Graphene for Robust

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Energy, Environmental, and Catalysis Applications

A Framework of Cytochrome/Vitamin B2 Linker/ Graphene for Robust Microbial Electricity Generation Sheng-Song Yu, Lei Cheng, Jie-Jie Chen, Wen-Wei Li, Feng Zhao, Wen-Lan Wang, Dao-Bo Li, Feng Zhang, and Han-Qing Yu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10877 • Publication Date (Web): 24 Sep 2018 Downloaded from http://pubs.acs.org on September 29, 2018

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ACS Applied Materials & Interfaces

A Framework of Cytochrome/Vitamin B2 Linker/Graphene for Robust Microbial Electricity Generation

Sheng-Song Yu,a Lei Cheng,a Jie-Jie Chen,a,* Wen-Wei Li,a Feng Zhao,b Wen-Lan Wang,a Dao-Bo Li,a Feng Zhang,a and Han-Qing Yua,* a

CAS Key Laboratory of Urban Pollutant Conversion, Department of Applied

Chemistry, University of Science and Technology of China, Hefei 230026, China b

Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China.

*Corresponding Authors: Dr. Jie-Jie Chen, E-mail: [email protected] Prof. Han-Qing Yu, E-mail: [email protected]

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1

ABSTRACT

2

A bio-electrochemical system (BES) allows direct electricity production from wastes,

3

but its low power density, which is mainly associated with its poor anodic

4

performance, limits its practical applications. Here, the anodic performance of a BES

5

can be significantly improved by electrodepositing vitamin B2 (VB2) onto a graphene

6

(rGO)-modified

7

sulfurreducens as the model microorganisms. The VB2/rGO/GC electrode results in

8

200% higher electrochemical activity than a bare GC anode. Additionally, in

9

microbial electrolysis cells, the current density of this composite electrode peaks at

10

~210 µA cm-2 after 118 h and is maintained for 113 h. An electrochemical analysis

11

coupled with molecular simulations reveals that using VB2 as a linker between the

12

electrochemically active protein of this model strain and the rGO surface accelerates

13

the electron transfer, which further improves the bioelectricity generation and favors

14

the long-term stability of the BES. The VB2 bound with a flexible ribityl group as the

15

organic molecular bridge efficiently mediates energy conversion in microbial

16

metabolism and artificial electronics. This work provides a straightforward and

17

effective route to significantly enhance the bioenergy generation in a BES.

18

KEYWORDS: Bioelectrochemical system, energy conversion, extracellular electron

19

transfer, c-type cytochromes, vitamin B2, graphene

glassy

carbon

electrode

(VB2/rGO/GC)

2


with

Geobacter

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20

INTRODUCTION

21 22

A bioelectrochemical system (BES), a green energy device, shows promising

23

functions in terms of electricity generation, hydrogen formation, and valuable

24

chemical production from wastewater.1 Such a system exchanges information and

25

energy between microorganisms and an electrode by using electron transfer to

26

integrate biotic and abiotic components. This process allows waste to be a renewable

27

resource and offers opportunities to simultaneously address the future energy and

28

environmental challenges.1-2 Microbial fuel cell (MFC) and microbial electrolysis cell

29

(MEC) are two BES models that are exciting technologies and provide the possibility

30

for commercialization. In MFCs, the chemical energy in waste is directly converted

31

into electricity using microorganisms as the biocatalyst and an electrode as the

32

electron acceptor. Additionally, MEC employs microorganisms to convert organic

33

matter into hydrogen or various chemicals. The key step for driving the BES is

34

extracellular electron transfer (EET) at the biological/inorganic interface that links

35

microbial metabolism and artificial electronics.3-5 To exploit the electron sinks by

36

performing EET, two typical dissimilatory metal-reducing bacteria, Geobacter spp.

37

and Shewanella spp., can pass directly through the c-type cytochromes in the outer

38

membrane6 (OM c-Cyts) or bacterial filaments7 or be mediated by electron shuttles.8

39

Among these distinct EET modes, some electron shuttles could be self-excreted by

40

electricity-generating microorganisms and used repeatedly to relay bioelectrons from

41

outer membrane and achieve long-range EET for Shewanella spp.,8-9 or bound to OM 3



42

c-Cyt scaffolds as redox cofactors to promote EET for both Geobacter spp. and

43

Shewanella spp..10 Use of different electron shuttles can effectively facilitate the EET

44

process and provide more opportunities to reveal new electron transfer routes by

45

changing the external environment.

46

Vitamin B2 (VB2), as a cofactor of diverse oxidoreductases, catalyzes

47

biotransformation and energy transfer reactions and has many advantages over

48

artificial electron shuttles to enhance EET.9, 11 The versatility of the chemical behavior

49

of VB2 is largely ascribed to its unique ability to serve as a key center by undergoing

50

cycling redox at diazabutadiene of the isoalloxazine moiety.12 The standard redox

51

potential of VB2 is -400 mV (vs. Ag/AgCl), less than that of OM-c-Cyts. Thus,

52

electrons can be transferred from cells to VB2 and ultimately to a terminal acceptor.

53

The redox potential of VB2 usually varies from -400 mV to -60 mV as a result of its

54

interaction with OM proteins.10,

55

shuttle-dependent EET process by restoring the current after a dose of VB2.9 Density

56

functional theory (DFT) calculations have also confirmed the importance of redox

57

mediators in EET at the molecular level.14-15

13

Previous studies have proven an electron

58

Geobacter sulfurreducens with an abundance of OM c-Cyts is the most efficient

59

current-producing microorganism characterized to date, playing an important role in

60

energy conversion as a model microorganism. However, many previous works have

61

shown that Geobacter does not have the ability to deploy the diffusive

62

organic-molecule ESs to enhance EET, which is a main transfer route to solid.9 In all

63

these studies, free redox mediators were used. It remains unknown whether and how 4


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64

the composite system of OM c-Cyts linked by the ES molecule to an electrode would

65

affect the EET processes. Although the current generated by Shewanella with

66

riboflavin modified anodes was higher than that with blank anodes,16 the possible

67

mechanism of ES as the link between an electrochemically active protein and an

68

anode surface has not been revealed. VB2 might bind to an electrode with aromatic

69

isoalloxazine moiety or a ribityl “tail” to further activate the EET processes. VB2

70

might be similar to organic molecule wires, which have been used to efficiently

71

mediate electron transfer in photovoltaics and molecular electronics processes.17

72

However, the mechanism of biological proteins and inorganic electrodes linked by

73

small organic molecules for passing electrons remains poorly understood.

74

Therefore, this work aims to bridge the above knowledge gap by constructing a

75

biofilm/VB2 linker/graphene electrode and elucidate the EET mechanism by coupling

76

the

77

Graphene-based materials have been widely tested as a BES anode due to their

78

excellent properties to favor direct EET and energy conversion.18 Furthermore, due to

79

its two-dimensional structure consisting of sp2-hybridized carbon atoms, graphene

80

could also form a π-π conjugation with benzene-ring-like molecules to further

81

improve BES performance.19-21 In the present work, the composite electrode was

82

prepared by combining a thin layer of reduced graphene oxide (rGO) with VB2

83

through π-π stacking on the surface. The VB2/rGO electrode might have dual

84

advantages of reversible redox reactivity and high conductivity to favor electron

85

transfer from OM c-Cyts to electrodes. The electrochemical properties and microbial

electrochemical

characterization

of

biofilm

5


with

DFT

calculations.


86

biofilm on the electrode were examined and compared to VB2-free electrodes. The

87

OM c-Cyts-VB2-rGO interaction and EET mechanism for the modified electrodes

88

were elucidated by coupling bioelectrochemical experiments with molecular

89

modeling. This work may provide useful information and methods for designing more

90

efficient electrodes to favor BES application in energy recovery from wastes.

91 92

EXPERIMENTAL PROCEDURES

93 94

Electrode Preparation and Characterization. VB2 was purchased from Sigma Co.,

95

USA, and used without further purification. Other reagents, all purchased from

96

Sinopharm Chemical Reagent Co., China, were of analytical grade and used without

97

further purification. Aqueous solutions were prepared using deionized water.

98

Electrochemical measurements were conducted in a traditional three-electrode system

99

(GC electrode as working electrode, KCl-saturated Ag/AgCl as reference electrode,

100

platinum wire as counter electrode) with a CHI 660C electrochemical workstation

101

(Chenhua Instrument Co., China). Before the tests, the electrolyte was deoxygenated

102

with nitrogen. All experiments were conducted at an ambient temperature of ~25 °C.

103

All potentials reference the Ag/AgCl electrode, if not mentioned specifically. For

104

electrochemical cells, a constant potential of +0.1 V was applied to the working

105

electrode with a CHI 1030A potentiostat (Chenhua Instrument Co., China). Before

106

each test, the GC electrode (d = 3 mm) was polished with 0.3-µm and 0.05-µm

6


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107

alumina powder in succession and thoroughly sonicated in water and ethanol for 30

108

seconds.

109

To prepare the GC electrode, GO was first synthesized from graphite powder

110

using a modified method of Hummers and Offeman.22 The prepared GO (6 µl, 0.5 g

111

l-1) was cast homogeneously on the surface of a precleaned GC and dried under an

112

infrared lamp. Then, cyclic voltammetry (CV) was performed with the pretreated GC

113

electrode for 30 cycles in a potential window of -1.4~0 V and at a scan rate (v) of 100

114

mV s-1 in 0.1 M PBS (pH 7.0). The obtained electrode is noted as rGO/GC.

115

Additionally, VB2 (10 mg) was dissolved in 200 ml of PBS (100 mM, pH 3.0) and

116

stored at 4 °C in the dark. The electrodes of bare GC and rGO/GC were moved to the

117

VB2 solution (133 µmol l-1) and scanned between -0.8 V and 0.4 V for 30 cycles at a

118

scan rate of 50 mV s-1 to prepare the VB2/GC and VB2/rGO/GC electrodes. The

119

preparation process is illustrated in Scheme 1. The EIS measurements were conducted

120

in a frequency range of 10,000 to 0.01 Hz at an open current potential (OCP) with an

121

alternating current perturbation of 5 mV.

122

The contact angles of the electrodes were measured using a contact angle

123

approach (JC2000A, Powereach Co., China). Before the measurement, the electrodes

124

were placed on glass slides horizontally, and the advancing contact angles were

125

directly measured using the sessile drop technique with a drop of water. All contact

126

angle values were based on the arithmetic means of at least ten independent

127

measurements.

7



128

Microbial Cultivations. Geobacter sulfurreducens DL-1 strain was kindly

129

endowed by Prof. Derek Lovley from the University of Massachusetts (USA). The

130

bacterium was routinely cultivated in a modified growth medium without vitamins

131

and resazurin. The medium consisted of mineral solution, acetate and fumarate. The

132

composition of the mineral solution is consistent with the composition in our previous

133

work.23 20-mM acetate and 50-mM fumarate were individually supplemented as the

134

electron donor and acceptor. The medium was boiled for 15 min and bubbled with

135

N2:CO2 (80:20) to remove dissolved oxygen. After cooling to room temperature,

136

NaHCO3 was dosed to adjust the pH to 7.0. Then, the medium was dispensed in

137

serum bottles and sealed with butyl rubber stoppers and aluminum caps before

138

autoclaving.

139

MEC Operation. MECs (Figure S1a) with different working electrodes were

140

operated at a poised potential of +0.1 V using a three-electrode system. The MEC

141

chamber was sterilized and filled with 50 ml of sterile growth medium, which

142

contained 20 mM sodium acetate as an electron donor and electrolyte without

143

fumarate. The electrolyte was deaerated by bubbling with N2:CO2 (80:20), and the

144

solution pH was adjusted to 7.0. Geobacter cells were injected into the chamber to a

145

final concentration of 0.3 OD600. All microbial incubation and electrochemical tests

146

were conducted at 30 °C.

147

MFC Construction and Operation. Dual-chamber MFCs, consisting of two

148

glass bottles, were used (Figure S1b). Each chamber had a volume of 120 ml and was

149

separated by a proton exchange membrane (GEFC-10N, GEFC Co., China). Carbon 8


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150

felt (3×3 cm2) was used as the cathode. Pieces of carbon paper (CP, 2×3 cm2),

151

modified with VB2 and rGO as mentioned above or a bare piece (as the control), were

152

used as anodes. The anode chamber was inoculated with Geobacter cells at a

153

concentration of 0.3 OD600 and was fed with 100 ml of medium containing 20 mM

154

acetate as substrate. The cathode chamber contained 100 ml of 50 mM K3Fe(CN)6 in

155

PBS (50 mM, pH 7.0). The assembly process was conducted in an anaerobic glove

156

box (Whitley DG250, Don Whitley Scientific Co., UK). The voltage across an

157

external resistance of 1000 Ω was collected using a data acquisition/switch unit

158

(34970A, Agilent Inc., USA). Polarization curves were obtained by LSV at a scan rate

159

of 1 mV s-1 and used to calculate the maximum power density. All microbial

160

incubation and electrochemical tests were conducted at 30 °C.

161

Theoretical Calculations. To determine the optimal electrode modification

162

conditions that facilitate microbe-electrode interactions, the interaction affinity

163

between VB2 and graphene surface as well as the orientation of VB2 was

164

characterized using interaction energy (∆Eint), i.e., the difference between the total

165

energy of the stacking system (ET, VB2/GO) and the sum energy of the individual GO

166

surface (ET,GO) and VB2 (ET, VB2):

167

∆ = , / − (, + , )

(1)

168

For electron transfer between OM c-Cyt and the electrode surface, the quantum

169

mechanical (QM) region of minimum size contained iron-porphyrin, VB2 and some

170

carbon atoms of the GO structure for accepting electrons. Bishistidine axial ligation

171

was introduced to the heme groups to provide an environment for the active center in 9



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172

the OM c-Cyt. The remaining atoms composing the molecular mechanism region

173

were calculated in the General Utility Lattice Program (GULP)24 with the Universal

174

forcefield. The DFT calculations were performed with DMol3,25 using the

175

exchange-correlation function of Perdew, Bueke, and Ernzerhof (PBE)26 within the

176

generalized gradient approximation (GGA)27 in combination with double precision

177

numerical basis sets, including polarization (DNP) to analyze the thermodynamic

178

properties of the proton-coupled electron transfer process of VB2 on the rGO surface.

179

The electron transfer between the clusters of OM c-Cyt active center and the

180

VB2-rGO package only changes their charges, and obvious structural change does not

181

occur. Thus, the electron transfer rate constant (ket) can be described using Marcus

182

theory:28

183 184

=

exp [−

(∆ # $) %

]

(2)

185 186

where VDA is the value of electronic interaction between the heme and VB2/rGO (or

187

rGO) at the crossing point configuration, λ is the reorganization energy, ∆Go is Gibbs

188

free energy change of the electron transfer reaction, h is Planck’s constant, kB is the

189

Boltzmann constant, and T is the temperature. The calculation details of the three

190

unknown electron transfer parameters, VDA, λ, and ∆Go, are described in Note S2 of

191

Supplemental Information.

192

In the molecular dynamics simulations, the model cell contains domain II of MtrF

193

and 16 VB2 molecule-modified rGO surface in aqueous solution (3000 H2O 10


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194

molecules). The VB2 is in the oxidation state to accept electrons from hemes (Fe2+) in

195

domain II, and 16 H3O+ were introduced to provide protons for the proton-coupled

196

electron transfer. With the energy minimized models of the domain II-VB2-rGO in

197

aqueous solution, molecular dynamics simulations were performed in Forcite module

198

for equilibrating the models and obtaining the final structures (see Supplemental

199

Information for more details).

200 201

RESULTS AND DISCUSSION

202 203

Electrochemical and Surface Properties of Prepared Electrodes. The preparation

204

of VB2 immobilization onto the rGO-modified electrode is illustrated in Scheme 1.

205

After the continuous cyclic voltammetry scanning, VB2 was electrodeposited on the

206

high specific surface area of rGO with sp2 backbones of carbon atoms (Figure S2, and

207

detailed description in Note S1 in Supplemental Information). In addition, the SEM

208

images of the MFC anodes consisting of VB2-modified CP are shown in Figure 1a

209

(CP) and 1b (rGO/CP).

210

The properties of the prepared electrodes were examined using CV. Compared to

211

the results of bare GC and rGO/GC electrodes in a blank phosphate buffer (PBS),

212

only the VB2-immobilized electrodes exhibited a pair of quasi-reversible redox peaks

213

with a redox potential of approximately -0.4 V, which is attributed to VB2 (Figure 1c).

214

Additionally, there was a very slight change in the electrochemical response after 30

11



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215

scans (Figure 1d), suggesting a stable interaction between rGO and VB2 at the

216

VB2/rGO/GC electrode surface.

217

The electrochemical dynamic behaviors of VB2-immobilized electrodes were

218

analyzed using CV at different scan rates. The anodic and cathodic peak currents of

219

both electrodes increased simultaneously with the increasing scan rate in the applied

220

range (Figure 1e, f). The two-peak currents were proportional to the scan rate with

221

two linear regression equations. This result demonstrates that the electrochemical

222

response of VB2 on both electrodes is a typical surface-controlled process.29

223

Meanwhile, the surface coverage (Γ) of VB2 on the electrodes could be calculated

224

from the slope of Ip vs. scan rate (v):30

225 226

'( =

) *+Γ

(3)

%,

227 228

where v is the scan rate, A is the surface area of the modified electrode, and the other

229

symbols have the usual meanings. The surface coverage of VB2 was calculated as

230

9.58 nmol cm-2 for the VB2/rGO/GC and 4.53 × 10-2 nmol cm-2 for the VB2/GC.

231

To

estimate

the

interfacial

charge

transfer

resistance

(Rct)

at

the

232

electrode-electrolyte interface, the electrochemical impedance spectroscopy (EIS) of

233

all the electrodes was recorded at their open circuit potentials (Figure 1g),31 and the

234

data were fitted with a special equivalent circuit model.32 The Rct of VB2/GC

235

decreased substantially compared to the bare GC electrode, corresponding to the

236

smaller slope in the V-j curve of graphene electrode16 and indicating an accelerated 12


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237

interfacial electron transfer by VB2. This may be due to the rapid and reversible redox

238

reactions of VB2. For the rGO/GC electrode, Rct decreased distinctively to the lowest

239

level due to the high conductivity of the electrochemically reduced GO on the GC

240

surface. The Rct of VB2/rGO/GC was slightly greater than that of rGO/GC but still

241

much smaller than the Rct of VB2/GC and the bare GC electrodes. This result suggests

242

that the sp2 hybridized rGO was effective for enhancing the electron transfer on the

243

electrode surface.

244

Due to the decreased number of hydrophilic functional groups on GO, the contact

245

angle of the rGO/CP electrode (120°) was very close to the contact angle of the CP

246

electrode (128°) (Figure S3), indicating that the hydrophobicity of CP was unchanged.

247

In contrast, the contact angle was 100° for VB2/CP and 0° for VB2/rGO/CP,

248

suggesting that these electrodes had a higher hydrophilicity. The enhanced

249

hydrophilicity should be attributed to the alcoholic hydroxyl of VB2. VB2 was likely

250

linked to rGO by an isoalloxazine ring through π-π stacking as evidenced by Raman

251

while exposing its hydroxyl to the solution.16 The hydrophilic part of VB2 could bind

252

with the OM c-Cyts of DMRB and connect to the electrode via the isoalloxazine ring.

253

Such a configuration enabled VB2 to mediate the EET from DMRB to the electrode

254

more efficiently.

255 256

Bioelectricity Generation in MECs with Different Electrodes. The roles of rGO

257

and VB2 modification in enhancing EET at the microbe-electrode interface were

258

examined using MECs with Geobacter sulfurreducens as the model microbe. 13



259

Modification with both rGO and VB2 drastically increased the electricity output of the

260

MECs (Figure 2a). The catalytic current densities of all the electrodes were initially at

261

similar low levels due to the low bacterial adhesion and activity of using electrodes as

262

the sole electron acceptor.33 However, the current increasing point varied for the four

263

electrodes, indicating that different initial times were required for transferring

264

electrons from the microbe to the electrodes. The current density of the VB2/rGO/GC

265

electrode peaked at ~210 µA cm-2 after 118 h and maintained a high level for 113 h.

266

Such a result was not observed for the other three electrodes, indicating that the

267

electron transfer was accelerated due to the synergy between rGO and VB2. The

268

performance of the MEC system with the VB2/rGO/GC electrode is compared with

269

those in other reported works in Table S1. The bioelectricity generation in the MEC

270

with the VB2/rGO/GC electrode is comparable with those reported in other works,

271

and is higher than that of the MECs with pure strains.

272

The shorter start-up time, higher bioelectricity, and longer plateau period of the

273

modified electrodes demonstrate that rGO and VB2 could enhance EET by improving

274

bacterial adhesion on the electrode. VB2 has been frequently used as a diffusive

275

electron shuttle between OM c-Cyts and electron acceptors through a two-electron

276

redox reaction.34 In addition, VB2 may bind with the OmcA protein as a cofactor to

277

enhance EET through a one-electron process with a higher reaction rate than that of

278

free VB2.13 Therefore, immobilizing VB2 on electrodes is beneficial for EET. The

279

gradients of oxidized VB2 can attract microbes toward insoluble electron acceptors

280

via energy taxis, which has been proposed as a new EET mechanism.35 Thus, VB2 14


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281

could be concentrated at the electrolyte-electrode interface through π-π stacking to

282

form a rising gradient toward the electrode, which accelerates biofilm formation and

283

improves bioelectricity generation.

284

Moreover, VB2 immobilization increased the amount of microbes that adhere to

285

the electrode.16, 36 This could explain not only the 1.7-fold higher current density in

286

the MEC with VB2/GC than with the bare GC but also the high current density (210

287

µA cm-2) and longer duration of sustained current.

288 289

Bioelectricity Generation in MFCs with Electrodes. The remarkable performance

290

of the VB2 and/or rGO-modified CP electrodes was further verified by MFCs. Stable

291

voltages (~500 mV) were obtained within 40 h for all the electrodes (Figure 2b), and

292

the acclimation time increased in the order of VB2/rGO/CP < rGO/CP < VB2/CP.

293

However, the stable voltage of the MFC with the bare electrode was less than that

294

with the modified electrodes. This is consistent with the MEC results (Figure 2a),

295

indicating that both rGO and VB2 could accelerate the EET, and their synergetic

296

effect could further improve the bioelectricity generation.

297

The MFC with the VB2/rGO/CP anode had a higher maximum power density

298

than that with other anodes (Figure 2c). The overall trend in the current density was

299

the same as that in MECs. Previous studies have shown that bacterial adhesion to

300

anode surfaces is favored by graphene due to its large specific surface area and by

301

VB2 due to its binding affinity to OM c-Cyts.10 This observation is also confirmed by

302

the SEM of biofilm (Figure S4). This could explain the shorter acclimation time of the 15



303

MECs with the modified anodes. The total quantities of electric charge and electricity

304

generated by Geobacter in the MFCs were calculated (Table 1). The VB2/rGO/CP

305

electrode enabled 59% more electric charge production than the bare CP electrode.

306

Less improvement was obtained by the rGO/CP (45%) and VB2/CP (28%). The total

307

electrical energy increased in the order of VB2/CP < rGO/CP < VB2/rGO/CP.

308 309

Electrochemical Characteristics of Electrode Biofilms. The electrochemical

310

activity of biofilms formed on the various electrodes was characterized under turnover

311

and nonturnover conditions after the current of the MECs dropped to the background

312

level. The electron transfer resistance of the electrode biofilm was measured using CV

313

at a low scan rate, which exhibited a sigmoidal shape (typical of electroactive biofilms

314

under turnover conditions) for all electrodes (Figure 3a). The use of rGO- and

315

VB2-modified electrodes resulted in a current nearly 3-times higher than the use of

316

bio-GC electrode in the MECs. The highest plateau current generated by the

317

VB2/rGO/GC electrode further confirms the acceleration of EET due to the synergy

318

between VB2 and rGO.

319

The first derivatives of the CVs from Figure 3a were obtained to visualize the

320

inflection point (Figure 3b). A dominant inflection point centered at -0.35 V was

321

detected for all electrodes, and the peak height increased after the modifications with

322

rGO and VB2. This inflection point should be ascribed to OM c-Cyts,37 as evidenced

323

by the nonturnover CV analysis in Figure 3c. For the VB2-modified electrodes, the

324

inflection points shifted positively compared to those of the non-VB2 electrodes, 16


Page 16 of 38

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325

indicating the binding behavior of VB2 to OM c-Cyts. This binding led to an

326

increased electron transfer rate.10, 13

327

To acquire the redox couple information at the microbe-electrode interface, CV

328

experiments were carried out under nonturnover conditions after culturing the

329

biofilms in substrate-free medium for 24 h. Several redox peaks of electroactive

330

cellular components, such as OM c-Cyts, redox mediators or their mixture, were

331

observed (Figure 3c). To clarify the redox behaviors of the possible components,

332

carbon oxide (CO) was used to suppress the redox peak of OM c-Cyts (Figure 3d). A

333

significant decrease in the peak intensity of redox couple with a mid-potential of -0.35

334

V was observed, indicating that this potential was due to the OM c-Cyts. For the

335

VB2/rGO/GC, a few new redox peaks appeared, which could be attributed to the

336

electron transfer reaction of VB2. The suppression of OM c-Cyts by CO led to a

337

negative shift of redox potential, which also proved the combination between VB2 and

338

OM c-Cyts. Interestingly, the midpoint potentials were the same as the inflection

339

points in Figure 3b, suggesting that both OM c-Cyts and VB2, especially the

340

VB2-bound OM c-Cyts, predominately contributed to the fast EET.

341 342

Mechanism of Interfacial EET in the Package of OM c-Cyts Active

343

Center/VB2/rGO. To improve the energy conversion efficiency at the BES anode and

344

tune the synergy of cytochrome-redox active molecules-rGO substrate, the molecular

345

mechanism behind the interfacial EET should be clarified. The EET process at the

346

interface between cytochrome and VB2/rGO was investigated using DFT calculations 17



347

and molecular dynamics simulations. The negative interaction energy change (∆Einter

348

= -0.10 eV, Table S2) between VB2 and rGO reveals that rGO tends to conjugate with

349

the N-heterocycle isoalloxazine of VB2 molecules (Figure 4a). Such a parallel

350

configuration would favor the maintenance of the biological activity of VB2 as well as

351

the electron transfer efficiency. Due to the increased delocalization of π electrons in

352

the conjugated structure, VB2 molecules could act as redox linker for sustainable

353

energy storage. To determine the redox state of the VB2 molecules conjugated on the

354

rGO surface, the thermodynamic properties of the proton-coupled electron transfer

355

reaction (Scheme S1) under standard conditions were studied using DFT calculations.

356

VB2 can mediate the electron transfer via a one- or two-electron pathway.38 This

357

unique feature allows VB2 to act as an intermediate between heme (the active center

358

of OM c-Cyts) that donates only one electron and rGO that permits free-movement of

359

many electrons at a time.

360

To investigate the electron transfer chain that bridges the proteins and inorganic

361

nanomaterials, the thermodynamic properties of a one-proton-coupled one-electron

362

transfer process for VB2 reduction (Scheme S1) were investigated. The standard

363

Gibbs free energy (∆GӨ) of VB2 reduction on the rGO surface is -6.02 eV (Table S3),

364

which is more negative than that of the free VB2 in aqueous solution (-4.95 eV).

365

Therefore, VB2 is more thermodynamically favorable to maintaining a reduction state

366

(semiquinone intermediate), and rGO substrate could affect the redox reactions of the

367

mediator co-factors for energy transduction. The theoretical electron transfer rate at

368

the interface of the OM c-Cyts active center and VB2/rGO was calculated using 18


Page 18 of 38

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369

Marcus theory.39 The OM c-Cyts of G. sulfurreducens DL-1, such as OmbB/OmbC,

370

OmaB/OmaC and OmcB/OmcC proteins, are similar to the MtrABC proteins of

371

S. oneidensis MR-1 in terms of their function and localization at the outer

372

membrane40-41 with the same redox active center known as heme: iron containing

373

protoporphyrin. Thus, because of the lack of sufficient crystal structure information

374

for the OM c-Cyts of G. sulfurreducens DL-1, MtrF (PDB code: 3PMQ, Figure S5a)

375

purified from S. oneidensis can be used as a model representative of the larger

376

multiheme cytochromes.42 MtrF (PDB code: 3PMQ), a homolog of MtrC, are

377

organized by four domains and 10 hemes in the crystal structure (Figure S5a). The

378

cluster model for the donor-acceptor pair (Figure 4b) was used to calculate the inner

379

reorganization energy. The electron donor site of OM c-Cyts is the Fe atom in the

380

porphyrin plane (heme). Bishistidine axial ligation is also introduced to explain the

381

effect of amino acid residues on the electron transfer.

382

The thermodynamic driving force of the electron transfer reaction at reaction

383

equilibrium, ∆Go, in Equation 2 can be estimated from our experimental data of the

384

redox potential of OM c-Cyts (-0.35 V vs. Ag/AgCl, Figure 4b) by using the Nernst

385

equation. The calculation details are described in Supplemental Information. The

386

calculation results show that the electron transfer rate constant of the heme-VB2/rGO

387

system is 1023 times higher than that of heme-rGO (Table S4), indicating a drastically

388

accelerated EET at the electrode surface from VB2 modification. This also provides

389

molecular-level evidence that VB2 could act as a molecular bridge to connect the OM

390

c-Cyts and rGO for highly efficient electron conduction.43 Although the actual 19



391

bioenergy conversion performance of BES might be influenced by various factors

392

under the experimental conditions, the performance promotion could still be observed

393

by using the anode of VB2-coupled rGO (Figure 2).

394

To better understand the synergy between VB2 and rGO that accelerates EET,

395

molecular dynamics simulations were carried out to determine the optimal

396

configurations for OM c-Cyts on the VB2/rGO surface. For the EET process, heme 5

397

in domain II (aa 187-318) is the solvent-exposed terminus for electron output to

398

electron acceptors, such as solid substrates or electron shuttles (Figure S5b). The

399

equilibrium state of the domain II/VB2/rGO system (Figure S5c, d) in a water

400

environment indicates that the rGO surface changed to a wave plane but still

401

conjugated with the VB2 molecules (Figure 5c). Moreover, the ribityl “tail” of the

402

VB2 with the hydroxyl groups was oriented toward the aqueous solution to form

403

hydrogen bonds with the water molecules or residues at the surface of domain II. This

404

favored a decreased electron transfer distance between the Fe atom of heme 5 and the

405

substrate surface to enhance EET. The ribityl “tail” of the VB2 also increased the

406

surface roughness and hydrophilicity of the electrode. Thus, the electrode

407

modification with a redox mediator provided opportunities for better EET control by

408

tuning the electrochemical and physical properties of the electrode surface at the

409

molecular level. These molecular dynamics simulation results imply that various

410

unexplored redox active molecules from natural energy conversion systems or after

411

rational artificial modification could be used to fabricate BES electrodes for

412

outstanding performance. 20


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413

With the development of cheap electrode materials, appropriate MFC

414

configurations and better understanding of the behaviors of electricity-generating

415

microorganisms at the molecular level and biofilm level, further improvement of

416

MFC performance can be expected in the future. One distinct advantage of MFC over

417

chemical fuel cells is the mild-condition operation, which significantly decreases

418

energy consumption. Energy consumption could be further reduced by optimizing the

419

reactor configuration and hydraulics in the future.44 Another attractive feature of MFC

420

is its convenience for real-time monitoring and facile control; thus, a robust process

421

can be achieved.45 Although the direct power output of MFC is low at the present

422

stage, such bioelectricity can be utilized in situ for applications such as MEC and

423

microbial desalination cell or function as a power source for low-power devices.46 In

424

addition, integrating MFCs with other decontamination technologies such as

425

anaerobic digestion and photoelectrocatalysis could greatly improve the conversion

426

efficiency of pollutants.47 In the future, energy production with MFC could be

427

increased by enlarging reactors and tuning microbes. With the development of

428

microbiology, engineering and chemistry, MFC-centered technologies have great

429

potential for achieving energy-neutral or -productive waste management processes.

430 431

CONCLUSIONS

432 433

Electrode modification with VB2 was found to significantly accelerate the EET and

434

improve bioelectricity generation in a BES. The improvement was attributed mainly 21



435

to the increased affinitive contact between the biofilm and the electrode, which

436

resulted from the strengthened biocompatibility and increased specific surface area of

437

the electrode. The high conductivity of graphene, efficient redox reactions of VB2,

438

and the associated structure of VB2-OM c-Cyts also contributed to the low potential

439

polarization and enhanced EET. The immobilized VB2 on the electrode acted as a

440

molecular bridge to mediate the EET between the OM c-Cyts and the anode surface.

441

This work shows that electricity generation in BESs could be enhanced by tuning the

442

functional groups of mediators and mixing two mediators with different redox

443

potentials at the electrode surface to form a redox gradient from OM c-Cyts to the

444

anode. Our results also further the understanding of the EET process and may provide

445

a promising approach for tuning the electron transfer routes in favor of more efficient

446

waste-to-bioenergy conversion.

447 448

ASSOCIATED CONTENT

449

Supporting Information

450

Experimental details of immobilization of VB2 onto the rGO-modified electrode

451

(Note S1), calculation methods of rate constant of electron transfer (Note S2),

452

Pictures of MEC and MFC (Figure S1), Electrochemical modification of GC

453

electrode (Figure S2), Contact angles of different electrodes (Figure S3), SEM

454

image of biofilm grown on VB2/rGO/CP electrode and CP electrode in the MFCs

455

(Figure S4), Structure of MtrF and VB2/rGO (Figure S5), VB2 reduction

456

mechanism (Scheme S1), Performance comparison of the MEC system with the 22


Page 22 of 38

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457

VB2/rGO/GC electrode with other related works (Table S1), Interaction energy

458

(∆Einter) between VB2 and rGO (Table S2), Thermodynamic properties of VB2

459

reduction on rGO surface and in aqueous solution (Table S3), and Rate constant

460

of electron transfer from heme to VB2/rGO or rGO surfaces (Table S4). This

461

material is available free of charge via the Internet at http://pubs.acs.org/.

462 463

ACKNOWLEDGMENTS

464

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

465

(51508545, and 21477120), China Postdoctoral Science Foundation (2014M560522),

466

and the Collaborative Innovation Center of Suzhou Nano Science and Technology of

467

Ministry of Education of China for the partial support of this work. The DFT

468

calculations were performed on the supercomputing system in the Supercomputing

469

Center of the University of Science and Technology of China.

470 471 472

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Figure Captions Figure 1. Characterization of modified electrodes: (a-b) SEM of CP and rGO/CP, (c) CV responses of electrodes in PBS with a scan rate of 100 mV/s, (d) Repeated CVs of VB2-modified electrodes in PBS. (e-f) CVs of VB2/GC and VB2/rGO/GC in 100 mM PBS (pH 7.0) at scan rates between 100-500 mV/s; Insets show the linear relationship between peak current and scan rate, for VB2/GC: Ipa = 0.0688 + 0.0017v (R2 = 0.993) and Ipc = -0.5104 – 0.0014v (R2 = 0.99); for VB2/rGO/GC: Ipa = 19.96 + 0.36v (R2 = 0.999) and Ipc = -18.06 - 0.36v (R2 = 0.999), (g) EIS plots of working electrodes scanned at 0.01~100 kHz and open circuit potential with a perturbation signal of 5 mV. Figure 2. Energy production of MECs and MFCs using modified electrodes: (a) Evolution of current density with time for MECs with electrodes poised at +0.1 V vs Ag/AgCl, (b) Cell voltage of MFCs, (c) Maximum power density of MFCs. Figure 3. Electrochemical behavior of biofilm: (a) CV of electroactive biofilms in turnover conditions at a scan rate of 5 mV/s, (b) First derivatives of CVs from Figure 3a showing the midpoint potential detectable in catalytic waves of mature biofilms, (c) CV of electroactive biofilms under nonturnover conditions at a scan rate of 5 mV/s, (d) CV of electroactive biofilms under nonturnover conditions after aerating with CO for 20 min at a scan rate of 5 mV/s. Figure 4. Energy-minimized structures of electron transfer systems: (a) Conjugation structure of VB2 and rGO surface. The structure in the dashed box is N-heterocycle isoalloxazine in the VB2, and the hydrogen atoms are added to saturate the graphene edges, (b) Cluster model of electron donor-acceptor pair: heme-VB2/rGO, the bishistidine axial ligation bounded to the Fe atom of heme, (c) Snapshots of the solvated domain II/VB2/rGO system, VB2 molecules are still conjugated on the wave rGO surface at the equilibrium state, and the ribityl “tail” of the VB2 is oriented to domain II of the electrochemical active protein.

31



Page 32 of 38

Tables

Table 1. Generated charge and electrical energy by MFCs with different anode materials. All double-chamber MFCs were separated by a proton exchange membrane. The anode chamber was inoculated with Geobacter cells at a concentration of 0.3 OD600. The cathode chamber contained 100 ml of 50 mM K3Fe(CN)6 in PBS (50 mM, pH 7.0) with carbon felts as electrodes.

Charge/C

vs. CP

Electrical energy/J

vs. CP

CP

29

---

11

---

VB2/CP

37

28%

16

45%

rGO/CP

42

45%

18

64%

VB2/rGO/CP

46

59%

20

82%

32


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Scheme 1. Schematic of the VB2/rGO/GC electrode preparation process: rGO was cast on GC surface followed by electrochemical reduction. Then, VB2 was immobilized on electrodes through electrodepositing as described above.

33



(c)

GC VB2/GC

40

rGO/GC VB2/rGO/GC

0 -20 0.0 Current (µA)

(b)

60

20

Curent (µA)

(a)

-40 -60

-2.5 -5.0 -7.5 -0.8 -0.6 -0.4 -0.2 0.0

-80

E (V vs. Ag/AgCl)

-0.8

(e) 3 2 1

Current (µA)

Current (µA)

10 0 -10

last cycle

-20 -30

0

-0.6

-0.4

100 mV s -1 200 mV s -1 300 mV s -1 400 mV s -1 500 mV s

VB2/GC

-1

1.0 0.5

-2 -3

Ipa = 0.0688 + 0.0017v

0.0

2

R = 0.993 Ipc = - 0.5104 - 0.0014v

-0.5

2

R = 0.998

-1.0 -1.5

0 100 200 300 400 500 -1

v (mV s )

-5 300 200 100

-0.4

-0.2

-0.8

0.0

100 mV s -1 200 mV s -1 300 mV s -1 400 mV s -1 500 mV s

-0.6

VB2/rGO/GC

(g) 320 280

GC VB2/GC

240

rGO/GC VB2/rGO/GC

200

-Z'' (Ω )

0 -100

200 100

-200

-0.4

-0.2

0.0

E (V vs. Ag/AgCl)

E-1 (V vs. Ag/AgCl)

Ip (µA)

(f)

-0.6

-Z'' (Ω)

-0.8

0.0

E (V vs. Ag/AgCl)

-4

1st cycle

-0.2

-1

Ip (µA)

(d) 30 Short dash dot - VB /GC 2 20 Solid - VB2/rGO/GC

Current (µA)

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 34 of 38

Ipa= 19.96 + 0.36v

6 5 4 3 2 1 0

160

8

10 12 14 16 18 Z' (Ω)

120

2

0

R = 0.999 Ipc= -18.06 - 0.36v

-100

80

2

R = 0.999

-200

-300

100 200 300 400 500 -1

40

v (mV s )

-400 -0.8

-0.6

-0.4

-0.2

0

0.0

0

E (V vs. Ag/AgCl)

50

100

150

200

250

300

Z' (Ω)

Figure 1. Characterization of modified electrodes: (a-b) SEM of CP and rGO/CP, (c) CV responses of electrodes in PBS with a scan rate of 100 mV/s, (d) Repeated CVs of VB2-modified electrodes in PBS. (e-f) CVs of VB2/GC and VB2/rGO/GC in 100 mM PBS (pH 7.0) at scan rates between 100-500 mV/s; Insets show the linear relationship between peak current and scan rate, for VB2/GC: Ipa = 0.0688 + 0.0017v (R2 = 0.993) and Ipc = -0.5104 – 0.0014v (R2 = 0.99); for VB2/rGO/GC: Ipa = 19.96 + 0.36v (R2 = 0.999) and Ipc = -18.06 - 0.36v (R2 = 0.999), (g) EIS plots of working electrodes scanned at 0.01~100 kHz and open circuit potential with a perturbation signal of 5 mV. 34


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Figure 2. Energy production of MECs and MFCs using modified electrodes: (a) Evolution of current density with time for MECs with electrodes poised at +0.1 V vs Ag/AgCl, (b) Cell voltage of MFCs, (c) Maximum power density of MFCs. 35



Figure 3. Electrochemical behavior of biofilm: (a) CV of electroactive biofilms in turnover conditions at a scan rate of 5 mV/s, (b) First derivatives of CVs from Figure 3a showing the midpoint potential detectable in catalytic waves of mature biofilms, (c) CV of electroactive biofilms under nonturnover conditions at a scan rate of 5 mV/s, (d) CV of electroactive biofilms under nonturnover conditions after aerating with CO for 20 min at a scan rate of 5 mV/s.

36


Page 36 of 38

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Figure 4. Energy-minimized structures of electron transfer systems: (a) Conjugation structure of VB2 and rGO surface. The structure in the dashed box is N-heterocycle isoalloxazine in the VB2, and the hydrogen atoms are added to saturate the graphene edges, (b) Cluster model of electron donor-acceptor pair: heme-VB2/rGO, the bishistidine axial ligation bounded to the Fe atom of heme, (c) Snapshots of the solvated domain II/VB2/rGO system, VB2 molecules are still conjugated on the wave rGO surface at the equilibrium state, and the ribityl “tail” of the VB2 is oriented to domain II of the electrochemical active protein.

37



Table of Contents (TOC)

38


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Graphene for Robust

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