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Engineering electrode-attached microbial consortia for high-performance xylose-fed microbial fuel cell Yun Yang, Yichao Wu, Yidan Hu, Yingxiu Cao, Chueh Loo Poh, Bin Cao, and Hao Song ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.5b01733 • Publication Date (Web): 09 Oct 2015 Downloaded from http://pubs.acs.org on October 11, 2015

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Engineering electrode-attached microbial consortia for high-performance xylose-fed microbial fuel cell Yun Yang1,2, Yichao Wu2,3, Yidan Hu2, Yingxiu Cao4, Chueh Loo Poh1, Bin Cao2,3 *, Hao Song4 * 1

School of Chemical and Biomedical Engineering, Nanyang Technological University,

70 Nanyang Drive, 637457, Singapore 2

Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological

University, 60 Nanyang Drive, 637551, Singapore 3

School of Civil and Environmental Engineering, Nanyang Technological University, 50

Nanyang Drive, 637798, Singapore 4

Key Laboratory of Systems Bioengineering (Ministry of Education), SynBio Research

Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P.R. China

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ABSTRACT. Microbial fuel cell (MFC) is a promising technology for energy harvesting from biomass, however, reported MFCs with wild-type or biologically modified exoelectrogenic bacteria such as Shewanella oneidensis often exhibited poor performance in generating electricity from sugars. Herein, a synthetic fermenter-exoelectrogen (Escherichia coli-S. oneidensis) microbial consortium was developed to expand the spectrum of carbon sources for MFC through establishing a highly electroactive anodic biofilm by rationally tuning its microbial community profile to favor efficient electron transfer. Specifically, a synthetic riboflavin pathway from Bacillus subtilis was incorporated into E. coli to overproduce flavins to facilitate flavin-mediated electron transfer, and a highly hydrophobic S. oneidensis strain CP2-1-S1 was adopted as the exoelectrogen to increase its adhesion to the carbon electrode. The highly hydrophobic interactions between S. oneidensis and the anode along with the overproduced flavins (increased from 3.3 µM to 115.2 µM) by the recombinant E. coli provided a definite advantage for S. oneidensis over E. coli in the attachment to the anode surface. Compared with the structure of the wild-type community immobilized on the anode, the cell number of S. oneidensis increased by ~3 times, while the cell number of E. coli decreased by 93.3% in the engineered electrode-attached community. Such rationally engineered anodic biofilm with the tuned microbial community profile (the percentage of S. oneidensis cells in the anodic biofilm increased from 48.2% to 98.2%) showed a much higher catalytic current (from 0.19 to 1.84 A/m2 at 0 V vs. SHE). The xylose-fed MFC inoculated with our engineered microbial consortium generated a maximum power density of 728.6 mW/m2, which was 6.8 times higher than that inoculated with wild-type co-culture (92.8 mW/m2).


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KEYWORDS Microbial consortia, Shewanella oneidensis MR-1, Escherichia coli, biofilm engineering, synthetic biology, extracellular electron transfer, flavin, hydrophobic interaction

Introduction

Microbial fuel cell (MFC) is a green and sustainable technology using exoelectrogenic microorganisms as the biocatalyst for energy harvesting and electricity generation from organic matter.1-6 Shewanella oneidensis, a well-studied exoelectrogen, is capable of transferring electrons extracellularly through its metal-reducing (Mtr) pathway,7-9 being widely studied for MFC performance10. However, the wild-type (WT) S. oneidensis can only use limited spectrum of carbon sources such as lactate and pyruvate, while sugars, the most abundant composition of biomass, could not be utilized by WT S. oneidensis owing to its incomplete sugar utilization pathways.11-15

A few strategies have been studied in enabling sugar-powered MFC catalyzed by S. oneidensis. A recombinant S. oneidensis strain was constructed by harboring the glucose facilitator and glucokinase genes from Zymomonas mobilis, which could generate electricity by growing on glucose anaerobically. However, this system exhibited rather poor extracellular electron transfer (EET) efficiency.16 In addition, microbial consortia were studied for sugar-fed MFC, in which a nonelectrogenic microbe digested sugars and provided metabolite as the electron donor for S. oneidensis to generate electricity.17,18 These researchers focused mainly on the establishment of metabolite exchange-based 3 ACS Paragon Plus Environment

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interactions among multiple species in the microbial consortia to achieve sugar-powered MFCs. However, the electroactivity of the anodic biofilm is actually a most crucial factor determining the power generation in MFCs, which was largely neglected in the previous engineering efforts of microbial consortia in MFCs.19,20

The EET rates in the synthetic S. oneidensis-involved microbial consortia were limited likely due to several reasons. Firstly, flavins, as the electron shuttles or cofactors of outer membrane (OM) c-type cytochromes (c-Cyts), not only determine the interfacial electron transfer rate between S. oneidensis OM and the electrode,21-27 but also could facilitate biofilm formation on the electrode and the electroactivity of single immobilized cell of S. oneidensis28,29. However, the physiological concentration of flavins secreted by the microbes in the consortia was rather low, which significantly limited the EET efficiency of microbial consortia. Secondly, the overall catalytic current is positively correlated with the cell number of electrode-attached S. oneidensis, whereas the biofilm formation capacity of S. oneidensis was relatively poor. In addition, the fermentative and the exoelectrogenic microbes were nearly equal in population in the anodic biofilm formed by the mix species, which could significantly impair the conductivity of the anodeattached biofilm and limit the MFC performance. All of these challenges impede the practical application of S. oneidensis-based microbial consortium in efficient energy harvesting from sugars.

In the current work, a synthetic microbial consortium composed of genetically modified E. coli and S. oneidensis was rationally designed and constructed to efficiently harvest energy from xylose, a common monosaccharide derided from vegetables and fibers


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(Figure 1). Rational design of microbial consortia is an efficient engineering strategy to improve system function by the division of overall labor among several species within a microbial community, in which each species in the microbial community is designed to execute what it is expert at.30-32 Herein, a synthetic riboflavin pathway from Bacillus subtilis was introduced into E. coli to facilitate EET of S. oneidensis and improve its initial attachment onto the electrode. Furthermore, a highly hydrophobic S. oneidensis mutant was employed in the consortium to allow the exoelectrogen to better interact with the hydrophobic carbon electrode. With the integrated effects of overproduced flavins by E. coli and strong hydrophobic interaction between S. oneidensis and the carbon anode, the attachment of S. oneidensis onto the anode was greatly favored over that of E. coli, resulting in a distinct microbial community profile of the anodic biofilm, in which the proportion of S. oneidensis was increased from 48.2% to 98.2%. Consequently, our rationally engineered microbial consortium formed a highly electroactive anodic biofilm, and the corresponding xylose-fed MFC generated a maximum power density of 728.6 mW/m2, which was 6.8 times higher than that inoculated with wild-type co-culture (92.8 mW/m2). In addition, the electricity output by our synthetic consortium was, to the best of our knowledge, among the highest in the reported MFCs with biologically engineered S. oneidensis (Table 1).

Materials and Methods

In vitro gene synthesis. The coding sequences of ribADEH genes of B. subtilis were extracted from BioCyc database33 and adapted for expression in S. oneidensis by a java 5 ACS Paragon Plus Environment

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codon adaption tool (JCAT)34. Each gene component was synthesized as a biobrick, and restriction enzyme sites of EcoRI, XbaI, SpeI and SbfI were avoided in the codon optimized sequences. The optimized gene sequence was flanked by an upstream prefix (contain EcoRI and XbaI), a RBS site (BBa_B0034, iGEM) 6 bp ahead of the start codon and a downstream suffix (contain SpeI and SbfI). The oligonucleotide sets for in vitro gene synthesis were displayed in Table S1 (Supporting Information).

Plasmid construction and transformation. Plasmid constructions for riboflavin synthesis pathway were implemented in E. coli BL21 (DE3). Synthesized ribA, ribD, ribE, ribH biobricks were inserted into the vector pSB1AK3 (iGEM) sequentially, and formed the resulting expression plasmid pSB1AK3-A4. Two Plac promoters (BBa_R0011, iGEM) were placed ahead of ribA and ribE, respectively (Figure 2a). To confer kanamycin resistant of S. oneidensis (ATCC 700550) that grew in the co-culture medium supplemented with 50 µg/ml kanamycin, an empty vector pYYD28 was firstly transformed into E. coli WM3064, which was subsequently transformed into S. oneidensis strains by conjugation.35 The highly hydrophobic S. oneidensis strain CP2-1S1 was obtained in our previous study36. Both WT and mutant S. oneidensis CP2-1-S1 strains used in the following experiments contained the empty vector pYYD. For growth of E. coli WM3064, 100 µg/ml 2,6-diaminopimelic acid (DAP) was supplemented.

Culture condition and flavin measurement. E. coli BL21 (DE3) strain containing pSB1AK3 or pSB1AK3-A4 from -80 °C freezer stock was inoculated into LB broth supplemented with 50 µg/ml kanamycin shaking at 37 °C for ~12 h. The 1.6 ml E. coli culture suspension was inoculated into 40 ml M9 medium supplemented with trace


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amounts (1% v/v) of vitamin stock solution, mineral stock solution and amino acids stock solution29. 20 mM xylose was added as the carbon source. For both E. coli strains harboring empty vector or pSB1AK3-A4, 0.01 mM IPTG and 50 µg/ml kanamycin were added into the culture medium. After growing 15 h at 37 °C with 200 rpm, the supernatant was filtered (0.2 µm, Acrodisc Syringe Filter, Pall Corporation, USA) and subjected to flavin measurements using high-performance liquid chromatography (HPLC) as previously described.28

MFC setup. E. coli BL21 (DE3) harboring pSB1AK3/pSB1AK3-A4 and S. oneidensis harboring the empty vector pYYD (from -80 °C freezer stock) was inoculated into 20 ml LB broth supplemented with 50 µg/ml kanamycin before being shaken for ~12 h at 37 °C or 30 °C, respectively. 16 ml E. coli culture suspension was inoculated into 400 ml M9 medium supplemented with trace amounts (1% v/v) of vitamin stock solution, mineral stock solution and amino acids stock solution. 20 mM xylose, 0.01 mM IPTG and 50 µg/ml kanamycin were also added to enable aerobic riboflavin overproduction by E. coli strains. 2 ml S. oneidensis suspension was transferred to 200 ml LB with 50 µg/ml kanamycin. After growing 15 h at 37 °C with 200 rpm, the E. coli culture was mixed with 200 ml fresh M9 based medium, and the cell density was adjusted to OD600 ~ 0.3. Calculated volume of S. oneidensis culture were centrifuged and the cell pellet was resuspended in the adjusted E. coli solution, the ratio of inoculated cell densities of E. coli and S. oneidensis was 3/5. The co-culture solutions were dispersed into the anode chambers (140 ml working volume) of three H cell reactors for parallel experiments. The anodes were purged with nitrogen gas to remove oxygen. Carbon cloth (Gashub, Singapore) was used both as the anodic electrode (2.5 cm × 2.5 cm) and cathodic 7 ACS Paragon Plus Environment

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electrode (2.5 cm × 3 cm). The Nafion 117 membrane (Gashub, Singapore) was pretreated at 80 °C for 0.5 h with 3% hydrogen peroxide, distilled water and 0.5 M sulphuric acid sequentially. After washing with sterile distilled water, the Nafion 117 membrane was clamped to separate the anode and the cathode. The cathodic electrolyte was made of 50 mM K3[Fe(CN)6], 50 mM K2HPO4 and 50 mM KH2PO4. The anode and cathode was connected by a 1 kΩ external resistor, and incubated at 32 °C. The voltage outputs of MFCs were continuously recorded by data acquisition cards MPS-110001 (Morpheus Electronics Technology Co. Ltd, China).

Metabolite quantification. The metabolites in the anolyte were analyzed using a HPLC system equipped with a diode array detector.18 5 mM sulfuric acid was employed as the mobile phase flowing at 0.6 ml/min through the Aminex HPX-87H column (Bio-Rad, USA) which was incubated at 50 °C. Signals at 210 nm were used to quantify organic acids. Signals at 190 nm was used to quantify xylose concentration.

Electrochemical analysis. Cyclic voltammetry (CV) with a low scan rate (1 mV/s) was conducted on a three-electrode configuration with an Ag/AgCl reference electrode (CH Instruments, USA) using a CHI 1000C multichannel potentiostat (CH Instruments, USA). Linear sweep voltammetry (LSV) with a slow scan rate (0.1 mV/s) was applied to obtain the polarization curves and the potential decreased from the open circuit potential (OCP) to ~-0.4 V controlled by CHI 1000C.

Analysis of the electrode-attached community. The anode was placed in a 50 ml test tube containing 5 ml of 0.9% NaCl solution, vortexed for 2 min. A series of dilutions were spread onto LB agar plates and incubated at 30 °C for 24 h before the CFU counting 8 ACS Paragon Plus Environment

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of S. oneidensis. Colonies of S. oneidensis were dark orange in color. For the CFU counting of E. coli, the diluted cell suspension were spread on LB agar supplemented with 50 µg/ml kanamycin and ampicillin, incubated at 37 °C.

Quantitative biofilm assay. Biofilm assay was conducted in a 96-well plate as previously reported.37 In brief, 1.5 µl overnight LB culture of E. coli with pSB1AK3 or pSB1AK3-A4 were inoculated in 150 µl M9 medium (supplemented with 1% v/v of vitamin stock solution, mineral stock solution and amino acids stock solution; 20 mM xylose, 0.01 mM IPTG and 50 µg/ml kanamycin). The plate was shaking at 30 °C, with the growth of planktonic biomass monitored at OD600 by a Tecan microplate reader Infinite 200 PRO (Tecan, Männedorf, Switzerland). After 24 h incubation, the surfaceassociated biomass was stained by 1 % crystal violet after the planktonic cells were removed. After adding 200 µL of 95% ethanol, the amount of biofilm biomass was quantified by OD590 normalized to OD600. Eight replicates were carried out for each strain.

Fluorescent in situ hybridization (FISH). After the voltage was stabilized, the anodic carbon cloth was subjected to FISH. Fixation, hybridization and washing of the FISH sample were performed sequentially as described previously with slight modifications38. After washed by PBS buffer, the sample was fixed in 4% paraformaldehyde for 4 hours at 4 °C, and then stored at -20 °C in a solution with 1:1 (v/v) of PBS buffer and 96% ethanol. Before hybridization, the fixed sample was transferred to a 6-well microplate and dried at 46 °C for 15 min. Then the sample was dehydrated in a gradient of 50%, 80%, and 95% ethanol for 3 min each. After drying at 46 °C, hybridizations with


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oligonucleotide probes were performed in the hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl, 0.01% SDS and 40% formamide) at 46 °C for 2 hours. Probe ECO45A which targeted 23S rRNA of E. coli was 5’-labeled with Cy5 fluorescence39, and probe SHEW227 targeting 16s rRNA of genus Shewanella was labelled with FITC fluorescence40. After FISH hybridization, the hybridization buffer was washed away with pre-warmed washing buffer (46 mM NaCl, 20 mM Tris-HCl and 5 mM EDTA), then the sample was incubated at 48 °C for 10 min. Following the incubation, the sample was rinsed with ice-cold ddH2O, air dried, and mounted in Citifluor AF1 Antifadent Solution to reduce photo-bleaching. Sample visualization was performed by Carl Zeiss Confocal Laser Scanning Microscopy (CLSM) LSM 780 with a 20 × objective.

Results and Discussion

Characterization of xylose-fed co-culture MFC

WT E. coli and WT S. oneidensis were inoculated into the anodic chamber of the MFC, and the current generation using xylose as the sole carbon source was examined. Considerable and stable voltage was detected during the discharge of the co-culture inoculated MFC (Figure 2a). The metabolite concentrations in the anolyte during MFC discharge were quantified to study the metabolite communication between E. coli and S. oneidensis in the co-culture (Figure 2b). Xylose was quickly consumed within 42 h. Acetate and succinate were found to accumulate during the MFC discharge, which were most likely the anaerobic fermentation products of E. coli. However the concentration of


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formate firstly increased during the MFC discharge, but significantly dropped at the end of the discharge. This observation suggests that formate is the electron donor for S. oneidensis provided by E. coli, as the quick digestion of xylose by E. coli led to an initial accumulation of the metabolite, which subsequently decreased by S. oneidensis upon xylose depletion. This observation is in accordance with our reported result when glucose was used as the carbon source for the co-culture.18 Formate has a relatively lower redox potential (formate/CO2, -0.42V vs. SHE) than other organic acids, thus would provide electrons with higher energy. However, the high proportion (51.8%) of E. coli cells on the electrode (Figure 2c) limited the electricity generation by the exoelectrogen S. oneidensis.

A synthetic riboflavin synthesis pathway incorporated into E. coli

Flavins play crucial roles in extracellular electron transfer of S. oneidensis. When intracellular electrons transfer from quinone pools, CymA, MtrCAB and arrive at the OM,41-47 the concentration of flavins determines the interfacial electron transfer rate between OM of S. oneidensis and the electrode as electron shuttle or cofactor.23,25-27,48-50 However, the physiological concentration of flavins secreted by S. oneidensis was rather low (~1 µM), hence caused much potential loss during the EET process.25,29,51,52 In addition, it is reported that the concentration of flavins could also facilitate the biofilm formation of S. oneidensis on the anode probably driven by accelerated electrode respiration.28,29 Hence, flavin overproduction was targeted not only to accelerate


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interfacial electron transfer between S. oneidensis and the electrode, but also to specifically enhance attachment of S. oneidensis to the electrode surface.

Considering the higher metabolic activity of E. coli than S. oneidensis and ribulose 5phosphate (R5P), one of the precursors for riboflavin synthesis, is the intermediate of xylose isomerization, a riboflavin synthesis pathway originated from Bacillus subtilis was incorporated into E. coli BL21 (DE3). This riboflavin synthesis pathway contained four genes ribADEH (Figure 3a), which enabled the overproduction of riboflavin from the precursors guanosine 5-triphosphate (GTP) and R5P (Figure 3b). Two Plac promoters controlled the expression of ribADEH in E. coli BL21 (DE3) strain. Compared with E. coli harboring empty vector pSB1AK3 as the control, the recombinant E. coli incorporated with this synthetic riboflavin pathway could secreted 34.1 times’ higher concentration of flavins aerobically for 16 h, from 3.3 ± 0.2 µM to 115.2 ± 2.1 µM (Figure 3c), secreted via transporters during the active growth of E. coli 53,54. The synthetic riboflavin pathway could enable further accumulation of more riboflavin in the MFC anolyte anaerobically with intact anaerobic growth rate of recombinant E. coli compared with WT E. coli with empty vector (Figure S1b, Supporting Information).

The recombinant E. coli harboring the riboflavin synthesis module along with WT S. oneidensis were inoculated into the anodic chamber of MFC, and generated much higher voltage than that of WT E. coli co-cultured with WT S. oneidensis. (Figure 3d), indicating decreased potential loss benefited by the overproduced flavins. Moreover, the overproduced flavins greatly altered the composition of the microbial community in the electrode-attached biofilm. As shown in Figure 3e, by co-culturing WT S. oneidensis


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with the recombinant E. coli, the cell number of immobilized S. oneidensis in the anodic biofilm increased to 83.3 ± 8.2 ×107 from 30.2 ± 5.1 ×107 (co-culture of WT S. oneidensis with WT E. coli), and the cell number of anode-attached E. coli greatly decreased by 93.3% to 1.7 ± 0.2 ×107 from 32.2 ± 3.7 ×107. Such decrease in immobilized E. coli was a result of strong competition from S. oneidensis, as the biofilm formation ability of E. coli itself was not influenced by the incorporated riboflavin synthesis pathway (shown in Figure S1a, Supporting information). Consequently, the ratio of cell number of S. oneidensis to E. coli in the biofilm elevated from 48.2% to 98.0% by the overproduced flavins.

Surface modification of S. oneidensis further increased cell number of immobilized S. oneidensis

Surface properties of microorganism are also crucial factors determining biofilm formation. Since S. oneidensis is highly hydrophilic, modification of carbon electrode to be more hydrophilic has been reported to increase the immobilization and EET of S. oneidensis.55,56 Meanwhile, biologically engineering the surface of S. oneidensis to be more hydrophobic has also been proved feasible to enhance biofilm formation on carbon electrode.57-59 A putrescine biosynthesis disruption mutant of S. oneidensis CP2-1-S1 previously developed by us was found to be highly hydrophobic and cohesive (its hydrophobicity was increased from 24.0% to 89.0%), and formed more stable biofilm than WT S. oneidensis in a flow cell.36 This CP2-1-S1 mutant was employed in the present work to further increase the biofilm formation of S. oneidensis on the carbon electrode.


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When the recombinant E. coli containing the riboflavin synthesis pathway was cocultured with mutant S. oneidensis CP2-1-S1, the voltage output was further improved (Figure 4a). The cell number of S. oneidensis in the anodic biofilm was further increased from 83.3 ± 8.2 × 107 to 120.2 ± 8.3 × 107 attributed to the surface modification of S. oneidensis, and the proportion of S. oneidensis reached 98.2% of the total anodeimmobilized cells with minimal presence of E. coli (Figure 4b). Such successfully tuned microbial community profile in the anodic biofilm was further verified by fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) imaging (Figure 4c, d). E coli was probed by ECO45A which targeted E. coli 23S rRNA and 5’labeled with Cy5 fluorescence.39 S. oneidensis was probed by SHEW227 targeting 16s rRNA of genus Shewanella and labelled with FITC fluorescence.40 As shown in the CLSM images, the overproduced flavins and hydrophobic interaction significantly altered the interspecies interaction between E. coli and S. oneidensis on the anode. For anodic biofilm formed by WT E. coli and WT S. oneidensis, E. coli cells were mostly observed in aggregates packaged with S. oneidensis cells (Figure 4c). However, such aggregates of E. coli with S. oneidensis disappeared, and E. coli cells were barely seen on the anode in the presence of overproduced flavins and highly hydrophobic interactions between S. oneidensis and the anode (Figure 4d), which was most likely a consequence of intense competition for electrode attachment from S. oneidensis. Such rationally tuned composition of anodic biofilm directed by our artificial purpose is more favorable for efficient electron transfer in the biofilm towards the anode.

Electrochemical analysis and MFC performances


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Electrochemical analysis was conducted to study the electron transfer capacity of our rationally tuned biofilm. Cyclic voltammetry (CV) at a low scan rate was applied to reveal redox reaction kinetics at the cell-electrode interfaces at pseudo-steady state. As shown in Figure 5a, the catalytic current of these biofilms enhanced greatly as the ratio and cell numbers of immobilized S. oneidensis on the electrode increased, starting from around -0.2 V (vs. SHE) that is the typical redox peaks of flavins. To further reveal whether the increase in the catalytic current was attributed to more immobilized cells or more electroactive S. oneidensis cell, the current signals in Figure 5a were normalized to the corresponding cell numbers of attached S. oneidensis, giving rise to Figure 5b. It was shown that the presence of overproduced flavins by recombinant E. coli significantly enhanced the turnover electricity per single cell of WT S. oneidensis (Figure 5b), probably due to accelerated flavin-mediated EET and higher efficiency of electron transfer in the biofilm with dramatically excluded E. coli cells. In addition, the EET rates of WT S. oneidensis and CP2-1-S1 mutant were fairly similar, while S. oneidensis CP2-1S1 contributed to the further improved overall catalytic current mainly by its increased cell number in the anodic biofilm. Thus, interfered by the overproduced flavins and hydrophobic interaction, our rationally tuned biofilm contained more electroactive S. oneidensis cell and more immobilized S. oneidensis cells, thus, the MFC with the synthetic microbial consortia composing recombinant E. coli and S. oneidensis CP2-1-S1 generated the highest catalytic current.

The MFC performances were evaluated by conducting linear sweep voltammetry (LSV) at a low scan rate (0.1 mV/s) from their open circuit potentials (OCPs). Our engineering efforts of the microbial consortia did not significantly alter the OCP of MFCs 15 ACS Paragon Plus Environment

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(~-0.76V). The polarization curves were shown in Figure 5c. The slope of the linear part in the polarization curve represents the internal resistance of the corresponding MFC. It was implied that the increased flavin biosynthesis in E. coli and hydrophobicity in S. oneidensis could significantly decrease the internal resistance of the anodic biofilm formed by the synthetic microbial consortia. The power density output curves were calculated by multiplying the current with its corresponding voltage in the polarization curves (as shown in Figure 5d). For E. coli harboring empty vector co-cultured with WT S. oneidensis, the maximum power density was 92.8 ± 5.9 mW/m2 and the maximum current density was 0.17 ± 0.01 A/m2. Upon flavin was overproduced by the recombinant E. coli, the co-culture with WT S. oneidensis generated maximum power density of 491.1 ± 8.3 mW/m2 and maximum current density of 0.99 ± 0.05 A/m2. Finally the maximum power density was elevated to 728.6 ± 36.8 mW/m2 with the maximum current density being 1.44 ± 0.08 A/m2 when the recombinant E. coli was co-cultured with CP2-1-S1 mutant. To the best of our knowledge, this MFC power density is the highest record reported for MFCs with biologically engineered S. oneidensis16,28,57-60 (Table 1). In addition, xylose was for the first time utilized as the electron donor for S. oneidensisinvolved MFCs. This high-performance xylose-fed MFC with synthetic microbial consortia focused on programming the electrode-associated microbial community, paving a new way for further development of biomass-fed MFC technology catalyzed by synthetic microbial consortia. ASSOCIATED CONTENT Supporting Information


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The following file is available free of charge on the ACS Publications website at DOI. Quantitative analysis of the biofilm formation and anaerobic growth curve with monitored flavin concentration by the WT/A4 E. coli. Primer sequences of the genes for riboflavin biosynthesis. AUTHOR INFORMATION Corresponding Authors Hao Song, Tel.: +86-22-60977327, E-mail: [email protected]; and Bin Cao, Tel.: +656790-5277, E-mail: [email protected]. Author Contributions YY, BC and HS designed experiments; YY, YW, YH and YC conducted experiments; all authors analyzed data and wrote the paper. ACKNOWLEDGMENTS

This research was supported by the National Basic Research Program of China (“973” Program: 2014CB745100), Chinese National High Technology Research and Development Program (“863” Program: 2012AA02A701), National Natural Science Foundation of China (NSFC 21376174), a grant from Singapore Centre on Environmental Life Sciences Engineering (SCELSE), and an AcRF Tier-2 grant (MOE2011-T2-2-035, Singapore). We acknowledge Prof. Dianne Newman (Caltech) for providing E. coli WM3064, and Prof. Ying-Jin Yuan (Tianjin University) for help in gene synthesis. ABBREVIATIONS MFC, microbial fuel cell; EET, extracellular electron transfer; OM, outer membrane; GTP, guanosine 5-triphosphate; R5P, riboluso 5-phosphate; WT, wild type; IPTG,


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isopropyl β-D-1-thiogalactopyranoside; CV, cyclic voltammetry; OCP, open circuit potential; LSV, linear sweep voltammetry. REFERENCES (1)

He, C. S.; Mu, Z. X.; Yang, H. Y.; Wang, Y. Z.; Mu, Y.; Yu, H. Q. Chemosphere.

2015, 140, 12-17. (2)

Li, W. W.; Yu, H. Q.; He, Z. Energy Environ. Sci. 2014, 7, 911-924.

(3)

Lovley, D. R. Nat. Rev. Microbiol. 2006, 4, 497-508.

(4)

Logan, B. E. Nat. Rev. Microbiol. 2009, 7, 375-381.

(5)

Qiao, Y.; Bao, S. J.; Li, C. M. Energy Environ. Sci. 2010, 3, 544-553.

(6)

Wang, H. M.; Luo, H. P.; Fallgren, P. H.; Jin, S.; Ren, Z. J. Biotechnol. Adv.

2015, 33, 317-334. (7)

Okamoto, A.; Nakamura, R.; Hashimoto, K. Electrochim. Acta. 2011, 56, 5526-

5531. (8)

Jiao, Y.; Qian, F.; Li, Y.; Wang, G.; Saltikov, C. W.; Gralnick, J. A. J. Bacteriol.

2011, 193, 3662-5. (9)

Coursolle, D.; Baron, D. B.; Bond, D. R.; Gralnick, J. A. J. Bacteriol. 2010, 192,

467-474. (10) Franks, A. E.; Nevin, K. P. Energies. 2010, 3, 899-919.


Page 23 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(11) Pinchuk, G. E.; Geydebrekht, O. V.; Hill, E. A.; Reed, J. L.; Konopka, A. E.; Beliaev, A. S.; Fredrickson, J. K. Appl. Environ. Microbiol. 2011, 77, 8234-8240. (12) Pinchuk, G. E.; Rodionov, D. A.; Yang, C.; Li, X. Q.; Osterman, A. L.; Dervyn, E.; Geydebrekht, O. V.; Reed, S. B.; Romine, M. F.; Collart, F. R.; Scott, J. H.; Fredrickson, J. K.; Beliaev, A. S. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 2874-2879. (13) Pinchuk, G. E.; Hill, E. A.; Geydebrekht, O. V.; De Ingeniis, J.; Zhang, X. L.; Osterman, A.; Scott, J. H.; Reed, S. B.; Romine, M. F.; Konopka, A. E.; Beliaev, A. S.; Fredrickson, J. K.; Reed, J. L. PLoS Comput. Biol. 2010, 6, e1000822. (14) Fredrickson, J. K.; Romine, M. F.; Beliaev, A. S.; Auchtung, J. M.; Driscoll, M. E.; Gardner, T. S.; Nealson, K. H.; Osterman, A. L.; Pinchuk, G.; Reed, J. L.; Rodionov, D. A.; Rodrigues, J. L. M.; Saffarini, D. A.; Serres, M. H.; Spormann, A. M.; Zhulin, I. B.; Tiedje, J. M. Nat. Rev. Microbiol. 2008, 6, 592-603. (15) Rodionov, D. A.; Yang, C.; Li, X.; Rodionova, I. A.; Wang, Y.; Obraztsova, A. Y.; Zagnitko, O. P.; Overbeek, R.; Romine, M. F.; Reed, S.; Fredrickson, J. K.; Nealson, K. H.; Osterman, A. L. BMC Genomics. 2010. 11, 494-512. (16) Choi, D.; Lee, S. B.; Kim, S.; Min, B.; Choi, I. G.; Chang, I. S. Bioresour. Technol. 2014, 154, 59-66. (17) Rosenbaum, M. A.; Bar, H. Y.; Beg, Q. K.; Segre, D.; Booth, J.; Cotta, M. A.; Angenent, L. T. Bioresour. Technol. 2011, 102, 2623-2628. (18) Wang, V. B.; Sivakumar, K.; Yang, L.; Zhang, Q.; Kjelleberg, S.; Loo, S. C.; Cao, B. Sci. Rep.-Uk. 2015, 5, 11222. 19 ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 37

(19) Renslow, R.; Babauta, J.; Kuprat, A.; Schenk, J.; Ivory, C.; Fredrickson, J.; Beyenal, H. Phys. Chem. Chem. Phys. 2013, 15, 19262-19283. (20) Torres, C. I.; Marcus, A. K.; Lee, H. S.; Parameswaran, P.; Krajmalnik-Brown, R.; Rittmann, B. E. FEMS Microbiol. Rev. 2010, 34, 3-17. (21) Edwards, M. J.; White, G. F.; Norman, M.; Tome-Fernandez, A.; Ainsworth, E.; Shi, L.; Fredrickson, J. K.; Zachara, J. M.; Butt, J. N.; Richardson, D. J.; Clarke, T. A. Sci. Rep.-Uk. 2015, 5, 11677. (22) Wang, Z. M.; Shi, Z.; Shi, L.; White, G. F.; Richardson, D. J.; Clarke, T. A.; Fredrickson, J. K.; Zachara, J. M. Geochim. Cosmochim. Ac. 2015, 163, 299-310. (23) Okamoto, A.; Nakamura, R.; Nealson, K. H.; Hashimoto, K. ChemElectroChem. 2014, 1, 1808–1812. (24) Wu, C.; Cheng, Y. Y.; Li, B. B.; Li, W. W.; Li, D. B.; Yu, H. Q. Bioresour. Technol. 2013, 136, 711-4. (25) Okamoto, A.; Hashimoto, K.; Nealson, K. H.; Nakamura, R. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 7856-7861. (26) Brutinel, E. D.; Gralnick, J. A. Appl. Microbiol. Biotechnol. 2012, 93, 41-48. (27) Marsili, E.; Baron, D. B.; Shikhare, I. D.; Coursolle, D.; Gralnick, J. A.; Bond, D. R. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 3968-3973. (28) Yang, Y.; Ding, Y.; Hu, Y.; Cao, B.; Rice, S. A.; Kjelleberg, S.; Song, H. ACS Synth. Biol. 2015, 4, 815-823.


Page 25 of 37

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(29) Baron, D.; LaBelle, E.; Coursolle, D.; Gralnick, J. A.; Bond, D. R. J. Biol. Chem. 2009, 284, 28865-28873. (30) Zhou, K.; Qiao, K. J.; Edgar, S.; Stephanopoulos, G. Nat. Biotechnol. 2015, 33, 377-383. (31) Song, H.; Ding, M. Z.; Jia, X. Q.; Ma, Q.; Yuan, Y. J. Chem. Soc. Rev. 2014, 43, 6954-6981. (32) Fredrickson, J. K. Science. 2015, 348, 1425-1427. (33) Caspi, R.; Altman, T.; Billington, R.; Dreher, K.; Foerster, H.; Fulcher, C. A.; Holland, T. A.; Keseler, I. M.; Kothari, A.; Kubo, A.; Krummenacker, M.; Latendresse, M.; Mueller, L. A.; Ong, Q.; Paley, S.; Subhraveti, P.; Weaver, D. S.; Weerasinghe, D.; Zhang, P. F.; Karp, P. D. Nucleic Acids Res. 2014, 42, D459-D471. (34) Grote, A.; Hiller, K.; Scheer, M.; Munch, R.; Nortemann, B.; Hempel, D. C.; Jahn, D. Nucleic Acids Res. 2005, 33, W526-W531. (35) Saltikov, C. W.; Newman, D. K. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 109838. (36) Ding, Y. Z.; Peng, N.; Du, Y. H.; Ji, L. H.; Cao, B. Appl. Environ. Microbiol. 2014, 80, 1498-1506. (37) Wu, Y. C.; Ding, Y. Z.; Cohen, Y.; Cao, B. Appl. Microbiol. Biotechnol. 2015, 99, 1967-1976.


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Page 26 of 37

(38) Kiely, P. D.; Call, D. F.; Yates, M. D.; Regan, J. M.; Logan, B. E. Appl. Microbiol. Biotechnol. 2010, 88, 371-380. (39) Neef, A.; Amann, R.; Schleifer, K.-H. Syst. Appl. Microbiol. 1995, 18, 113-122. (40) Huggett, M. J.; Crocetti, G. R.; Kjelleberg, S.; Steinberg, P. D. Aquat. Microb. Ecol. 2008, 53, 161-171. (41) Marritt, S. J.; McMillan, D. G. G.; Shi, L.; Fredrickson, J. K.; Zachara, J. M.; Richardson, D. J.; Jeuken, L. J. C.; Butt, J. N. Biochem. Soc. Trans. 2012, 40, 1217-1221. (42) Marritt, S. J.; Lowe, T. G.; Bye, J.; McMillan, D. G. G.; Shi, L.; Fredrickson, J.; Zachara, J.; Richardson, D. J.; Cheesman, M. R.; Jeuken, L. J. C.; Butt, J. N. Biochem. J. 2012, 444, 465-474. (43) Shi, L.; Rosso, K. M.; Zachara, J. M.; Fredrickson, J. K. Biochem Soc. Trans. 2012, 40, 1261-1267. (44) Shi, L.; Rosso, K. M.; Clarke, T. A.; Richardson, D. J.; Zachara, J. M.; Fredrickson, J. K. Front. Microbiol. 2012, 3, 50. (45). Hartshorne, R. S.; Reardon, C. L.; Ross, D.; Nuester, J.; Clarke, T. A.; Gates, A. J.; Mills, P. C.; Fredrickson, J. K.; Zachara, J. M.; Shi, L.; Beliaev, A. S.; Marshall, M. J.; Tien, M.; Brantley, S.; Butt, J. N.; Richardson, D. J. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 22169-22174. (46) Meitl, L. A.; Eggleston, C. M.; Colberg, P. J. S.; Khare, N.; Reardon, C. L.; Shi, L. Geochim. Cosmochim. Ac. 2009, 73, 5292-5307.


Page 27 of 37

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(47) Shi, L. A.; Richardson, D. J.; Wang, Z. M.; Kerisit, S. N.; Rosso, K. M.; Zachara, J. M.; Fredrickson, J. K. Environ. Microbiol. Rep. 2009, 1, 220-227. (48) von Canstein, H.; Ogawa, J.; Shimizu, S.; Lloyd, J. R. Appl. Environ. Microbiol. 2008, 74, 615-623. (49) Okamoto, A.; Hashimoto, K.; Nealson, K. H. Angew. Chem. Int. Ed. Engl. 2014, 53, 10988–10991. (50) Okamoto, A.; Saito, K.; Inoue, K.; Nealson, K. H.; Hashimoto, K.; Nakamura, R. Energy Environ. Sci. 2014, 7, 1357-1361. (51) Kotloski, N. J.; Gralnick, J. A. Mbio. 2013, 4, e00553-12. (52) Ross, D. E.; Brantley, S. L.; Tien, M. Appl. Environ. Microbiol. 2009, 75, 52185226. (53) Gutiérrez-Preciado A.; Torres A. G.; Merino E.; Bonomi H. R.; Goldbaum F. A.; García-Angulo V. A. PLoS One. 2015, 10, e0126124. (54) McAnulty, M. J.; Wood, T. K. Bioengineered. 2014, 5, 386-392. (55) Guo, K.; Freguia, S.; Dennis, P. G.; Chen, X.; Donose, B. C.; Keller, J.; Gooding, J. J.; Rabaey, K. Environ. Sci. Technol. 2013, 47, 7563-7570. (56) Ding, C. M.; Lv, M. L.; Zhu, Y.; Jiang, L.; Liu, H. Angew. Chem. Int. Ed. Engl. 2015, 54, 1446-1451. (57) Kouzuma, A.; Meng, X. Y.; Kimura, N.; Hashimoto, K.; Watanabe, K. Appl. Environ. Microbiol. 2010, 76, 4151-4157. 23 ACS Paragon Plus Environment

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(58). Tajima, N.; Kouzuma, A.; Hashimoto, K.; Watanabe, K. Biosci. Biotechnol. Biochem. 2011, 75, 2229-2233. (59) Kouzuma, A.; Oba, H.; Tajima, N.; Hashimoto, K.; Watanabe, K. BMC Microbiol. 2014, 14, 190. (60) Liu, T.; Yu, Y. Y.; Deng, X. P.; Ng, C. K.; Cao, B.; Wang, J. Y.; Rice, S. A.; Kjelleberg, S.; Song, H. Biotechnol. Bioeng. 2015, 112, 2051-2059.


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Table 1. Summary of the reported performances of MFCs catalyzed by biologically modified S. oneidensis. Modification

Anode

Disruption of SO3177 by transposon mutagenesis Disruption of SO3350 by transposon mutagenesis Disruption of SO1860 by transposon mutagenesis Cloning of glf and glk genes from Z. mobilis Overexpression of a c-di-GMP biosynthesis gene yedH Overproduction of flavins by a synthetic flavin synthesis pathway A synthetic microbial consortium composed of recombinant E. coli and mutant S. oneidensis CP2-1-S1

MFC configura tion Graphite S;450 ml felt Graphite S; 450 ml felt Graphite S; 450 ml felt Graphite D; 25 ml felt Carbon D; 50 ml cloth Carbon D; 140 ml cloth Carbon D; 140 ml cloth

Power density (mW/m2) 65.3

Carbon source

Refere nce

Lactate

57

~110

Lactate

58

87.4

Lactate

59

*

Glucose

16

167.6

Lactate

60

233.0

Lactate

28

728.6

Xylose

This work

* The power density of the marked work was not characterized in the published article. However, indicated by the current density, the power density of recombinant S. oneidensis consuming glucose most likely would not be significantly improved compared with that of WT S. oneidensis consuming lactate.


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Figure 1. Schematic view of the rational designed synthetic microbial consortia in bioelectrochemical systems. (a) Metabolic pathway of xylose fermentation in E. coli under anaerobic condition. E. coli plays a role as the fermenter, digests xylose and generates mix organic acids. A synthetic riboflavin pathway is incorporated into E. coli to overproduce riboflavin. (b) Extracellular electron transfer pathway in S. oneidensis. Formate, which is one of the mix organic acids produced through pyruvate metabolism in E. coli, is the primary electron donor for S. oneidensis. The electrons extracted from formate oxidation catalyzed by Fdh (formate dehydrogenase) go through quinone pool, CymA, MtrCAB and finally to the electrode. Flavins are the electron shuttles or cofactors of OM c-Cyts, mediating EET of S. oneidensis, the overproduced riboflavin by E. coli could accelerate the electrode respiration of S. oneidensis. (c) Initial attachment process of E. coli and S. oneidensis to the electrode. Attachment of S. oneidensis to the electrode could be promoted by the overproduced riboflavin. In addition, engineering the surface property of S. oneidensis to be more hydrophobic could also facilitate the attachment through hydrophobic interaction. (d) Composition of the electrode-associated microbial community. After the initial attachment of S. oneidensis is enhanced, the majority of bacteria on the electrode would be S. oneidensis.


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Figure 2. MFC discharge curve (a), metabolite quantification (b), and analysis of microbial community immobilized on the anode (c) for MFC inoculated with WT E. coli and WT S. oneidensis. (a) “WT+WT” represents E. coli harboring an empty vector mixed with WT S. oneidensis. The data were representative of three independent replicates. (b) Metabolites in anolytes at the initial time (0 h), after discharging for 42 h, and at the time when the voltage dropped ~30% in a short time (indicating exhaust of electron donor) were quantified, respectively. (c) CFU counts of E. coli and S. oneidensis were displayed on the left y axis, and the relative ratio of S. oneidensis in the anodic biofilm was displayed on the right y axis.


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Figure 3. The synthetic riboflavin synthesis pathway incorporated in E. coli. (a) Plasmid map of the vector expressing ribADEH from B. subtilis in E. coli. For coordinated expression, two Plac promoters were placed to monitor the expression of whole riboflavin synthesis operon. (b) Intracellular riboflavin was overproduced from the precursors of guanosine 5-phosphate (GTP) and ribulose 5-phosphate (R5P) catalyzed by RibADEH. The intracellular riboflavin was secreted through unknown exporters. (c) Quantification of flavins (RF: riboflavin; FMN: flavin mononucleotide) produced by E. coli harboring pSB1AK3 or pSB1AK3-A4. (d) MFC voltage output curves of recombinant E. coli with the synthetic riboflavin synthesis module co-cultured with WT S. oneidensis (“A4+WT”, blue line) compared with that of WT E. coli mixed with WT S. oneidensis (“WT+WT”, black line). At ~67h, when the “A4+WT” consortium exhausted the electron donor, 35 ml of the anodic electrolyte (total 140 ml) was replaced with fresh M9 based medium (xylose concentration in the anodic chamber was ~5 mM at the beginning of the 2nd run of the MFC). Data were representative of three replicates. (e) 28 ACS Paragon Plus Environment

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Analysis of microbial community attached on the anode influenced by the synthetic riboflavin synthesis pathway.

Figure 4. Impact of mutant S. oneidensis CP2-1-S1 on the MFC voltage output (a) and composition of the microbial community attached on the anode. (a) “A4+WT” stands for recombinant E. coli harboring pSB1AK3-A4 co-cultured with WT S. oneidensis; “A4+CP” stands for recombinant E. coli harboring pSB1AK3-A4 co-cultured with mutant S. oneidensis CP2-1-S1. The voltage output curve of MFC inoculated with WT E. coli and WT S. oneidensis (“WT+WT”) was displayed here again for better comparison. At ~67h, when the “A4+WT” and “A4+CP”consortia depleted the electron donors, 35 ml of the anodic electrolyte was replaced with fresh M9 based medium (xylose concentration was ~5 mM at the beginning of the 2nd run of the MFC) (b) CFU counts of E. coli and S. oneidensis (according to left y axis) and the relative ratios of S. oneidensis on the anode electrodes (according to the right y axis) were displayed and compared among the three combinations. Fluorescence in situ hybridization (FISH) and 29 ACS Paragon Plus Environment

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confocal laser scanning microscopy (CLSM) images of “A4+CP” (d) and “WT+WT” (c) were compared. Fluorescent rRNA oligonucleotide probes were used to differentiate E. coli (ECO45A which targeted E. coli 23S rRNA was 5’-labeled with Cy5 fluorescence39, red) and S. oneidensis (SHEW227 which targeted 16s rRNA of genus Shewanella was labelled with FITC fluorescence40, green) cells. Aggregates of E. coli and S. oneidensis showed yellow color. Long thin darkness is the shape of carbon fiber. Scale bar is 20 µm.

Figure 5. Electrochemical analysis of the microbial consortia in MFCs. (a) Turnover cyclic voltammetry (CV) at a low scan rate of 1 mV/s. (b) Normalized CV signals by the corresponding cell numbers of attached S. oneidensis on the anodes. (c) MFC polarization curves got by linear sweep voltammetry (LSV) with a low scan rate of 0.1 mV/s. (d) MFC power density output curves, which were calculated based on the corresponding polarization curves in (c). All data were representative of three independent replicates.


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