Buisman CJN. Microbial solar cells: applying photosynthetic and electrochemically active organisms. Trends Biotechnol 2011;29:41–9.  Rabaey K, Rozendal RA. Microbial electrosynthesis—revisiting the electrical route for microbial production. Nat Rev Microbiol 2010;8:706–16.  Logan BE, Call D, Cheng S, Hamelers HVM, Sleutels THJA, Jeremiasse AW, et al. Microbial electrolysis cells for high yield hydrogen gas production from organic matter. Environ Sci Technol 2008;42:8630–40.  Nielsen LP, Risgaard-Petersen N, Fossing H, Christensen PB, Sayama M. Electric currents couple spatially separated biogeochemical processes in marine sediment. Nature 2010;463:1071–4.  Kaden J, Galushko AS, Schink B. Cysteine-mediated electron transfer in syntrophic acetate oxidation by cocultures of Geobacter sulfurreducens and Wolinella succinogenes. Arch Microbiol 2002;178:53–8.  Seeliger S, Cord-Ruwisch R, Schink B. A periplasmic and extracellular c-type cytochrome of Geobacter sulfurreducens acts as a ferric iron reductase and as an electron carrier to other acceptors or to partner bacteria. J Bacteriol 1998;180:3686–91.  Kato S, Hashimoto K, Watanabe K. Methanogenesis facilitated by electric syntrophy via (semi)conductive iron-oxide minerals. Environ Microbiol 2012;14:1646–54.  Gregory KB, Bond DR, Lovley DR. Graphite electrodes as electron donors for anaerobic respiration. Environ Microbiol 2004;6:596–604.  Rosenbaum M, Aulenta F, Villano M, Angenent LT. Cathodes as electron donors for microbial metabolism: which extracellular electron transfer mechanisms are involved? Bioresour Technol 2011;102:324–33.  Strycharz SM, Glaven RH, Coppi MV, Gannon SM, Perpetua LA, Liu A, et al. Gene expression and deletion analysis of mechanisms for electron transfer from electrodes to Geobacter sulfurreducens. Bioelectrochemistry 2011;80:142–50.  Freguia S, Masuda M, Tsujimura S, Kano K. Lactococcus lactis catalyses electricity generation at microbial fuel cell anodes via excretion of a soluble quinone. Bioelectrochemistry 2009;76:14–8.  Zhang TT, Zhang LX, Su WT, Gao P, Li DP, He XH, et al. The direct electrocatalysis of phenazine-1-carboxylic acid excreted by Pseudomonas alcaliphila under alkaline condition in microbial fuel cells. Bioresour Technol 2011;102:7099–102.  Deng LF, Li FB, Zhou SG, Huang DY, Ni JR. A study of electron-shuttle mechanism in Klebsiella pneumoniae based-microbial fuel cells. Chin Sci Bull 2010;55:99–104.  Xia X, Cao XX, Liang P, Huang X, Yang SP, Zhao GG. Electricity generation from glucose by a Klebsiella sp. in microbial fuel cells. Appl Microbiol Biotechnol 2010;87:383–90.  Qiao Y, Li CM, Bao SJ, Lu ZS, Hong YH. Direct electrochemistry and electrocatalytic mechanism of evolved Escherichia coli cells in microbial fuel cells. Chem Commun 2008;11:1290–2.  Nimje VR, Chen CY, Chen CC, Jean JS, Reddy AS, Fan CW, et al. Stable and high energy generation by a strain of Bacillus subtilis in a microbial fuel cell. J Power Sources 2010;195:5427–8.  Bond DR, Lovley DR. Evidence for involvement of an electron shuttle in electricity generation by Geothrix fermentans. Appl Environ Microbiol 2005;71:2186–9.