Enhancing Extracellular Electron Transfer of Shewanella oneidensis ...

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Enhancing Extracellular Electron Transfer of Shewanella oneidensis MR‑1 through Coupling Improved Flavin Synthesis and MetalReducing Conduit for Pollutant Degradation Di Min,†,‡,# Lei Cheng,†,# Feng Zhang,† Xue-Na Huang,† Dao-Bo Li,† Dong-Feng Liu,*,† Tai-Chu Lau,‡,§ Yang Mu,† and Han-Qing Yu*,† †

CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei, 230026, China ‡ USTC-CityU Joint Advanced Research Center, Suzhou, China § Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, China S Supporting Information *

ABSTRACT: Dissimilatory metal reducing bacteria (DMRB) are capable of extracellular electron transfer (EET) to insoluble metal oxides, which are used as external electron acceptors by DMRB for their anaerobic respiration. The EET process has important contribution to environmental remediation mineral cycling, and bioelectrochemical systems. However, the low EET efficiency remains to be one of the major bottlenecks for its practical applications for pollutant degradation. In this work, Shewanella oneidensis MR-1, a model DMRB, was used to examine the feasibility of enhancing the EET and its biodegradation capacity through genetic engineering. A flavin biosynthesis gene cluster ribD-ribC-ribBA-ribE and metal-reducing conduit biosynthesis gene cluster mtrC-mtrA-mtrB were coexpressed in S. oneidensis MR-1. Compared to the control strain, the engineered strain was found to exhibit an improved EET capacity in microbial fuel cells and potentiostat-controlled electrochemical cells, with an increase in maximum current density by approximate 110% and 87%, respectively. The electrochemical impedance spectroscopy (EIS) analysis showed that the current increase correlated with the lower interfacial charge-transfer resistance of the engineered strain. Meanwhile, a three times more rapid removal rate of methyl orange by the engineered strain confirmed the improvement of its EET and biodegradation ability. Our results demonstrate that coupling of improved synthesis of mediators and metal-reducing conduits could be an efficient strategy to enhance EET in S. oneidensis MR-1, which is essential to the applications of DMRB for environmental remediation, wastewater treatment, and bioenergy recovery from wastes.



INTRODUCTION Dissimilatory metal-reducing bacteria (DMRB) can couple the oxidation of organic or inorganic compounds to the reduction of metal compounds as a part of their energy generating strategy.1−3 Close attention has been paid to DMRB for their important influence on the biogeochemical cycling of metals4 in sediments, submerged soils, and the terrestrial subsurface.5 Furthermore, microbial metal reduction has been utilized in various biotechnological processes,6−9 such as bioenergy recovery with microbial fuel cells (MFCs), biosynthesis with microbial electrosynthesis (MES) and pollutant degradation in environmental remediation.10−12 In recent years, many types of bacteria, including members of the genera Shewanella,13−15 Geobacter,16,17 Desulf uromonas,18 Aeromonas,19 and Pelobacter,20 have been identified as DMRB. Among DMRB, S. oneidensis MR-1 is becoming a research focus due to its respiration versatility,21 metabolic diversity,22 and genetic accessibility. Shewanella can transfer electrons extracellularly to various electron acceptors for respiration, including iron and manganese oxides, sulfur species, NO3−, NO2−, trimethylamine © XXXX American Chemical Society

N-oxide (TMAO), dimethyl sulfoxide (DMSO), fumarate, O2, and even, radionuclides and toxic metals, such as Tc, U, Cr.5,22 These characteristics make it a model organism for microbial extracellular electron transfer (EET) investigations. Extensive studies have been conducted to understand microbial EET pathways in strain MR-1. Direct EET, including the physical contact of outer membrane cytochromes (mainly through metal-reducing conduit),14 conductive nanowires,23 and flavinmediated EET24 have been identified as main mechanism of EET in S. oneidensis MR-1. The kinetics and efficiency of EET in S. oneidensis MR-1 are key factors in determining its performance in bioelectrochemical systems and environmental remediation. Efforts have been made to improve the EET by optimizing the electrode materials,25 operation parameters,26 and components of Received: Revised: Accepted: Published: A

September 13, 2016 April 4, 2017 April 17, 2017 April 17, 2017 DOI: 10.1021/acs.est.6b04640 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology MFCs27 or addition of metal ions, such as Cu2+,28 Cd2+,28 and Ca2+.29 Another logical method is genetic modification to increase the amount of releasable electrons and improve the efficiency of transferring released electrons to extracellular electron acceptors. It was reported that a transposon mutant of S. oneidensis MR-1 deficient in the biosynthesis of cell surface polysaccharides showed an increased ability to adhere to a graphite anode and to generate 50% more current in an MFC than the control strain.30 In addition, an engineered S. oneidensis MR-1, heterologously overexpressed a cyclic diguanylate monophosphate (c-di-GMP) biosynthesis gene ydeH, significantly enhanced biofilm formation and EET.31 However, the efficiency of these methods to enhance EET is relatively low. A feasible approach that a flavin biosynthesis pathway from Bacillus subtilis was heterologously expressed in S. oneidensis MR-1 was adopted with a high efficiency.32 The synthetic flavin module enabled enhancing bidirectional EET rate of MR-1. Since metal-reducing conduit and electron shuttles have been identified to play central roles in EET, it is reasonable to assume that the expression level of metalreducing conduit may become another bottleneck for further improvement of EET in S. oneidensis MR-1 in the presence of sufficient flavins. To date, there is no report about the coupling of improved synthesis of flavins and metal-reducing conduit in S. oneidensis MR-1. Therefore, this work aims to elevate the EET in S. oneidensis MR-1 and its pollutant degradation capacity by using genetic engineering approaches. For this purpose, a serial of engineered strains were constructed and expression of mtrC-mtrA-mtrB and ribD-ribC-ribBA-ribE in S. oneidensis MR-1 was analyzed (Figure 1). Then, the EET performance of the engineered strain via coupling improved synthesis of metal-reducing conduit and flavins in bioelectrochemical systems was evaluated. Finally, the engineered strain was applied to decolorate methyl orange (MO), a typical organic pollutant. In this way, the feasibility of elevating EET in S. oneidensis MR1 by a synergy between direct EET and mediated EET was explored. The engineered strains could be used for potential applications in environmental remediation and power generation from wastes in electrochemical systems.

Figure 1. Schematic illustration of the flavin and metal-reducing conduit mediated EET pathway in S. oneidensis MR-1. Intracellular electrons flow through CymA and MtrA and come to outer membrane cytochrome c (OmcA and MtrC). The interfacial electron transfer between outer membrane and extracellular electron acceptors may occur by direct contact-based EET, via outer membrane cytochrome c or nanowires, or indirect EET mediated by flavin as electron shuttles. Flavin adenine dinucleotide (FAD) is synthesized from the precursors guanosine 5′-triphosphate (GTP) and D-ribulose 5′-phosphate (R5P) by flavin biosynthesis gene cluster ribD-ribC-ribBA-ribE. S. oneidensis MR-1 secretes FAD into the periplasmic space, where it is hydrolyzed by UshA to flavin mononucleotide (FMN) and adenosine monophosphate (AMP). Moreover, FAD is also used as cofactor of fumarate reductase flavoprotein subunit (FccA). FMN diffuses through outer membrane porins and hydrolyses into riboflavin (RF).

needed, 100 μg/mL 2,6-diaminopimelic acid was dosed for the growth of E. coli WM3064. Microbial Cultivation Conditions for Flavins Production. S. oneidensis MR-1 from −80 °C freezer stock was inoculated into 30 mL of LB broth shaking at 30 °C overnight aerobically. For the flavin synthesis under aerobic conditions, 1 mL of S. oneidensis MR-1 culture suspension was inoculated into 50 mL of Shewanella mineral medium with 20 mM lactate as elector donor. The composition of Shewanella mineral medium was referred to a previous report.33 For the flavin synthesis under anaerobic conditions, 1 mL of S. oneidensis MR1 (harboring pYYDT or recombination plasmids) culture suspension was inoculated into 50 mL of Shewanella mineral medium (with 20 mM lactate and 40 mM sodium fumarate) in a sealed 100 mL serum vial. S. oneidensis MR-1 strains were cultured at 30 °C with 200 rpm. The flavin concentration in the vials was monitored by periodically sampling and analysis. Bacterial culture of 1 mL was withdrawn from each serum at given time intervals and immediately centrifuged at 6000 rpm (5000g) for 90 s. The flavin concentration in the supernatants was determined using a high-performance liquid chromatography (HPLC, Agilent Co., USA) following a method reported previously.33 RNA Extraction and qRT-PCR Analysis. The RNAiso Plus Kit (Takara Co., China) was used for extracting total cellular RNA from Shewanella cultures. The absorption of light at 230, 260, and 280 nm was exploited to characterize the concentration and purity of the final extracted RNA. The PrimeScript II first Strand cDNA Synthesis Kit (Takara Co., China) and the SYBR Premix Ex Taq (Takara Co., China) were used for the cDNA synthesis and the qRT-PCR analysis,



EXPERIMENTAL SECTION Plasmid Construction and Transformation. All plasmid constructions were performed in Escherichia coli. E. coli strains were cultured in Luria−Bertani (LB) medium at 37 °C with 200 rpm. Whenever needed, 50 μg/mL kanamycin was added into the culture medium. The sequence coding for flavin biosynthesis gene cluster ribD-ribC-ribBA-ribE was amplified from S. oneidensis MR-1, purified, and treated with SpeI and Sbf I. The fragment was cloned into pYYDT expression plasmid32 and form the resulting expression plasmids pYYDT-Rib. Similarly, the sequence coding for metal-reducing conduit biosynthesis gene cluster mtrC-mtrA-mtrB was amplified from S. oneidensis MR-1 and cloned into pYYDT expression plasmid to form the resulting expression plasmids pYYDT-Mtr. An additional promoter Ptac and metal-reducing conduit biosynthesis gene cluster mtrC-mtrA-mtrB were added into plasmid pYYDT-Rib to form the resulting expression plasmid pYYDT-RM (Figure 2a). Plasmids to be introduced into S. oneidensis MR-1 were first transformed into the plasmid donor strain E. coli WM3064 and transferred into S. oneidensis MR-1 by conjugation. When B

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Figure 2. Multigene assembly in pYYDT and functional expression of mtrC-mtrA-mtrB and ribD-ribC-ribBA-ribE in S. oneidensis MR-1. (a) Schematic plasmid maps of expression vectors. (b) Riboflavin concentration secreted by the control strain and recombinant S. oneidensis MR-1 strains under anaerobic and aerobic conditions.

NaOH for 10 min at 95 °C to solubilize the attached cells. The supernatant was analyzed using a BCA protein assay kit (Beyotime Co., China) according to the manufacturer’s instructions. MO Bioreduction Tests. The control strain and the strain MR-1/pYYDT-RM were cultured in LB medium at 30 °C. After 12 h cultivation, cells in LB medium were harvested, washed and inoculated in Shewanella mineral medium under aerobic conditions. The overnight cultures were used for the anaerobic MO decolorization experiments. Lactate and MO were added into Shewanella mineral medium and used as the sole carbon source and the electron acceptor, respectively. The medium in each serum vial was sparged with N2 to ensure an anaerobic atmosphere. The initial concentration of cells was OD600 of 0.1. The MO concentration was measured using a UV−vis spectrophotometer (UV-2401PC, Shimadzu Co., Japan) at 465 nm.

respectively, according to the manufacturer’s instruction. All real-time RT-PCR reactions were conducted using the StepOne real-time PCR system (Applied Biosystems Inc., USA). The relative quantity of tested cDNA normalized to the abundance of 16s cDNA was automatically calculated by this system. Primers used for qRT-PCR analysis are listed in Table S1. Electrochemical Tests. Dual-chamber MFCs (Figure S1a), with the electrodes connected via a 1 kΩ external resistor, were used to record voltage output of Shewanella strains every 10 min using a data acquisition system (USB2801, ATD Co., China). Carbon felt (Beijing Sanye Carbon Co., China) with a specific surface area of 12 cm2 and a proton exchange membrane (GEFC-10N, GEFC Co., China) were used as the electrode materials and the separator, respectively. The composition of catholyte was 50 mM potassium ferricyanide in 50-mM phosphate buffer solution at pH 7.0. Prior to the experiment, an anaerobic atmosphere of the anode and cathode chambers was achieved by flushing with high-purity nitrogen gas. S. oneidensis MR-1 strains were incubation into the MFC anode chamber at 30 °C. All tests were conducted in triplicate. To evaluate the performance of MFCs, linear sweep voltammetry (LSV) at 1 mV/s voltage scan rate was used to measure the polarization curves. The power density output curves could be calculated by multiplying the current with its corresponding voltage. Electrochemical impedance spectroscopy (EIS) was used to evaluate the internal resistance of the MFCs over a frequency range from 0.01 Hz to 100 kHz at an open circuit potential with a perturbation signal of 5 mV. To investigate the EET ability of S. oneidensis MR-1 in a constant potential, S. oneidensis MR-1 strains were cultured in anaerobic mineral salts medium (including 20 mM lactate and 40 mM sodium fumarate) with filter-sterilized casamino acids (0.05% vol/vol) until an OD600 of 0.4 was reached, then transferred into a conventional three-electrode microbial electrolysis cell (MEC, Figure S1b) under anaerobic atmosphere with a CHI1030B electrochemical workstation (Chenhua Instrument Co., China) served as a potentiostat, and lactate was added (20 mM) to ensure excess electron donor. A constant potential of 0.2 V (vs Ag/AgCl) was applied to the carbon paper electrodes (1.5 × 2 cm2) and monitored the change of the currents with time. To quantify the attached biomass on electrodes, the total protein concentration on the electrodes was determined. Carbon felt electrodes were removed from the electrochemical cells, washed twice in PBS buffer, and incubated in 2 mL of 1 N



RESULTS Multigene Assembly in pYYDT. The plasmid pYYDT has been used as a standardized molecular building block for facilitating convenient and fast assembly of genetic modules in S. oneidensis MR-1.32 The mtrC-mtrA-mtrB gene cluster from S. oneidensis MR-1 was cloned into pYYDT, which was named as pYYDT-Mtr for the improved expression of metal-reducing conduit in S. oneidensis MR-1. The flavin biosynthesis pathway (clustered in sequential order of ribD-ribC-ribBA-ribE) of S. oneidensis MR-1 was assembled into pYYDT, which was named as pYYDT-Rib for the enhanced synthesis of flavins. The plasmids containing both of mtrC-mtrA-mtrB genes and ribDribC-ribBA-ribE genes were named as pYYDT-RM. One additional promoter Plac was placed after ribE to achieve coordinated efficient expression of all genes. Plasmid pYYDT served as control. All plasmids are shown in Figure 2a. Functional Expression of mtrC-mtrA-mtrB and ribDribC-ribBA-ribE in S. oneidensis MR-1. The control strain (MR-1/pYYDT) and all recombinant S. oneidensis MR-1 strains (MR-1/pYYDT-Rib, MR-1/pYYDT-Mtr and MR-1/pYYDTRM) were aerobically cultured. The flavin concentration secreted by these strains was measured after 64-h incubation (Figure 2b). The S. oneidensis MR-1 strain bearing the flavin biosynthesis gene cluster ribD-ribC-ribBA-ribE (strain MR-1/ pYYDT-Rib and strain MR-1/pYYDT-RM) produced 0.50 μM riboflavin/g protein and 0.49 μM riboflavin/g protein, respectively, which was 2 times and 1.96 times higher than C

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Figure 3. Current output (a) and power density (b) of the control strain and recombinant strains in MFCs.

that of the control strain (0.25 μM riboflavin/g protein). The strain MR-1/pYYDT-Mtr excluding the flavin biosynthesis genes (0.21 μM riboflavin/g protein) achieved a similar riboflavin level as the control strain. This result confirms the functional expression of the flavin biosynthesis pathway genes in the recombinant S. oneidensis MR-1 strains. The riboflavin biosynthesized by the control strain and the recombinant S. oneidensis MR-1 strain under anaerobic conditions exhibited a similar trend but relatively lower levels than those under aerobic conditions (Figure 2b). The strain MR-1/pYYDT-Rib produced the highest level of riboflavin (0.065 μM riboflavin/g protein), while the riboflavin concentration of the other strains was 0.060 μM riboflavin/g protein (strain MR-1/pYYDT-RM), 0.013 μM riboflavin/g protein (strain MR-1/pYYDT-Mtr), 0.020 μM riboflavin/g protein (strain MR-1/pYYDT), respectively. The extracellular flavin mononucleotide (FMN) concentration of all the four strains after 64-h incubation was also determined (Figure S2). Under aerobic conditions, the FMN concentration secreted by the strains MR-1/pYYDT, MR-1/pYYDT-Rib, MR-1/pYYDTMtr, and MR-1/pYYDT-RM was 0.83, 1.29, 0.70, and 1.61 μM FMN/g protein, respectively. Under anaerobic conditions, the value was 0.14, 0.31, 0.10, and 0.34 μM FMN/g protein, respectively. The transcription of flavin biosynthesis genes (ribD, ribC, ribBA, and ribE) and metal-reducing conduits genes (mtrC, mtrA, and mtrB) of the control strain and the recombinant S. oneidensis MR-1 were detected using qRT-PCR (Figure S3). The expression of ribD, ribC, ribBA, and ribE in the strain MR1/pYYDT-Rib was enhanced by about 90-, 74-, 65-, and 57fold, respectively, compared with the control strain. The expression of mtrC, mtrA, and mtrB in the strain MR-1/ pYYDT-Rib showed a similar level as the control strain. The levels of mtrC, mtrA, and mtrB in the strain MR-1/pYYDT-Mtr were increased by about 37-, 26-, and 19-fold, respectively, compared to the levels of the control strain. Meanwhile, there was no significant difference in the expression of ribD, ribC, ribBA, and ribE between the strain MR-1/pYYDT-Mtr and the control strain. Compared with the control strain, the expression levels of ribD, ribC, ribBA, and ribE in the strain MR-1/pYYDT-RM were increased by about 46-, 34-, 23-, and 20-fold, respectively, which were lower than those of the strain MR-1/pYYDT-Rib. The expression levels of mtrC, mtrA, and mtrB in the strain MR-1/pYYDT-RM exhibited a similar trend, which were lower than those of the strain MR-1/pYYDT-Mtr. A possible explanation for this phenomenon was that a larger plasmid (the size of plasmid pYYDT-RM was greater than that of

pYYDT-Rib and pYYDT-Mtr) exhibited a lower replication and transcription efficiency. Interestingly, the induction of the ribD, ribC, ribBA, and ribE genes was not of the same level, and ribD (close to the tac promoter) and ribE (distant from the tac promoter), respectively, exhibited the highest and the lowest fold changes in expression levels (Figure 2a). Such a difference might be attributed to the polar expression effect, as observed with the operons in other bacteria.34 The expression levels of mtrC, mtrA, and mtrB also displayed the same polar effect. Electricity-Generating Capacities of the MFC Cultivated with the Strains. The current densities of the dualchamber MFCs cultivated with the control strain and the recombinant S. oneidensis MR-1 strains were measured (Figure 3a). All the MFCs achieved a current density after about 20 h and remained above 40 mA/m2 for 120 h. The MFCs with the strain MR-1/pYYDT-Rib and MR-1/pYYDT-Mtr could generate a higher current density (126 and 133 mA/m2, respectively) than that of the MFC with the control strain (89 mA/m2). The current density of the MFC inoculated with the strain MR-1/pYYDT-RM reached its maximum value of 188 mA/m2, which was 2.1 times higher than that of the control strain. The polarization and power output curves (Figure 3b) show that MFCs inoculated with the three recombinant S. oneidensis MR-1 strains had higher power densities than the control strain. The MFC inoculated with the strain MR-1/pYYDT-RM achieved a maximum power density of 0.037 W/m2, which was 3.50-fold as much as that of the control MFC. The values for the MFCs inoculated with other two recombinant S. oneidensis MR-1 strains were 0.015 (strain MR-1/pYYDT-Rib) and 0.020 W/m2 (MR-1/pYYDT-Mtr), respectively, which were 1.45- and 1.90-fold higher than that of the control MFC. This result indicates that both the improved synthesis of flavins or metal-reducing conduit could enhance the EET in S. oneidensis MR-1. Coupling improved synthesis of flavins and metal-reducing conduit in S. oneidensis MR-1 offered a far more efficient means of achieving a higher current density compared to the individually improved synthesis of flavins or metalreducing conduit. The mechanism behind the enhancement of power output and density in the MFCs cultivated with the recombinant S. oneidensis MR-1 strains was explored. EIS was used to determine the electron transfer resistance of the MFCs inoculated with the control strain and recombinant S. oneidensis MR-1 strains. The measured EIS results showed the welldefined single semicircles over the high frequency range for the control strain and recombinant S. oneidensis MR-1 strains (Figure S4). The diameter of the semicircle corresponds to the D

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Environmental Science & Technology interfacial charge-transfer resistance (Rct), which usually represents the resistance of electrochemical reactions on the electrode. A smaller Rct indicates a faster electron-transfer rate. The Rct values of the MFCs with the strain MR-1/pYYDT-Rib (Rct = 2169) and the strain MR-1/pYYDT-Mtr (Rct = 1888) were remarkably lower compared to the value of the MFC with the control strain (Rct = 4537), implying that the individually improved synthesis of flavins or metal-reducing conduit accelerated the electron transfer. This might be ascribed to the enhanced electron transfer rate by the synthesis of more flavins or formation of more biomass on the electrode in MFCs. The minimum Rct value (449) was obtained for the MFC with the strain MR-1/pYYDT-RM), which was only 10% of the value for the control strain. This result demonstrates that coupling improved synthesis of flavins and metal-reducing conduit in Shewanella substantially lowered the resistance of electrochemical reactions on the electrode, ultimately leading to the better performance of the MFC inoculated with the engineering strains. An MEC system was also used to compare the microbial electric current generation from the control strain and each recombinant S. oneidensis MR-1 strains at a constant potential. The S. oneidensis MR-1 strains were inoculated into the MEC systems, and the anode was poised at 0.2 V (vs Ag/AgCl). After initiation of MECs, oxidation current was immediately observed (Figure 4). This current reflected oxidation of lactate by

Figure 5. Cyclic voltammetry (CV) characterization of the MECs with inoculations of the control strain and recombinant strains under turnover condition, respectively. The scanning rate of the CV curves was 5 mV/s. Inset: Close-up of the catholic peak in the range of −0.5 V to −0.1 V vs Ag/AgCl.

whose redox peak position often varies slightly, depending on microenvironments.35,36 The strain MR-1/pYYDT-Mtr exhibited a much higher peak intensity centered at −0.25 V vs Ag/AgCl than that of the MR-1/pYYDT. This result demonstrated that more outermembrane c-type cytochromes in the strain MR-1/pYYDT-Mtr were involved in EET, implying the improved MFC performance and the enhanced direct contact-based catalytic current. In addition, there was another pair of peaks centered at −0.43 V vs Ag/AgCl, generating a flavin-mediated catalytic current as reported previously.24,37 Specifically, the strain MR-1/pYYDT-Rib showed a much higher peak intensity centered at −0.43 V vs Ag/AgCl than that of the MR-1/pYYDT, implying an enhanced electron transfer rate by the synthesis of more flavins in the strain MR-1/pYYDT-Rib. The intensities of redox peaks at −0.25 V vs Ag/AgCl and −0.43 V vs Ag/AgCl in the strain MR-1/pYYDT-RM were much higher than those in the MR-1/ pYYDT. This result further demonstrated that both flavinmediated and contact-based EET pathways were increased in the strain MR-1/pYYDT-RM. MO Decoloration by the Engineered Strains. Given the excellent performance of the strain MR-1/pYYDT-RM in the electrochemical systems, it was selected for the decoloration of MO, which is reported to be extracellularly reduced by S. oneidensis MR-1.38 The MO removal efficiencies obtained for the control strain and the strain MR-1/pYYDT-RM are illustrated in Figure 6. A more rapid MO removal process was observed for the strain MR-1/pYYDT-RM compared to the control strain. Within the initial 10 h, the strain MR-1/ pYYDT-RM completely decolorized MO, while color removal by the control strain was 47% (Figure 6a). Meanwhile, the firstorder rate constants (k) were calculated to evaluate their MO removal rates (Figure 6b). Within the initial 12 h, the k values increased by three times from 0.0852 h−1 for the control strain to 0.259 h−1 for the strain MR-1/pYYDT-RM. These results are consistent with the above electrochemical measurements. All of these demonstrate that coupling improved synthesis of mediators and metal-reducing conduits was an efficient approach to enhance EET in S. oneidensis MR-1.

Figure 4. Amperometric data from the MECs inoculated with the control strain and recombinant strains.

microbes and electrons transfer from cells to electrodes. The anodic current increased rapidly in the subsequent 10 h and decreased slowly thereafter. The maximum oxidation current density of the strain MR-1/pYYDT-RM was approximately 0.43 A/m2, while it was about 0.23 A/m2 for the MEC with the control strain. Such an improvement in the anode performance implies that the EET process was enhanced by using the engineering strains. Cyclic voltammetry (CV) analysis could provide useful information on the mechanism of EET. The CV results of MECs were shown in Figure 5. For MR-1, a pair of anodic and cathodic peaks centered at −0.25 V vs Ag/AgCl was identified in the CV under turnover conditions. This pair of peaks corresponded to the outer membrane c-type cytochromes, E

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that more biomass attached on electrode leads to the elevated catalytic current via outer membrane cytochrome c.31,32 To further investigate the impact of the engineered strains on the direct EET, the biomass of the strain MR-1/pYYDT-RM, MR1/pYYDT-Rib, MR-1/pYYDT-Mtr, and the control strain on the electrodes in the MFCs was measured as 13.4 ± 0.3, 11.7 ± 0.2, 12.3 ± 0.2 and 10 ± 0.2 μg cm−2, respectively. Moreover, the smallest Rct was observed for the MFC with the strain MR1/pYYDT-RM, further suggesting the fastest electron-transfer rate of electrochemical reactions on the electrode. These results demonstrate that the elevated EET ability of the strain MR-1/ pYYDT-RM is owing to the synergetic effect between the incremental shuttle-mediated EET and direct EET. Flavins are synthesized de novo by plants and microorganisms.40 Recently, a number of bacteria, such as S. oneidensis, Campylobacter jejuni, Helicobacter pylori, and three species of methanotrophic bacteria and Geothrix fermentans, have been found to use secreted flavins as electron shuttles to accelerate respiration of insoluble minerals and electrodes.41 Eleven phylogenetically distinct Shewanella strains have also been reported to secrete flavins and utilize them as electron shuttles under anaerobic conditions.37 A survey of the recently sequenced microbial genomes shows that the homologues of the metal-reducing conduits pathway of S. oneidensis MR-1 exist in the Fe(III)-reducing bacteria, Aeromonas hydrophila, Ferrimonas balearica, and Rhodoferax ferrireducens, and the Fe(II)-oxidizing bacteria, Dechloromonas aromatica RCB, Gallionella capsiferriformans ES-2, and Sideroxydans lithotrophicus ES-1.42 It is assumed that coupling the improved synthesis of flavins with metal-reducing conduits could enhanc EET in strains bearing two pathways genes. Moreover, this method might also be applicable for the DMRB capable of both direct and mediated EET. This warrants further investigations. After coupling improved synthesis of flavins and metalreducing conduit of S. oneidensis MR-1, we presented a novel strategy to enhance its EET significantly. However, it should be noticed that the efficiency of EET in the strain MR-1/pYYDTRM is still low. On one hand, the low yield of flavins in the engineering strains under anaerobic conditions still restrict the further improvement of EET, which might be caused by the lower fluxes of the essential metabolic pathways for flavin biosynthesis under anaerobic conditions.32 On the other hand, a polar expression effect results in an unbalance of the flavin biosynthesis gene clusters or metal-reducing conduit biosynthesis gene transcriptions. In S. oneidensis MR-1, one guanosine triphosphate (GTP) and two ribulose-5-phosphate molecules are converted into one riboflavin molecule in a stepwise manner by the enzymes encoded by the ribA, ribB, ribD, ribH, and ribE genes.41 The unbalancing transcription of the flavin biosynthesis gene clusters might lead to the accumulation of intermediate metabolites, which finally reduce the titer of flavins secreted by the engineering strains. In S. oneidensis MR1, MtrABC can be isolated as a protein complex with a stoichiometry of 1:1:1 and serve as an electron conduit between the periplasm of S. oneidensis MR-1 cells and its extracellular environments.43,44 The unbalancing transcription of the mtr gene cluster might lead to the formation of improper protein complexes, which may affect the electron transfer from the cell interior to the outer membrane. Thus, efforts should be made to optimize the recombinant strains through metabolic engineering to further enhance EET. For instance, several approaches, such as the optimization of gene codons, tuning promoter strengths and balancing the flavin biosynthesis gene

Figure 6. (a) Anaerobic reduction of MO at 45 mg L−1 by the control strain and the strain MR-1/pYYDT-RM. (b) Kinetic curves of MO reduction by Shewanella related strains.



DISCUSSION S. oneidensis MR-1 has the ability to breathe a wide variety of extracellular electron receptors, such as insoluble metal oxide, radionuclides and toxic metals, and even electrode.22 The concurrence of direct EET via outer membrane cytochromes and flavin-mediated EET was proven both in metal reduction and bioelectricity production.14,39 Our MFC results showed that either the enhanced synthesis of flavins or improved metalreducing conduit in S. oneidensis MR-1 could enhance the current output and power density of the MFCs inoculated with the engineered strains. Furthermore, a synergy was achieved when the flavin biosynthesis gene cluster ribD-ribC-ribBA-ribE and metal-reducing conduit biosynthesis gene cluster mtrCmtrA-mtrB approaches were coexpressed. The CV tests for the biofilm in the MECs demonstrated a synergy between the flavin biosynthesis genes and metal-reducing conduit biosynthesis genes in the engineering strain (Figure 5). The strain MR-1/ pYYDT-Rib showed a much higher peak intensity centered at −0.43 V vs Ag/AgCl (flavin-mediated catalytic current) than that of the MR-1/pYYDT. Interestingly, a much higher peak intensity centered at −0.25 V vs Ag/AgCl (outer membrane ctype cytochromes-mediated catalytic current) was also found for the strain MR-1/pYYDT-Rib. Similarly, the improved expression of metal-reducing conduit in the strain MR-1/ pYYDT-Mtr also showed a much higher peak intensity centered at −0.43 V vs Ag/AgCl (flavin-mediated catalytic current) than that of the MR-1/pYYDT. In addition, the redox peaks in the strain MR-1 with the overexpressions of both flavin synthesis genes and metal-reducing genes (MR-1/pYYDT-RM) exhibited much higher peak intensities centered at −0.25 V vs Ag/AgCl and −0.43 V vs Ag/AgCl than those in the MR-1/ pYYDT. A similar observation was reported in a previous study.31 All of these results demonstrate that there was synergy between the flavin-mediated and metal-reducing conduitmediated EET. Such an enhancement could be attributed to several reasons. The elevated flavin concentration could increase the concentration gradient of either oxidized or reduced electron shuttles, and thus accelerate the diffusion process of free flavins between cell−electrode interfaces. Since the diffusion process is a ratelimiting step of the shuttle-mediated EET,32 the elevated flavin concentration could finally enhance the electron shuttlemediated EET. In addition, a number of studies have shown F

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(7) Logan, B. E. Extracting Hydrogen Electricity from Renewable Resources. Environ. Sci. Technol. 2004, 38 (9), 160a−167a. (8) Xing, D. F.; Zuo, Y.; Cheng, S. A.; Regan, J. M.; Logan, B. E. Electricity Generation by Rhodopseudomonas palustris DX-1. Environ. Sci. Technol. 2008, 42 (11), 4146−4151. (9) Logan, B. E.; Call, D.; Cheng, S.; Hamelers, H. V. M; Sleutels, T. H. J. A.; Jeremiasse, A. W.; Rozendal, R. A. Microbial Electrolysis Cells for High Yield Hydrogen Gas Production from Organic Matter. Environ. Sci. Technol. 2008, 42 (23), 8630−8640. (10) Lloyd, J. R. Microbial Reduction of Metals and Radionuclides. FEMS Microbiol. Rev. 2003, 27 (2−3), 411−25. (11) Cummings, D. E.; Caccavo, F.; Fendorf, S.; Rosenzweig, R. F. Arsenic Mobilization by the Dissimilatory Fe(III)-reducing Bacterium Shewanella alga BrY. Environ. Sci. Technol. 1999, 33 (5), 723−729. (12) Lu, X.; Liu, Y. R.; Johs, A.; Zhao, L. D.; Wang, T. S.; Yang, Z. M.; Lin, H.; Elias, D. A.; Pierce, E. M.; Liang, L. Y.; Barkay, T.; Gu, B. H. Anaerobic Mercury Methylation and Demethylation by Geobacter bemidjiensis Bem. Environ. Sci. Technol. 2016, 50 (8), 4366−4373. (13) Bretschger, O.; Obraztsova, A.; Sturm, C. A.; Chang, I. S.; Gorby, Y. A.; Reed, S. B.; Culley, D. E.; Reardon, C. L.; Barua, S.; Romine, M. F.; Zhou, J.; Beliaev, A. S.; Bouhenni, R.; Saffarini, D.; Mansfeld, F.; Kim, B. H.; Fredrickson, J. K.; Nealson, K. H. Current Production and Metal Oxide Reduction by Shewanella oneidensis MR-1 Wild Type and Mutants. Appl. Environ. Microbiol. 2008, 74 (2), 553− 553. (14) Coursolle, D.; Baron, D. B.; Bond, D. R.; Gralnick, J. A. The Mtr Respiratory Pathway Is Essential for Reducing Flavins and Electrodes in Shewanella oneidensis. J. Bacteriol. 2010, 192 (2), 467−474. (15) Kim, B. H.; Kim, H. J.; Hyun, M. S.; Park, D. H. Direct Electrode Reaction of Fe(III)-reducing Bacterium, Shewanella putrefaciens. J. Microbiol. Biotechnol. 1999, 9 (2), 127−131. (16) Bond, D. R.; Lovley, D. R. Electricity Production by Geobacter sulf urreducens Attached to Electrodes. Appl. Environ. Microbiol. 2003, 69 (3), 1548−1555. (17) Geelhoed, J. S.; Stams, A. J. M. Electricity-Assisted Biological Hydrogen Production from Acetate by Geobacter sulf urreducens. Environ. Sci. Technol. 2011, 45 (2), 815−820. (18) Coates, J. D.; Lonergan, D. J.; Philips, E. J. P.; Jenter, H.; Lovley, D. R. Desulfuromonas palmitatis sp nov, a Marine Dissimilatory Fe[III] Reducer That Can Oxidize Long-Chain Fatty Acids. Arch. Microbiol. 1995, 164 (6), 406−413. (19) Pham, C. A.; Jung, S. J.; Phung, N. T.; Lee, J.; Chang, I. S.; Kim, B. H.; Yi, H.; Chun, J. A Novel Electrochemically Active and Fe(III)reducing Bacterium Phylogenetically Related to Aeromonas hydrophila, Isolated from a Microbial Fuel Cell. FEMS Microbiol. Lett. 2003, 223 (1), 129−134. (20) Richter, H.; Lanthier, M.; Nevin, K. P.; Lovley, D. R. Lack of Electricity Production by Pelobacter carbinolicus Indicates That the Capacity for Fe(III) Oxide Reduction Does Not Necessarily Confer Electron Transfer Ability To Fuel Cell Anodes. Appl. Environ. Microbiol. 2007, 73 (16), 5347−53. (21) Myers, C. R.; Nealson, K. H. Bacterial Manganese Reduction and Growth with Manganese Oxide as the Sole Electron-Acceptor. Science 1988, 240 (4857), 1319−1321. (22) 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. Towards Environmental Systems Biology of Shewanella. Nat. Rev. Microbiol. 2008, 6 (8), 592−603. (23) Gorby, Y. A.; Yanina, S.; McLean, J. S.; Rosso, K. M.; Moyles, D.; Dohnalkova, A.; Beveridge, T. J.; Chang, I. S.; Kim, B. H.; Kim, K. S.; Culley, D. E.; Reed, S. B.; Romine, M. F.; Saffarini, D. A.; Hill, E. A.; Shi, L.; Elias, D. A.; Kennedy, D. W.; Pinchuk, G.; Watanabe, K.; Ishii, S.; Logan, B.; Nealson, K. H.; Fredrickson, J. K. Electrically Conductive Bacterial Nanowires Produced by Shewanella oneidensis Strain MR-1 and Other Microorganisms. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (30), 11358−11363.

cluster and metal-reducing conduit biosynthesis genes transcription to avoid misregulation of the post-transcriptional modifications, could be used to engineer the strains. In summary, we demonstrate that coupling improved synthesis of mediators with metal-reducing conduits is an efficient strategy to enhance EET in S. oneidensis MR-1. In addition to Shewanella, this strategy may be used as a broadspectrum approach for other DMRB because of its several advantages, such as easy manipulation, effectiveness and good expansibility. The engineering strains with an enhanced EET and higher reduction efficiency have potential applications in environmental remediation including bioremediation and treatment of heavy metal contaminated soil, groundwater and azo dyes-rich wastewaters in practice.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.6b04640. Fuel cell systems, the extracellular FMN concentration of all the four strains after 64-h incubation, qRT-PCR results, and nyquist plots by the control strain and recombinant Shewanella oneidensis MR-1 strains, and information about strains, plasmids, and primers used in this work (PDF)



AUTHOR INFORMATION

Corresponding Authors

*Fax: +86 551 63601592. E-mail: dfl@ustc.edu.cn. *Fax: +86 551 63601592. E-mail: [email protected] ORCID

Tai-Chu Lau: 0000-0002-0867-9746 Han-Qing Yu: 0000-0001-5247-6244 Author Contributions #

D.M. and L.C. contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors wish to thank the National Natural Science Foundation of China (21477120, 51538012, 21590812, and 21607146), and the Collaborative Innovation Center of Suzhou Nano Science and Technology of the Ministry of Education of China for the support.



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