Bacterial extracellular electron transfer in bioelectrochemical systems

Bacterial extracellular electron transfer in bioelectrochemical systems

Process Biochemistry 47 (2012) 1707–1714 Contents lists available at SciVerse ScienceDirect Process Biochemistry journal homepage:

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Process Biochemistry 47 (2012) 1707–1714

Contents lists available at SciVerse ScienceDirect

Process Biochemistry journal homepage:


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 [1]. 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.

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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 [16]. Despite the pyruvate fermentative capacity [17], 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) [16]. 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 (, 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 [18]. 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 [21]. 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 ( 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 [22]. 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 [25]. 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 [28]. 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 [29]. 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 [31]. 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 [33]. 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 [33]. The electron transfer rate constant (k0 ) of S. oneidensis MR-1 MtrABC complex on a graphite electrode was estimated to be 195 s−1 [29], which is at the same order of magnitude with the constant of each individual protein in the complex [34] (Table 1). Those values are also comparable to the k0 of S. loihica PV-4 OMCs (150 ± 10 s−1 ) [34], 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 [37]. 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 [24]. 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 [38]. 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 [41]. 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) [7]. 3D structures and the electronic interaction of MacA and PpcA have been determined [42]. 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 [46]. Comparative genome analyses showed that omcZ is not conserved in all Geobacter species [41]. A deletion of omcZ resulted in >90% current decrease while showed no effect on the reductions of other electron acceptors including Fe(III) oxide [47]. Although no significant difference was observed for the transcription level of several OMC genes throughout the G. sulfurreducens biofilm based on microarray data [48], immunogold labeling showed that OmcZ were mostly accumulated at the biofilm–electrode interface in MFCs for electron transferring [41], 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.

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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 [34] −100/300 mV [34] −138/275 mV [34] −175/300 mV [34]

G. sulfurreducens

OmcZ OmcS

Outer membrane Outer membrane

8 6

Unknown Unknown

−180/360 mV [53] −212/320 mV [54]

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 [50]. 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 [55]. Subsequently, bacterial nanowires were observed in S. oneidensis MR-1 and some other bacteria, or between different bacterial species [56], 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.

Heme number

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 [58], 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 [56]. 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 [61]. The deletion of pilA gene could diminish the nanowire production and significantly decrease the current generation capacity of thick biofilms (>50 ␮m) [44] and showed less effect on the current generation by thin biofilms (<10 ␮m) [57], 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 [62]. 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 [62]. It is possible that some other redox proteins such as OMCs


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participate in and facilitate the electron transfer along the nanowires. The electronic conductivity of G. sulfurreducens nanowires was measured to be 6 mS/cm [63], which is comparable to the synthetic metallic nanostructures and higher than the proposed threshold (10−3 mS/cm) for efficient current generation in BES [52]. 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 [63]. 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 [68]. 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 [37]. 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 [70]. 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 [36]. 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 [69]. 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 [73]. Phenazines generated by a diverse of bacteria, particularly Pseudomonas species, were also intensively studied as intrinsic electron shuttles in MESs [50]. The presence of phenazines enhanced the current-generating capacity of a gram positive bacterium Brevibacillus sp. PTH1 [74], 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 [75]. 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 [76]. 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 [21].

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 [79]. It was estimated that an electrode colonized by a single-layer of Shewanella biofilm has 10–30% surface coverage of OMCs [36], 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 [2]. Likewise, the expression of RF biosynthesis enzyme (RibB) was also up-regulated when S. oneidensis MR-1 was grown in a biofilm [80], 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 [79]. 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) [32]. 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 [82]. Mobility and chemotaxis are also important for biofilm formation. Interestingly, deletions of the chemotaxis-related genes (e.g., cheA,

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Table 2 Bacteria-secreted electron shuttles in BES. Bacteria

Gram +/−



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 [69] 2-Amino-3-dicarboxy-1,4-naphthoquinone [109] Pyocyanin [68] Phenazine-1-carboxamide [74] Phenazine-1-carboxylic acid [110] 2,6-Di-tertbutyl-p-benzoquinon [111] Quinone [112] Hydroquinone [113] Unknown [114] Unknown [115]

−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 [76], and pH could decrease from 7.0 to 6.0 due to proton accumulation in biofilms [93]. 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 [59]. 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 [86]. 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 [63], 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 [87], methanogenic aggregates [88] and multispecies currentgenerating biofilms [86]. 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 [63], suggested that the conductivity was specifically contributed by Geobacter species, even though many biomaterials (e.g., DNA, cell wall) have comparable conductivity [89]. In biofilms with metal-like conductivity, electrons are delocalized and can be transferred without thermal activation [63]. 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 [92]. Extracellular polysaccharide network in biofilm EPS has been shown to be essential for anchoring and arranging of OMCs in G. sulfurreducenus biofilm [67], which is in contrast with the suggested impeding role of polysaccharide in S. onidensis MR-1 biofilm [82]. Spectral and electrochemical analyses showed that the composition of the Geobacter biofilm OMC network varied with the electrode potential [49]. Compared with the efficient EET process in biofilms, substrate oxidation by exoelectrogens was presumed to be the rate-limiting step in bacterial electrode reduction [91]. 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 [78]. 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 [96]. The low electron transfer rate of the biofilm cells with a distance greater than 20 ␮m from electrode would decrease the biofilm growth and


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conductivity [95]. However, Geobacter biofilm across a 100 ␮m gap showed a comparable conductivity with that of a thinner biofilm (50 ␮m) [86]. 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 [52]. Similar to the Geobacter biofilm lacking nanowires [97], the outer layer cells in T. potens biofilms showed low viability [52], 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 [102]). 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 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 [87]. 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 [108]. 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 [25]. However, in vitro voltammetry suggested that the Shewanella CTCs could not transfer electrons in the reversed direction [27]. 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.

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