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Enhancing Extracellular Electron Transfer of Shewanella oneidensis MR-1 through Coupling Improved Flavin Synthesis and Metal-Reducing 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 Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 17 Apr 2017 Downloaded from http://pubs.acs.org on April 17, 2017

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Environmental Science & Technology

Enhancing Extracellular Electron Transfer of Shewanella oneidensis MR-1 through Coupling Improved Flavin Synthesis and Metal-Reducing Conduit for Pollutant Degradation

Di Min1,2*, Lei Cheng1,*, Feng Zhang1, Xue-Na Huang1, Dao-Bo Li1, Dong-Feng Liu1,**, Tai-Chu Lau2,3, Yang Mu1, Han-Qing Yu1,** 1

CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei, 230026, China 2

3

USTC-CityU Joint Advanced Research Center, Suzhou, China

Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, China

1

ACS Paragon Plus Environment


1

Dissimilatory metal reducing bacteria (DMRB) are capable of extracellular electron

2

transfer (EET) to insoluble metal oxides, which are used as external electron acceptors

3

by DMRB for their anaerobic respiration. The EET process has important contribution

4

to environmental remediation mineral cycling, and bioelectrochemical systems.

5

However, the low EET efficiency remains to be one of the major bottlenecks for its

6

practical applications for pollutant degradation. In this work, Shewanella oneidensis

7

MR-1, a model DMRB, was used to examine the feasibility of enhancing the EET and

8

its biodegradation capacity through genetic engineering. A flavin biosynthesis gene

9

cluster ribD-ribC-ribBA-ribE and metal-reducing conduit biosynthesis gene cluster

10

mtrC-mtrA-mtrB were co-expressed in S. oneidensis MR-1. Compared to the control

11

strain, the engineered strain was found to exhibit an improved EET capacity in

12

microbial fuel cells and potentiostat-controlled electrochemical cells, with an increase

13

in maximum current density by approximate 110% and 87%, respectively. The

14

electrochemical impedance spectroscopy (EIS) analysis showed that the current

15

increase correlated with the lower interfacial charge-transfer resistance of the

16

engineered strain. Meanwhile, a three times more rapid removal rate of methyl orange

17

by the engineered strain confirmed the improvement of its EET and biodegradation

18

ability. Our results demonstrate that coupling of improved synthesis of mediators and

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metal-reducing conduits could be an efficient strategy to enhance EET in S.

20

oneidensis MR-1, which is essential to the applications of DMRB for environmental

21

remediation, wastewater treatment, and bioenergy recovery from wastes.

2


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INTRODUCTION

23 24

Dissimilatory metal-reducing bacteria (DMRB) can couple the oxidation of organic or

25

inorganic compounds to the reduction of metal compounds as a part of their energy

26

generating strategy.1-3 Close attention has been paid to DMRB for their important

27

influence on the biogeochemical cycling of metals4 in sediments, submerged soils,

28

and the terrestrial subsurface.5 Furthermore, microbial metal reduction has been

29

utilized in various biotechnological processes,6-9 such as bioenergy recovery with

30

microbial fuel cells (MFCs), biosynthesis with microbial electrosynthesis (MES) and

31

pollutant degradation in environmental remediation.10-12 In recent years, many types

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of bacteria, including members of the genera Shewanella,13-15 Geobacter,16,

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Desulfuromonas,18 Aeromonas,19 and Pelobacter,20 have been identified as DMRB.

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Among DMRB, S. oneidensis MR-1 is becoming a research focus due to its

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respiration versatility,21 metabolic diversity,22 and genetic accessibility. Shewanella

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can transfer electrons extracellularly to various electron acceptors for respiration,

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including iron and manganese oxides, sulfur species, NO3−, NO2−, trimethylamine

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N-oxide (TMAO), dimethylsulfoxide (DMSO), fumarate, O2, even radionuclides and

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toxic metals such as Tc, U, Cr.5, 22 These characteristics make it a model organism for

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microbial extracellular electron transfer (EET) investigations. Extensive studies have

41

been conducted to understand microbial EET pathways in strain MR-1. Direct EET,

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including the physical contact of outer membrane cytochromes (mainly through

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metal-reducing conduit),14 conductive nanowires,23 and flavin-mediated EET24 have 3


17


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been identified as main mechanism of EET in S. oneidensis MR-1.

45

The kinetics and efficiency of EET in S. oneidensis MR-1 are key factors in

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determining its performance in bioelectrochemical systems and environmental

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remediation. Efforts have been made to improve the EET by optimizing the electrode

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materials,25 operation parameters26 and components of MFCs27 or addition of metal

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ions, such as Cu2+,28 Cd2+,28 and Ca2+.29 Another logical method is genetic

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modification to increase the amount of releasable electrons and improve the

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efficiency of transferring released electrons to extracellular electron acceptors. It was

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reported that a transposon mutant of S. oneidensis MR-1 deficient in the biosynthesis

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of cell surface polysaccharides showed an increased ability to adhere to a graphite

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anode and to generate 50% more current in an MFC than the control strain.30 In

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addition, an engineered S. oneidensis MR-1, heterologously over-expressed a cyclic

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diguanylate monophosphate (c-di-GMP) biosynthesis gene ydeH, significantly

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enhanced biofilm formation and EET.31 However, the efficiency of these methods to

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enhance EET is relatively low. A feasible approach that a flavin biosynthesis

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pathway from Bacillus subtilis was heterologously expressed in S. oneidensis MR-1

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was adopted with a high efficiency.32 The synthetic flavin module enabled enhancing

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bidirectional EET rate of MR-1. Since metal-reducing conduit and electron shuttles

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have been identified to play central roles in EET, it is reasonable to assume that the

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expression level of metal-reducing conduit may become another bottleneck for

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further improvement of EET in S. oneidensis MR-1 in the presence of sufficient

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flavins. To date, there is no report about the coupling of improved synthesis of 4


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flavins and metal-reducing conduit in S. oneidensis MR-1.

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Therefore, this work aims to elevate the EET in S. oneidensis MR-1 and its

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pollutant degradation capacity by using genetic engineering approaches. For this

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purpose, a serial of engineered strains were constructed and expression of

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mtrC-mtrA-mtrB and ribD-ribC-ribBA-ribE in S. oneidensis MR-1 was analyzed

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(Figure 1). Then, the EET performance of the engineered strain via coupling

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improved synthesis of metal-reducing conduit and flavins in bioelectrochemical

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systems was evaluated. Finally, the engineered strain was applied to decolorate

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methyl orange (MO), a typical organic pollutant. In this way, the feasibility of

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elevating EET in S. oneidensis MR-1 by a synergy between direct EET and mediated

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EET was explored. The engineered strains could be used for potential applications in

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environmental remediation and power generation from wastes in electrochemical

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systems.

79 80

EXPERIMENTAL SECTION

81 82

Plasmid Construction and Transformation. All plasmid constructions were

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performed in Escherichia coli. E. coli strains were cultured in Luria-Bertani (LB)

84

medium at 37 oC with 200 rpm. Whenever needed, 50 µg/mL kanamycin was added

85

into the culture medium.

86

The sequence coding for flavin biosynthesis gene cluster ribD-ribC-ribBA-ribE

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was amplified from S. oneidensis MR-1, purified and treated with SpeI and SbfI. The 5



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fragment was cloned into pYYDT expression plasmid32 and form the resulting

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expression plasmids pYYDT-Rib. Similarly, the sequence coding for metal-reducing

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conduit biosynthesis gene cluster mtrC-mtrA-mtrB was amplified from S. oneidensis

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MR-1 and cloned into pYYDT expression plasmid to form the resulting expression

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plasmids pYYDT-Mtr. An additional promoter Ptac and metal-reducing conduit

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biosynthesis gene cluster mtrC-mtrA-mtrB were added into plasmid pYYDT-Rib to

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form the resulting expression plasmid pYYDT-RM (Figure 2a).

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Plasmids to be introduced into S. oneidensis MR-1 were first transformed into the

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plasmid donor strain E. coli WM3064 and transferred into S. oneidensis MR-1 by

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conjugation. When needed, 100 µg/mL 2,6-diaminopimelic acid was dosed for the

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growth of E. coli WM3064.

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Microbial Cultivation Conditions for Flavins Production. S. oneidensis MR-1

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from -80 oC freezer stock was inoculated into 30 mL LB broth shaking at 30 oC

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overnight aerobically. For the flavin synthesis under aerobic conditions, 1 mL S.

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oneidensis MR-1 culture suspension was inoculated into 50 mL Shewanella mineral

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medium with 20 mM lactate as elector donor. The composition of Shewanella mineral

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medium was referred to a previous report.33 For the flavin synthesis under anaerobic

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conditions, 1 mL S. oneidensis MR-1 (harboring pYYDT or recombination plasmids)

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culture suspension was inoculated into 50 mL Shewanella mineral medium (with 20

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mM lactate and 40 mM sodium fumarate) in a sealed 100 mL serum vial. S.

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oneidensis MR-1 strains were cultured at 30 oC with 200 rpm.

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The Flavin concentration in the vials was monitored by periodically sampling and 6


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analysis. Bacterial culture of 1 mL was withdrawn from each serum at given time

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intervals and immediately centrifuged at 6000 rpm (5000 g) for 90 s. The flavin

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concentration in the supernatants was determined using a high-performance liquid

113

chromatography (HPLC, Agilent Co., USA) following a method reported

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previously.33

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RNA Extraction and qRT-PCR Analysis. The RNAiso Plus Kit (Takara Co.,

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China) was used for extracting total cellular RNA from Shewanella cultures. The

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absorption of light at 230, 260 and 280 nm was exploited to characterize the

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concentration and purity of the final extracted RNA. The PrimeScript II 1st Strand

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cDNA Synthesis Kit (Takara Co., China) and the SYBR Premix Ex Taq (Takara Co.,

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China) were used for the cDNA synthesis and the qRT-PCR analysis, respectively,

121

according to the manufacturer’s instruction. All real-time RT-PCR reactions were

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conducted using the StepOne real-time PCR system (Applied Biosystems Inc., USA).

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The relative quantity of tested cDNA normalized to the abundance of 16s cDNA was

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automatically calculated by this system. Primers used for qRT-PCR analysis are

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listed in Table S1.

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Electrochemical Tests. Dual-chamber MFCs (Figure S1a), with the electrodes

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connected via a 1 kΩ external resistor, were used to record voltage output of

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Shewanella strains every 10 min using a data acquisition system (USB2801, ATD Co.,

129

China). Carbon felt (Beijing Sanye Carbon Co., China) with a specific surface area of

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12 cm2 and a proton exchange membrane (GEFC-10N, GEFC Co., China) were used

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as the electrode materials and the separator, respectively. The composition of 7



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catholyte was 50 mM potassium ferricyanide in 50-mM phosphate buffer solution at

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pH 7.0. Prior to the experiment, an anaerobic atmosphere of the anode and cathode

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chambers was achieved by flushing with high-purity nitrogen gas. S. oneidensis MR-1

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strains were incubation into the MFC anode chamber at 30 oC. All tests were

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conducted in triplicate.

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To evaluate the performance of MFCs, linear sweep voltammetry (LSV) at 1

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mV/s voltage scan rate was used to measure the polarization curves. The power

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density output curves could be calculated by multiplying the current with its

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corresponding voltage. Electrochemical impedance spectroscopy (EIS) was used to

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evaluate the internal resistance of the MFCs over a frequency range from 0.01 Hz to

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100 kHz at an open circuit potential with a perturbation signal of 5 mV.

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In order to investigate the EET ability of S. oneidensis MR-1 in a constant

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potential, S. oneidensis MR-1 strains were cultured in anaerobic mineral salts

145

medium (including 20 mM lactate and 40 mM sodium fumarate) with filter-sterilized

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casamino acids (0.05% vol/vol) until an OD600 of 0.4 was reached, then transferred

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into a conventional three-electrode microbial electrolysis cell (MEC, Figure S1b)

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under anaerobic atmosphere with a CHI1030B electro-chemical workstation

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(Chenhua Instrument Co., China) served as a potentiostat, and lactate was added (20

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mM) to ensure excess electron donor. A constant potential of 0.2 V (vs. Ag/AgCl)

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was applied to the carbon paper electrodes (1.5 × 2 cm2) and monitored the change

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of the currents with time.

8


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To quantify the attached biomass on electrodes, the total protein concentration

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on the electrodes was determined. Carbon felt electrodes were removed from the

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electrochemical cells, washed twice in PBS buffer, and incubated in 2 mL of 1 N

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NaOH for 10 min at 95 °C to solubilize the attached cells. The supernatant was

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analyzed using a BCA protein assay kit (Beyotime Co., China) according to the

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manufacturer’s instructions.

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MO Bioreduction Tests. The control strain and the strain MR-1/pYYDT-RM

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were cultured in LB medium at 30 °C. After 12 h cultivation, cells in LB medium

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were harvested, washed and inoculated in Shewanella mineral medium under aerobic

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conditions. The overnight cultures were used for the anaerobic MO decolorization

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experiments. Lactate and MO were added into Shewanella mineral medium and used

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as the sole carbon source and the electron acceptor, respectively. The medium in each

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serum vial was sparged with N2 to ensure an anaerobic atmosphere. The initial

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concentration of cells was OD600 of 0.1. The MO concentration was measured using a

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UV-Vis spectrophotometer (UV-2401PC, Shimadzu Co., Japan) at 465 nm.

168 169

RESULTS

170 171

Multigene Assembly in pYYDT. The plasmid pYYDT has been used as a

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standardized molecular building block for facilitating convenient and fast assembly of

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genetic modules in S. oneidensis MR-1.32 The mtrC-mtrA-mtrB gene cluster from S.

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oneidensis MR-1 was cloned into pYYDT, which was named as pYYDT-Mtr for the 9



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improved expression of metal-reducing conduit in S. oneidensis MR-1. The flavin

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biosynthesis pathway (clustered in sequential order of ribD-ribC-ribBA-ribE) of S.

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oneidensis MR-1 was assembled into pYYDT, which was named as pYYDT-Rib for

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the enhanced synthesis of flavins. The plasmids containing both of mtrC-mtrA-mtrB

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genes and ribD-ribC-ribBA-ribE genes were named as pYYDT-RM. One additional

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promoter Plac was placed after ribE to achieve coordinated efficient expression of all

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genes. Plasmid pYYDT served as control. All plasmids are shown in Figure 2a.

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Functional Expression of mtrC-mtrA-mtrB and ribD-ribC-ribBA-ribE in S.

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oneidensis MR-1. The control strain (MR-1/pYYDT) and all recombinant S.

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oneidensis

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MR-1/pYYDT-RM) were aerobically cultured. The flavin concentration secreted by

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these strains was measured after 64-h incubation (Figure 2b). The S. oneidensis MR-1

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strain bearing the flavin biosynthesis gene cluster ribD-ribC-ribBA-ribE (strain

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MR-1/pYYDT-Rib and strain MR-1/pYYDT-RM) produced 0.50 µM riboflavin/g

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protein and 0.49 µM riboflavin/g protein, respectively, which was 2 times and 1.96

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times higher than that of the control strain (0.25 µM riboflavin/g protein). The strain

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MR-1/pYYDT-Mtr excluding the flavin biosynthesis genes (0.21 µM riboflavin/g

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protein) achieved a similar riboflavin level as the control strain. This result confirms

193

the functional expression of the flavin biosynthesis pathway genes in the recombinant

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S. oneidensis MR-1 strains.

MR-1

strains

(MR-1/pYYDT-Rib,

MR-1/pYYDT-Mtr

and

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The riboflavin biosynthesized by the control strain and the recombinant S.

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oneidensis MR-1 strain under anaerobic conditions exhibited a similar trend but 10


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relatively lower levels than those under aerobic conditions (Figure 2b). The strain

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MR-1/pYYDT-Rib produced the highest level of riboflavin (0.065 µM riboflavin/g

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protein), while the riboflavin concentration of the other strains was 0.060 µM

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riboflavin/g protein (strain MR-1/pYYDT-RM), 0.013 µM riboflavin/g protein

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(strain MR-1/pYYDT-Mtr), 0.020 µM riboflavin/g protein (strain MR-1/pYYDT),

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respectively. The extracellular flavin mononucleotide (FMN) concentration of all the

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four strains after 64-h incubation was also determined (Figure S2). Under aerobic

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conditions, the FMN concentration secreted by the strains MR-1/pYYDT,

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MR-1/pYYDT-Rib, MR-1/pYYDT-Mtr and MR-1/pYYDT-RM was 0.83, 1.29, 0.70

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and 1.61 µM FMN/g protein, respectively. Under anaerobic conditions, the value

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was 0.14, 0.31, 0.10 and 0.34 µM FMN/g protein, respectively.

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The transcription of flavin biosynthesis genes (ribD, ribC, ribBA and ribE) and

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metal-reducing conduits genes (mtrC, mtrA and mtrB) of the control strain and the

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recombinant S. oneidensis MR-1 were detected using qRT-PCR (Figure S3). The

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expression of ribD, ribC, ribBA and ribE in the strain MR-1/pYYDT-Rib was

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enhanced by about 90-fold, 74-fold, 65-fold and 57–fold, respectively, compared with

213

the control strain. The expression of mtrC, mtrA and mtrB in the strain

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MR-1/pYYDT-Rib showed a similar level as the control strain. The levels of mtrC,

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mtrA and mtrB in the strain MR-1/pYYDT-Mtr were increased by about 37-fold,

216

26-fold and 19-fold, respectively, compared to the levels of the control strain.

217

Meanwhile, there was no significant difference in the expression of ribD, ribC, ribBA

218

and ribE between the strain MR-1/pYYDT-Mtr and the control strain. 11



219

Compared with the control strain, the expression levels of ribD, ribC, ribBA and

220

ribE in the strain MR-1/pYYDT-RM were increased by about 46-, 34-, 23- and

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20-fold, respectively, which were lower than those of the strain MR-1/pYYDT-Rib.

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The expression levels of mtrC, mtrA and mtrB in the strain MR-1/pYYDT-RM

223

exhibited a similar trend, which were lower than those of the strain

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MR-1/pYYDT-Mtr. A possible explanation for this phenomenon was that a larger

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plasmid (the size of plasmid pYYDT-RM was greater than that of pYYDT-Rib and

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pYYDT-Mtr) exhibited a lower replication and transcription efficiency. Interestingly,

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the induction of the ribD, ribC, ribBA and ribE genes was not of the same level, and

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ribD (close to the tac promoter) and ribE (distant from the tac promoter) respectively

229

exhibited the highest and the lowest fold changes in expression levels (Figure 2a).

230

Such a difference might be attributed to the polar expression effect, as observed with

231

the operons in other bacteria.34 The expression levels of mtrC, mtrA and mtrB also

232

displayed the same polar effect.

233

Electricity-Generating Capacities of the MFC Cultivated with the Strains.

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The current densities of the dual-chamber MFCs cultivated with the control strain and

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the recombinant S. oneidensis MR-1 strains were measured (Figure 3a). All the MFCs

236

achieved a current density after about 20 h and remained above 40 mA/m2 for 120 h.

237

The MFCs with the strain MR-1/pYYDT-Rib and MR-1/pYYDT-Mtr could generate

238

a higher current density (126 mA/m2 and 133 mA/m2, respectively) than that of the

239

MFC with the control strain (89 mA/m2). The current density of the MFC inoculated

240

with the strain MR-1/pYYDT-RM reached its maximum value of 188 mA/m2, which 12


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was 2.1 times higher than that of the control strain.

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The polarization and power output curves (Figure 3b) show that MFCs inoculated

243

with the three recombinant S. oneidensis MR-1 strains had higher power densities

244

than the control strain. The MFC inoculated with the strain MR-1/pYYDT-RM

245

achieved a maximum power density of 0.037 W/m2, which was 3.50-fold as much as

246

that of the control MFC. The values for the MFCs inoculated with other two

247

recombinant S. oneidensis MR-1 strains were 0.015 W/m2 (strain MR-1/pYYDT-Rib)

248

and 0.020 W/m2 (MR-1/pYYDT-Mtr), respectively, which were 1.45-fold and

249

1.90-fold higher than that of the control MFC. This result indicates that both the

250

improved synthesis of flavins or metal-reducing conduit could enhance the EET in S.

251

oneidensis MR-1. Coupling improved synthesis of flavins and metal-reducing conduit

252

in S. oneidensis MR-1 offered a far more efficient means of achieving a higher current

253

density compared to the individually improved synthesis of flavins or metal-reducing

254

conduit.

255

The mechanism behind the enhancement of power output and density in the

256

MFCs cultivated with the recombinant S. oneidensis MR-1 strains was explored. EIS

257

was used to determine the electron transfer resistance of the MFCs inoculated with the

258

control strain and recombinant S. oneidensis MR-1 strains. The measured EIS results

259

showed the well-defined single semicircles over the high frequency range for the

260

control strain and recombinant S. oneidensis MR-1 strains (Figure S4). The diameter

261

of the semicircle corresponds to the interfacial charge-transfer resistance (Rct), which

262

usually represents the resistance of electrochemical reactions on the electrode. A 13



263

smaller Rct indicates a faster electron-transfer rate. The Rct values of the MFCs with

264

the strain MR-1/pYYDT-Rib (Rct=2169) and the strain MR-1/pYYDT-Mtr (Rct=1888)

265

were remarkably lower compared to the value of the MFC with the control strain

266

(Rct=4537), implying that the individually improved synthesis of flavins or

267

metal-reducing conduit accelerated the electron transfer. This might be ascribed to the

268

enhanced electron transfer rate by the synthesis of more flavins or formation of more

269

biomass on the electrode in MFCs. The minimum Rct value (449) was obtained for the

270

MFC with the strain MR-1/pYYDT-RM), which was only 10% of the value for the

271

control strain. This result demonstrates that coupling improved synthesis of flavins

272

and metal-reducing conduit in Shewanella substantially lowered the resistance of

273

electrochemical reactions on the electrode, ultimately leading to the better

274

performance of the MFC inoculated with the engineering strains.

275

An MEC system was also used to compare the microbial electric current

276

generation from the control strain and each recombinant S. oneidensis MR-1 strains at

277

a constant potential. The S. oneidensis MR-1 strains were inoculated into the MEC

278

systems, and the anode was poised at 0.2 V (vs. Ag/AgCl). After initiation of MECs,

279

oxidation current was immediately observed (Figure 4). This current reflected

280

oxidation of lactate by microbes and electrons transfer from cells to electrodes. The

281

anodic current increased rapidly in the subsequent 10 h and decreased slowly

282

thereafter. The maximum oxidation current density of the strain MR-1/pYYDT-RM

283

was approximately 0.43 A/m2, while it was about 0.23 A/m2 for the MEC with the

284

control strain. Such an improvement in the anode performance implies that the EET 14


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process was enhanced by using the engineering strains.

286

Cyclic voltammetry (CV) analysis could provide useful information on the

287

mechanism of EET. The CV results of MECs were shown in Figure 5. For MR-1, a

288

pair of anodic and cathodic peaks centered at -0.25 V vs. Ag/AgCl was identified in

289

the CV under turnover conditions. This pair of peaks corresponded to the outer

290

membrane c-type cytochromes, whose redox peak position often varies slightly,

291

depending on microenvironments.35,

292

much higher peak intensity centered at -0.25 V vs. Ag/AgCl than that of the

293

MR-1/pYYDT. This result demonstrated that more outermembrane c-type

294

cytochromes in the strain MR-1/pYYDT-Mtr were involved in EET, implying the

295

improved MFC performance and the enhanced direct contact-based catalytic current.

296

In addition, there was another pair of peaks centered at -0.43 V vs. Ag/AgCl,

297

generating a flavin-mediated catalytic current as reported previously.24, 37 Specifically,

298

the strain MR-1/pYYDT-Rib showed a much higher peak intensity centered at -0.43 V

299

vs. Ag/AgCl than that of the MR-1/pYYDT, implying an enhanced electron transfer

300

rate by the synthesis of more flavins in the strain MR-1/pYYDT-Rib. The intensities

301

of redox peaks at -0.25 V vs. Ag/AgCl and -0.43 V vs. Ag/AgCl in the strain

302

MR-1/pYYDT-RM were much higher than those in the MR-1/pYYDT. This result

303

further demonstrated that both flavin-mediated and contact-based EET pathways were

304

increased in the strain MR-1/pYYDT-RM.

36

The strain MR-1/pYYDT-Mtr exhibited a

305

MO Decoloration by the Engineered Strains. Given the excellent performance

306

of the strain MR-1/pYYDT-RM in the electrochemical systems, it was selected for the 15



307

decoloration of MO, which is reported to be extracellularly reduced by S. oneidensis

308

MR-1.38 The MO removal efficiencies obtained for the control strain and the strain

309

MR-1/pYYDT-RM are illustrated in Figure 6. A more rapid MO removal process was

310

observed for the strain MR-1/pYYDT-RM compared to the control strain. Within the

311

initial 10 h, the strain MR-1/pYYDT-RM completely decolorized MO, while color

312

removal by the control strain was 47% (Figure 6a). Meanwhile, the first-order rate

313

constants (k) were calculated to evaluate their MO removal rates (Figure 6b). Within

314

the initial 12 h, the k values increased by three times from 0.0852 h-1 for the control

315

strain to 0.259 h-1 for the strain MR-1/pYYDT-RM. These results are consistent with

316

the above electrochemical measurements. All of these demonstrate that coupling

317

improved synthesis of mediators and metal-reducing conduits was an efficient

318

approach to enhance EET in S. oneidensis MR-1.

319 320

DISCUSSION

321 322

S. oneidensis MR-1 has the ability to breathe a wide variety of extracellular electron

323

receptors, such as insoluble metal oxide, radionuclides and toxic metals, and even

324

electrode.22 The concurrence of direct EET via outer membrane cytochromes and

325

flavin-mediated EET was proven both in metal reduction and bioelectricity

326

production.14,

327

flavins or improved metal-reducing conduit in S. oneidensis MR-1 could enhance the

328

current output and power density of the MFCs inoculated with the engineered strains.

39

Our MFC results showed that either the enhanced synthesis of

16


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329

Furthermore, a synergy was achieved when the flavin biosynthesis gene cluster

330

ribD-ribC-ribBA-ribE and metal-reducing conduit biosynthesis gene cluster

331

mtrC-mtrA-mtrB approaches were co-expressed. The CV tests for the biofilm in the

332

MECs demonstrated a synergy between the flavin biosynthesis genes and

333

metal-reducing conduit biosynthesis genes in the engineering strain (Figure 5). The

334

strain MR-1/pYYDT-Rib showed a much higher peak intensity centered at -0.43 V

335

vs. Ag/AgCl (flavin-mediated catalytic current) than that of the MR-1/pYYDT.

336

Interestingly, a much higher peak intensity centered at -0.25 V vs. Ag/AgCl (outer

337

membrane c-type cytochromes-mediated catalytic current) was also found for the

338

strain MR-1/pYYDT-Rib. Similarly, the improved expression of metal-reducing

339

conduit in the strain MR-1/pYYDT-Mtr also showed a much higher peak intensity

340

centered at -0.43 V vs. Ag/AgCl (flavin-mediated catalytic current) than that of the

341

MR-1/pYYDT. In addition, the redox peaks in the strain MR-1 with the

342

overexpressions of both flavin synthesis genes and metal-reducing genes

343

(MR-1/pYYDT-RM) exhibited much higher peak intensities centered at -0.25 V vs.

344

Ag/AgCl and -0.43 V vs. Ag/AgCl than those in the MR-1/pYYDT. A similar

345

observation was reported in a previous study.31 All of these results demonstrate that

346

there was synergy between the flavin-mediated and metal-reducing conduit-mediated

347

EET.

348

Such an enhancement could be attributed to several reasons. The elevated flavin

349

concentration could increase the concentration gradient of either oxidized or reduced

350

electron shuttles, and thus accelerate the diffusion process of free flavins between 17



351

cell−electrode interfaces. Since the diffusion process is a rate-limiting step of the

352

shuttle-mediated EET,32 the elevated flavin concentration could finally enhance the

353

electron shuttle-mediated EET. In addition, a number of studies have shown that more

354

biomass attached on electrode leads to the elevated catalytic current via outer

355

membrane cytochrome c.31, 32 To further investigate the impact of the engineered

356

strains on the direct EET, the biomass of the strain MR-1/pYYDT-RM,

357

MR-1/pYYDT-Rib, MR-1/pYYDT-Mtr and the control strain on the electrodes in the

358

MFCs was measured as 13.4 ± 0.3, 11.7 ± 0.2, 12.3 ± 0.2 and 10 ± 0.2 µg cm-2,

359

respectively. Moreover, the smallest Rct was observed for the MFC with the strain

360

MR-1/pYYDT-RM, further suggesting the fastest electron-transfer rate of

361

electrochemical reactions on the electrode. These results demonstrate that the elevated

362

EET ability of the strain MR-1/pYYDT-RM is owing to the synergetic effect between

363

the incremental shuttle-mediated EET and direct EET.

364

Flavins are synthesized de novo by plants and microorganisms.40 Recently, a

365

number of bacteria such as S. oneidensis, Campylobacter jejuni, Helicobacter pylori,

366

and three species of methanotrophic bacteria and Geothrix fermentans, have been

367

found to use secreted flavins as electron shuttles to accelerate respiration of insoluble

368

minerals and electrodes.41 Eleven phylogenetically distinct Shewanella strains have

369

also been reported to secrete flavins and utilize them as electron shuttles under

370

anaerobic conditions.37 A survey of the recently sequenced microbial genomes shows

371

that the homologues of the metal-reducing conduits pathway of S. oneidensis MR-1

372

exist in the Fe(III)-reducing bacteria Aeromonas. hydrophila, Ferrimonas. balearica 18


Page 18 of 36

Page 19 of 36


373

and Rhodoferax. ferrireducens and the Fe(II)-oxidizing bacteria Dechloromonas.

374

aromatica

375

lithotrophicus ES-1.42 It is assumed that coupling the improved synthesis of flavins

376

with metal-reducing conduits could enhanc EET in strains bearing two pathways

377

genes. Moreover, this method might also be applicable for the DMRB capable of both

378

direct and mediated EET. This warrants further investigations.

RCB,

Gallionella.

capsiferriformans

ES-2

and

Sideroxydans.

379

After coupling improved synthesis of flavins and metal-reducing conduit of S.

380

oneidensis MR-1, we presented a novel strategy to enhance its EET significantly.

381

However, it should be noticed that the efficiency of EET in the strain

382

MR-1/pYYDT-RM is still low. On one hand, the low yield of flavins in the

383

engineering strains under anaerobic conditions still restrict the further improvement

384

of EET, which might be caused by the lower fluxes of the essential metabolic

385

pathways for flavin biosynthesis under anaerobic conditions.32. On the other hand, a

386

polar expression effect results in an unbalance of the flavin biosynthesis gene

387

clusters or metal-reducing conduit biosynthesis gene transcriptions. In S. oneidensis

388

MR-1, one guanosine triphosphate (GTP) and two ribulose-5-phosphate molecules

389

are converted into one riboflavin molecule in a stepwise manner by the enzymes

390

encoded by the ribA, ribB, ribD, ribH, and ribE genes.41 The unbalancing

391

transcription of the flavin biosynthesis gene clusters might lead to the accumulation

392

of intermediate metabolites, which finally reduce the titer of flavins secreted by the

393

engineering strains. In S. oneidensis MR-1, MtrABC can be isolated as a protein

394

complex with a stoichiometry of 1:1:1 and serve as an electron conduit between the 19



395

periplasm of S. oneidensis MR-1 cells and its extracellular environments.43, 44 The

396

unbalancing transcription of the mtr gene cluster might lead to the formation of

397

improper protein complexes, which may affect the electron transfer from the cell

398

interior to the outer membrane. Thus, efforts should be made to optimize the

399

recombinant strains through metabolic engineering to further enhance EET. For

400

instance, several approaches, such as the optimization of gene codons, tuning

401

promoter strengths and balancing the flavin biosynthesis gene cluster and

402

metal-reducing conduit biosynthesis genes transcription to avoid misregulation of

403

the post-transcriptional modifications, could be used to engineer the strains.

404

In summary, we demonstrate that coupling improved synthesis of mediators with

405

metal-reducing conduits is an efficient strategy to enhance EET in S. oneidensis

406

MR-1. In addition to Shewanella, this strategy may be used as a broad-spectrum

407

approach for other DMRB because of its several advantages, such as easy

408

manipulation, effectiveness and good expansibility. The engineering strains with an

409

enhanced EET and higher reduction efficiency have potential applications in

410

environmental remediation including bioremediation and treatment of heavy metal

411

contaminated soil, groundwater and azo dyes-rich wastewaters in practice.

412 413

AUTHOR INFORMATION

414

* These authors contributed equally to this work.

415

**Corresponding authors.

416

Dr. Dong-Feng Liu, Fax: +86 551 63601592; E-mail: [email protected]; Prof. 20


Page 20 of 36

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417

Han-Qing Yu, Fax: +86 551 63601592; E-mail: [email protected]

418 419

Notes

420

The authors declare no competing financial interest.

421 422

ACKNOWLEDGEMENTS

423

The authors wish to thank the National Natural Science Foundation of China

424

(21477120, 51538012，21590812 and 21607146), and the Collaborative Innovation

425

Center of Suzhou Nano Science and Technology of the Ministry of Education of

426

China for the support.

427 428

ASSOCIATED CONTENT

429

Supporting Information Available. The images of the fuel cell systems (Figure S1),

430

the extracellular FMN concentration of all the four strains after 64-h incubation

431

(Figure S2), qRT-PCR results (Figure S3), and nyquist plots (Figure S4) by the

432

control strain and recombinant S. oneidensis MR-1 strains, information about strains,

433

plasmids and primers used in this work (Table S1). This information is available free

434

of charge via the Internet at http://pubs.acs.org/.

435 436

REFERENCES

437

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28


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Figure captions 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 hydrolysed 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). 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. Figure 3 Current output (a) and power density (b) of the control strain and recombinant strains in MFCs. Figure 4 Amperometric data from the MECs inoculated with the control strain and recombinant strains. 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. Insert: zoom in of the catholic peak in the range of -0.5 V ～ -0.1 V vs. Ag/AgCl. 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.

29



Figure 1

30


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Figure 2

31



Figure 3

32


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Figure 4

33



Figure 5

34


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Figure 6

35



Table of Contents (TOC) Art

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