Enzymatic electrosynthesis of formate from CO2 reduction in a hybrid biofuel cell system

Accepted Manuscript Enzymatic electrosynthesis of formate from CO2 reduction in a hybrid biofuel cell system

Lijuan Zhang, Jacky Ong, Junyi Liu, Sam Fong Yau Li PII:

S0960-1481(17)30190-8

DOI:

10.1016/j.renene.2017.03.009

Reference:

RENE 8602

To appear in:

Enzymatic electrosynthesis of formate from CO2 reduction in a hybrid biofuel cell system

Received Date:

20 June 2016

Revised Date:

15 February 2017

Accepted Date:

02 March 2017

Please cite this article as: Lijuan Zhang, Jacky Ong, Junyi Liu, Sam Fong Yau Li, Enzymatic electrosynthesis of formate from CO2 reduction in a hybrid biofuel cell system, Enzymatic

electrosynthesis of formate from CO2 reduction in a hybrid biofuel cell system (2017), doi: 10.1016 /j.renene.2017.03.009

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Highlights 

A MFC-EFC system was developed for bioelectrochemical CO2-to-formate conversion.



Electrocatalytic CO2 reduction was achieved with lowered overpotential by CbFDH.



Formate was bioelectro-synthesized from specific and sustainable CO2 reduction.



The hybrid system could be driven by bioelectric power from wastewater.

ACCEPTED MANUSCRIPT 1

Enzymatic electrosynthesis of formate from CO2 reduction in a

2

hybrid biofuel cell system

3

Lijuan Zhang a, Jacky Ong a, Junyi Liu a, Sam Fong Yau Li a,b*

4

a Department

5

117543, Singapore

6

b

7

117411, Singapore

of Chemistry, Faculty of Science, National University of Singapore, Singapore

NUS Environmental Research Institute, National University of Singapore, Singapore

8 9 10

*

11

Tel.: +65 65162681; fax: +65 67791691.

12

E-mail address: [email protected] (S.F.Y. Li).

Corresponding author.

1

ACCEPTED MANUSCRIPT 14

Abstract

15

To seek a sustainable way of CO2 sequestration and conversion, enzymatic electrosynthesis

16

(EES) of formate from CO2 reduction has been investigated in a hybrid biofuel cell system.

17

In an enzymatic fuel cell (EFC), waste CO2 species are specifically reduced to energy-rich

18

product of formate under mild biological conditions. Efficient formate production can be

19

achieved at lowered electrode potential owing to the electrochemically active participation

20

of formate dehydrogenase (CbFDH) as a biocatalyst. Electropolymerized neutral red

21

(PolyNR) is proven to be a promising modifier to enhance the electrochemical behavior of

22

enzymatic electrode, as well as a reducing reagent to regenerate mediator of NADH in

23

enzymatic CO2 reduction. Electrons for EES are extracted from the organic pollutants in

24

wastewater by microbial fuel cell (MFC) stacks arranged in series and/or parallel with

25

different unit numbers (n = 1, 2 and 3). The maximum formate production rate reaches around

26

60 mg L-1 h-1 with a Faraday efficiency of 70% in the EFC powered by a three-MFC stacked

27

in series. In view of practical applications, the hybrid MFC-EFC system has been

28

demonstrated to be advantageous in both product specificity and energy sustainability.

29

30 31

Keywords: Enzymatic fuel cell, Microbial fuel cell, CO2 reduction, Formate synthesis,

32

Formate dehydrogenase 2

ACCEPTED MANUSCRIPT 33

1. Introduction

34

With a rapid expansion of industrialization, anthropogenic carbon dioxide (CO2) that is

35

excessively emitted to atmosphere becomes an important greenhouse gas (GHG) in climate

36

change. GHG mitigation has been made a long-term commitment by many countries [1]. CO2

37

accounts for two thirds of the total GHG emitted by human activities, and is reported to be

38

the primary contributor to global warming. In the last century, global anthropogenic carbon

39

emission from fossil fuels has increased by around 10 times [2]. Among many industrial

40

emission sources, CO2 discharged from traditional wastewater treatment processes is of

41

particular interest to many environmental researchers. Tremendous amount of CO2, i.e. an

42

estimated 1.21 × 104 tons per day by 2025 [3], are released from the degradation of organic

43

pollutants in wastewater treatment plants worldwide. Although it is a causal factor in

44

warming the atmosphere and a waste product in treating wastewater, the inorganic CO2 can

45

be utilized as a substrate carbon source for synthesis of organic carbonates (e.g. formic acid,

46

acetate and methanol) [4, 5]. Thus far, efficient CO2 capture, sequestration and utilization

47

(CCSU) in wastewater treatment facilities is crucial to mitigate the potential impacts of CO2

48

on climate change and environment pollution.

49

To make the CCSU cycle virtuous, conversion of pollutants → CO2 → biofuels is a

50

desirable pathway to secure the molecular values of carbonaceous substrates in wastewater.

51

CO2 reduction to formate (or formic acid), which is the first step in methanol or methane

52

production route, is of particular interest in practical biofuel and bioenergy engineering.

53

Various CO2-to-formate conversion technologies have been well developed but are

54

challenged by different problems, such as the high energy consumption in electrochemical

55

reduction [6, 7] and the low reaction efficiency in bioconversion [8]. Recent studies illustrate

56

that efficient and specific synthesis of formate from CO2 reduction can be achieved by some 3

ACCEPTED MANUSCRIPT 57

biocatalysts in a bioelectrochemical system [9, 10]. Several whole microbial cells have

58

shown the ability to reduce CO2 to formate by decreasing the overpotential at electrodes.

59

However, the reaction rates were very low in most situations (experimental periods up to

60

days) [11]. Rapid and oriented electron movement from CO2 to formate has been proven to

61

be feasible by particular isolated enzymes such as formate dehydrogenase (FDH) in an

62

enzymatic fuel cell (EFC) [12, 13]. On the anode of an EFC, water or organic molecules are

63

oxidized, releasing protons and electrons. The electrons are transported from anode to

64

cathode where enzymatic electrosynthesis (EES) of formate takes place. On the cathode of

65

an EFC, the electrons are suggested to be transferred from solid electrode toward dissolved

66

CO2 species via the active sites of FDH, overcoming the high overpotential to activate CO2

67

reduction [14, 15]. Therefore, the energy input to an EFC can be decreased considerably

68

owing to the catalysis by FDH. Current FDH-reducing CO2 technology, however, suffers

69

from huge gaps in practical application. There are few well-applied systems in aqueous

70

conditions, especially in wastewater medium. And the limited studies on FDH reported only

71

partial CO2 reduction within deficient lifetime of enzyme. Moreover, there is a requirement

72

for electromotive force to drive EES for higher yield of formate. A driving force can be

73

expected from an external power supply such as a commonly used potentiostat [13, 16]. With

74

a growing demand for energy worldwide, however, a renewable electricity source is urgently

75

needed to be explored.

76

Microbial fuel cell (MFC) has been investigated to generate electricity by

77

electrochemically active bacteria (EAB) for more than a decade [17]. It has been extensively

78

considered as a promising source of power alternative to extract electrons from wastewater,

79

in which process the organic pollutants can be removed to produce clean water

80

simultaneously [18-20]. Nevertheless, as compared with the release of electrons, little

81

attention has been paid to the production of inorganic carbonaceous substrates such as 4

ACCEPTED MANUSCRIPT 82

carbonate/bicarbonate from organic pollutants degradation in which process CO2 might be

83

emitted to the atmosphere. Even less effort has been made to convert the produced CO2

84

wastes to valuable chemicals [21].

85

In this study, we aimed at enzymatic electrosynthesis of formate from CO2 reduction in a

86

hybrid MFC-EFC system. The electrons, i.e. CO2-reducing agents, were extracted from the

87

degradation of organic pollutants in wastewater by EAB in MFCs. A highly efficient and

88

reusable enzymatic cathode was fabricated for EES of formate in an EFC. FDH from

89

Candida boidinii (CbFDH) was immobilized on to surface of enzymatic cathode as

90

biocatalysts, and neutral red was deposited by electro-polymerization to enhance the

91

electrochemical properties of cathode. Specific and sustainable production of formate was

92

inspected at lowered electrode potentials in the EFC by implementing metallurgical MFC

93

stack in series or parallel connection as an external power supply.

94

2. Materials and methods

95

2.1 Hybrid MFC-EFC system

96

The hybrid biofuel cell system was constructed with an EFC and several series/parallel-

97

stacked metallurgical MFCs (unit number n = 1, 2 and 3 as shown in Fig. 1). Both EFC and

98

MFC were dual-chamber reactors separated by proton exchange membrane (PEM). The

99

chambers were sealed with silica gel to prevent any potential permeation of atmospheric CO2

100

and/or O2. All electrodes were carbon-based materials (Beijing Sanye Carbon Co., Ltd.,

101

China). They were firstly soaked in acetone overnight, thence in acid mixture of

102

H2SO4:HNO3 (1:3) for 6.0 h, and further cleaned in 0.20 M H2SO4 solution by cyclic

103

voltammetry (CV) between -0.50 V and +1.50 at 50 mV/s for 3 cycles. These clean electrodes

104

were subsequently annealed at 370 ℃ for 0.5 h, and equilibrated in 100 mM phosphate

5

ACCEPTED MANUSCRIPT 105

buffered saline (PBS, 11,472 mg L-1 Na2HPO4·2H2O and 4,904 mg L-1 NaH2PO4·H2O) prior

106

to use. The whole circuit was connected via pure titanium wires (> 99.9%).

107

The EFC reactor consisted of a cathodic and an anodic working volume of 20 mL each.

108

The anode was a single piece of highly porous graphite felt (2.5 cm length × 2.5 cm width ×

109

1.0 cm thickness). The enzymatic cathode was based on a simple graphite rod (GR, 1.5 mm

110

in diameter × 2.5 cm in length, Beijng Sanye Carbon Co., Ltd., China). Polymerized neutral

111

red (PolyNR) was electro-deposited onto the GR electrode in 100 mM PBS (pH 6.0) with

112

0.40 mM neutral red. CV was carried out between -0.80 V and +0.80 V at 50 mV/s for 100

113

cycles [22]. Enzyme (1.0 unit) was immobilized onto the surface of PolyNR-GR electrode

114

by modified Nafion micelles. The Nafion micelles (~5% in a mixture of lower aliphatic

115

alcohols and water) were pretreated with tetrabutyl ammonium bromide as reported

116

previously [23]. The fabricated enzymatic electrode was allowed to air dry at 4 ℃ for 4.0 h

117

and equilibrated in 100 mM PBS for at least 0.50 h before use.

118

The metallurgical MFC comprised an anodic working volume of 200 mL and a cathodic

119

working volume of 100 mL respectively. The anode was made of three identical graphite

120

felts (6.0 cm length × 5.5 cm width × 1.0 cm thickness) and the cathode was a graphite plate

121

with a working surface area of 15 cm2.

122

6

ACCEPTED MANUSCRIPT

123 124

Fig. 1. The schematic diagram of a hybrid MFC-EFC system.

125 126

2.2 Experimental design

127

To setup a hybrid MFC-EFC system, the microbial anodes in both MFCs and the EFC

128

were enriched with EAB from the effluent of MFCs that had been running on real domestic

129

wastewater from Ulu Pandan Reclamation Plant (Singapore) for more than two years in our

130

previous study [24]. The EAB were fed with simulated wastewater prepared in 100 mM PBS

131

(pH 7.0) containing (mg L-1): CH3COONa, 500; NH4Cl, 310; KCl, 130; mineral salts medium

132

and vitamins [25]. Stable electricity output could be obtained after a two-week startup

133

operation. The catholyte for EES of formate from CO2 reduction in EFC, on the basis of 100

134

mM PBS at pH 6.0, contained 10 mM NaHCO3 as CO2 substrates and 1.0 mM nicotinamide

135

adenine dinucleotide (NADH, reduced form) as mediators. The deionized (DI) water used

136

for EES was boiled for 30 min to expel any dissolved CO2 species, and then cooled down to

137

room temperature for immediate preparation of catholyte under N2 gas. 7

ACCEPTED MANUSCRIPT 138

The first experiment was to investigate the electrochemical behaviors of immobilized

139

CbFDH on enzymatic cathode. Catalytic CV measurement was carried out between 0 V and

140

-1.0 V at a scan rate of 25 mV s-1 for 4 cycles [10, 12]. Effects of PolyNR films on electron

141

transfer and regeneration of mediators were studied by monitoring the time dependency of

142

reduction current (chronoamperometry) at a controlled cathode potential of -0.80 V for a long

143

period of 480 min. Both electrocatalytic voltammogram and chronoamperogram were

144

recorded with or without CO2 substrates in catholyte. Formate formation was detected in the

145

catholyte at the end of each electrochemical measurement.

146

The second experiment was to manipulate the efficient yield of formate by PolyNR on

147

enzymatic electrode. Three different poised potentials, i.e. -0.60 V, -0.80 V and -1.0 V were

148

applied by a potentiostat for 2.0 h respectively. At the end of EES, reduction products were

149

analyzed to understand the bioelectrochemical conversion of CO2 in the EFC.

150

The third experiment was carried out to evaluate the bioelectricity-generating capacity of

151

metallurgical (cupric) MFC stacks from wastewater. The cathode chambers of metallurgical

152

MFCs were spiked with Cu2+-containing solution ([Cu2+] = 100 mg L-1 prepared with

153

CuSO4 5H2O, pH = 3.0 adjusted by H2SO4) with NaCl of 5,850 mg L-1 as supporting

154

catholyte. The catholyte was flushed with N2 gas for 2 h to remove dissolved oxygen as a

155

potential electron acceptor other than Cu2+. The MFC reactors were stacked in two modes:

156

series or parallel-connected MFC stacks as power supplies. In the series-connected mode,

157

the microbial anode of one MFC reactor was connected to the cathode of an adjacent MFC,

158

eventually producing a cathode potential negative enough for CO2 reduction in the EFC. The

159

overall output of a series-connected MFC stack was equal to the sum voltage of each

160

individual MFC. In the parallel-connected mode, the microbial anodes of each MFC reactor

161

were connected together, thus introducing an enhanced electron flux to the cathode of EFC.

●

8

ACCEPTED MANUSCRIPT 162

The overall current intensity of a parallel-connected MFC stack was equal to the sum current

163

of each individual MFC.

164

The last experiment was to test the feasibility and sustainability of formate synthesis from

165

CO2 reduction in EFC driven by bioelectric power extracted from wastewater by MFC stacks.

166

A series and/or parallel-connected MFC stack was implemented to replace the potentiostat

167

utilized in the first and the second experiments. An enzymatic electrode with both

168

immobilized CbFDH and PolyNR was employed in the EFC. The manipulation for the

169

bioelectrochemical yield of formate was identical to that in the second experiment, but with

170

1, 2 or 3 stacked MFCs to achieve different cathode potentials. Samples were taken at 10,

171

20, 30, 40, 60, 90 and 120 min from the start of EES.

172

All the chemicals were purchased from Sigma-Aldrich (St. Louis, USA) and prepared in

173

DI water (Milli-Q, Academic system, Millipore Co., USA). Each experiment was carried out

174

in parallel and repeated for three times in a temperature-controlled room at 20 oC. All samples

175

were taken in triplicate.

176

2.3 Analyses and calculations

177

The output voltage (U in V) from metallurgical MFC stacks and electrode potential in the

178

EFC (V vs. Ag/AgCl in 3.0 M KCl) were recorded at 30 s intervals via a data acquisition

179

system (Adam 4017, Advantech Co., Ltd., China). In the MFC stacks, power generation

180

(U2/R in mW) was calculated via an external resistance (R in Ω); Polarization curves were

181

obtained by changing the external resistance in 12 steps from open circuit to 50 Ω and

182

stabling the voltage for at least 10 min at each step; The electric quantity (Q in C) was by

183

integrating current as a function of time. In the EFC, the reduction current density (I/Scat in

184

mA cm-2) was based on the surface area of cathode (Scat). The Faraday efficiency (FE) for

185

the formation of formate was calculated as follows: 9

ACCEPTED MANUSCRIPT 186

FE 

2 F  nFormate t

100%

(1)

 Idt 0

187

where 2 is the number of electrons transferred for the formation of one molecule of formate

188

from CO2, F is the Faraday’s constant (96,485 C mol-1), nFormate is the moles of formate

189

harvested, I is the circuit current (A) and t is the reaction time (s).

190

The pH of aqueous solution was measured using a pH meter (PB-10, Sartorius AG,

191

Germany). Formate produced in EFC was qualitatively detected under automation on a 600

192

MHz NMR spectrometer (Premium Shielded Narrow Bore, Agilent Technologies, USA). An

193

HPLC (Agilent 1200 series, Agilent Technologies, USA), which was equipped with a C4

194

column (5.0 μm, 4.6 × 250 mm, GL Sciences Inc., Japan) and a DAD detector set at

195

wavelength of 210 nm, was used for the separation and quantitation of formate. Mobile phase

196

was 25 mM PBS (pH 2.0) at a constant flow rate of 0.80 mL min-1. All electrochemical

197

experiments were carried out on an IVIUMSTAT station (IVIUM Technologies, Eindhoven,

198

Netherlands) in three-electrode system with the fabricated enzymatic cathode as working

199

electrode, Ag/AgCl as reference electrode and Pt wire as counter electrode. For simplicity,

200

CO2 was used to denote all the dissolved species, i.e. [CO2]Total = [CO2]Gas + [H2CO3] +

201

[HCO3-] + [CO32-].

202

3. Results and discussion

203

3.1 Electrochemical behaviors of enzymatic cathode

204

Electrocatalytic voltammogram (Fig. 2a) shows that the enzymatic CO2 reduction initiates

205

at around -0.60 V, which is slightly higher than the calculated onset potential of -0.50 V (at

206

pH 6.0 in Fig. A.1). This finding implies that the overpotential required by

207

bioelectrochemical CO2 reduction can be reduced to as low as 0.10 V with the catalysis by 10

ACCEPTED MANUSCRIPT 208

CbFDH. Higher electromotive force is able to produce higher bioelectric current,

209

consequently contributing to higher yield of formate from CO2 reduction. The reduction

210

current increases rapidly as the driving force elevates from -0.60 V onwards, whose value

211

reaches -4.5 mA cm-2 at a poised potential as high as -1.0 V. There is no reduction current

212

observed in the absence of CO2 substrates. These above observations suggest that

213

electrocatalytic conversion of CO2 to formate can be efficiently catalyzed by CbFDH under

214

mild biological conditions.

215

Further, the bioelectrochemical behavior of GR electrode can be improved by the redox

216

active layers of PolyNR. Fig. 2b shows the enhanced catalytic current for bulk bioelectrolysis

217

trace on the PolyNR- coated cathode at a poised potential. In the chronoamperometry for

218

enzymatic CO2 reduction, an apparent reduction current, which cannot be detected without

219

CbFDH, is well maintained at around 2.0 mA cm-2 before fading away in the presence of

220

active PolyNR. The current density decreased by only 25% within an overall elapsed time of

221

480 min. In the absence of PolyNR layers, on the contrary, the reduction current gradually

222

disappears upon extended catalysis reactions. The increased current density indicated the

223

positive contribution from the bioelectrochemically active PolyNR layers. During the

224

electropolymerization process, an increase in current density for the redox peaks was

225

observed as PolyNR films formed on the surface of GR electrode (Fig. B.1). The redox active

226

polymeric layers are supposed to play an important role in efficient interfacial electron

227

transfer between the solid electrode surface of GR and the active site inside the CbFDH.

228

Hence the current density on a PolyNR-modified GR electrode is almost two times higher

229

than that on a simple GR as cathode material. Moreover, neutral red is a biocompatible redox

230

dye without inhibitory effect on enzymatic functions, unlike the commonly used toxic benzyl

231

viologen and methyl viologen in conventional assays [26]. These facts provide the evidences

232

that PolyNR can be confirmed as a promising promoter for the electrocatalytic CO2 11

ACCEPTED MANUSCRIPT 233

conversion to formate by CbFDH. When no CO2 substrates are added, negligible reduction

234

current can be detected on the enzymatic electrode. No formate was generated due to lack of

235

carbon sources. When bicarbonate was employed as CO2 substrates, there was an

236

accumulated formate concentration of 84.22 ± 6.53 mg L-1 with both PolyNR and CbFDH

237

after 480 min. These results proved the feasibility of CO2-to formate conversion in an MFC-

238

EFC system.

239

240

241 12

ACCEPTED MANUSCRIPT 242

Fig. 2. (a) Cyclic voltammetry of CO2 reduction on enzymatic electrode with immobilized

243

CbFDH. (b) Chronoamperometry of CO2 reduction on enzymatic electrode with immobilized

244

CbFDH and/or PolyNR at a poised potential of -0.80 V (vs. Ag/AgCl).

245 246

3.2 Specific and efficient formate generation in EFC

247

Fig. 3 illustrates the specific and efficient EES of formate from CO2 reduction in the EFC.

248

The only 1H NMR signal near 8.3 ppm (for all samples) is ascribed to the proton involved in

249

the molecular formula of HCOO-, suggesting formate being the specific reduction product

250

for EES. Using PolyNR as NADH-regenerators and CbFDH as biocatalysts, higher

251

production rates of formate could be obtained at more negative cathode potentials. When the

252

poised potentials increases from -0.60, -0.80 to -1.0 V, the average formate yield goes up

253

from 41.67 ± 4.16, 57.40 ± 4.60 to 64.71 ± 3.38 mg L-1 h-1 within a two-hour reaction period.

254

In comparison to the enzymatic electrode with PolyNR, the average formate yield reduces

255

nearly by half to 21.59 ± 4.56, 24.72 ± 4.03 and 28.06 ± 4.24 mg L-1 h-1 on the PolyNR-free

256

cathode under respective cathode potentials applied. Apart from the improvement on electron

257

transfer mentioned above, the deposited PolyNR exerts a lasting influence on the catalytic

258

electron flux for enzymatic CO2 reduction. NADH is a crucial mediator in EES, it is

259

consumed and oxidized to NAD+ coupled to the enzymatic reduction of CO2 to formate [13].

260

With the depletion of NADH, CO2-reduction course will be slowed down due to the limited

261

availability of mediators. The redox potential of PolyNR is slightly lower than that of NAD+

262

(-0.325 V vs. -0.320 V at pH 7.0, standard hydrogen electrode as reference) [27], it is an ideal

263

catalyst that can electrically reduce NAD+ to NADH. By using the electrochemically active

264

PolyNR as a reducing agent, the oxidized NAD+ can be recovered to NADH to further

265

mediate the CO2-reduction process [22]. More importantly, the fabricated electrode with 13

ACCEPTED MANUSCRIPT 266

immobilized CbFDH and coated PolyNR can be reused easily for more testing batches. A

267

net formate production rate higher than 20 mg L-1 h-1 could be insured after a long-term

268

running of 10 h (2.0 h per batch for 5 tests). This efficient CO2-to-formate capacity of

269

enzymatic cathode, due to the protected lifetime of immobilized CbFDH and regenerated

270

NADH mediator, contributes sustainably to formate generation from CO2.

271

272 273

Fig. 3. 1H NMR spectra for the CO2-reduction products and the yield of formate on the

274

enzymatic cathode at different poised potentials.

275 276

3.3 Electricity generation from metallurgical MFC stacks

277

To supply the EFC with a sufficient cathode potential and an input flux of electrons, MFCs

278

were connected in series and/or parallel to construct a MFC stack as an external power. As

279

shown in Fig 4a, an increased unit number tends to produce increasingly positive output

280

voltage from a series-connected MFC stack and more negative cathode potentials in the EFC.

281

When the MFC unit number is added up to n = 3, an overall output voltage as high as 1.59 ± 14

ACCEPTED MANUSCRIPT 282

0.12 V can be obtained to drive the CO2 reduction in EFC. The respective cathode potentials,

283

i.e. -0.74 ± 0.11, -0.93 ± 0.09 and -1.00 ± 0.11 V for n = 1, 2 and 3, are higher than the

284

practical onset potential of -0.60 V determined by CV measurement. This indicates that

285

sufficient motivation force can be harvested from the organic pollutants in wastewater to

286

support the lowered overpotential (around 0.10 V) for EES.

287

288

289 15

ACCEPTED MANUSCRIPT 290

Fig. 4. (a) Cathode potentials of EFC and output voltage produced by metallurgical MFC

291

stacks connected in series with different unit number. (b) Polarization curves of a 3-MFC

292

stack in series.

293 294

The maximum power output (Fig. 4b) from a three-MFC stack connected in series reaches

295

0.99 mW at a current intensity of 1.28 mA. Meanwhile, from a three-MFC stack in parallel

296

connection, an enhanced output flux of electrons (current intensity up to 8 mA) could be

297

acquired. Therefore, the metallurgic MFC stack is able to serve as an equivalent of an

298

external power supply for EES of formate from CO2 reduction in the EFC.

299

3.4 Sustainable EES driven by metallurgical MFC stacks

300

By accepting the electrons extracted from the organic matters in wastewater by MFCs,

301

considerable amount of CO2 wastes can be reduced to energy-rich formate in the EFC. As

302

presented in Fig. 5, the catalytic production of formate increases linearly as the CO2 reduction

303

reaction proceeds on the enzymatic electrode. The conversion course gradually slows down

304

after 60 min and eventually remains relatively constant in the last 30 min. A maximum

305

formate production rate of up to 100 mg L-1 h-1 was observed in the first 10 min of reaction.

306

Similar to the scenario in the potentiostat-EFC system, higher yield of formate could be

307

expected from the MFC-EFC system with more MFC units. At the end of 120-min testing

308

period, the accumulated concentration of formate from EES come to 61.88 ± 0.95, 56.82 ±

309

9.06 and 44.52 ± 9.82 mg L-1 powered by three, two and one-MFC stacks in series at the end

310

of EES (Fig. 5a). In the cathode chamber of EFC powered by a parallel-connected MFC

311

stack, the ascending formate concentration shares similar curve profiles to those in a series-

312

stacked MFC-EFC system (Fig. 5b). The formate productivity in EFC benefits less from the

313

increased unit number of MFC in a parallel stack, with a highest accumulated concentration 16

ACCEPTED MANUSCRIPT 314

of only 53.39 ± 8.47 mg L-1 at n = 3. This might be due to the reduced electrode potentials

315

when MFCs were parallel in a stack. The output bioelectric current rose twofold (n = 2) and

316

threefold (n = 3) as compared to a single MFC applied, but the electrode potential decreased

317

by posing more parallel MFCs accordingly. Based on these phenomena, a series-connected

318

MFC stack is more suitable to use in bioenergy generation for efficient CO2-to-formate

319

conversion, which makes an environmentally friendly way for specific formate production

320

from CO2 reduction and pollutant removal from wastewater.

321

17

ACCEPTED MANUSCRIPT

322

323 324

Fig. 5. Evolution of formate from bioelectrochemical CO2 reduction powered by (a) series-

325

connected and (b) parallel-connected MFC stacks with different unit number.

326 327

The MFC-EFC system is comparable in formate generation to a potentiostat-EFC system.

328

An average formate production rate of 59.13±3.85 mg L-1 h-1 could be achieved in the MFC-

329

EFC with a cathode potential of -1.00 ± 0.11 V imposed by a three-MFC stack connected in

330

series, which was slightly lower than that of 64.71 ± 3.38 mg L-1 h-1 in a potentiostat-EFC 18

ACCEPTED MANUSCRIPT 331

with a poised potential of -1.0 V. Hence the organic compounds in wastewater can be utilized

332

as efficient electron donors with MFCs for CO2 reduction in an EFC. To make the EES

333

process

334

carbonate/bicarbonate-medium feeding can be considered in the EFC for continuous formate

335

production.

336

3.5 Bioresource recovery and CO2 mitigation in hybrid MFC-EFC system

more

practical

for

application,

a

gaseous

CO2 source

or

constant

337

The hybrid MFC-EFC system has been proven to be energy-sustainable in specific

338

formate production for efficient CCSU. Table 1 summarizes the catalytic performance of

339

CbFDH in CO2 reduction to formate in the EFC by providing the enzymatic cathode with

340

electrons released from wastewater in MFC stacks. In the EFC powered by a three-MFC

341

stack in series, with a highest Faradaic efficiency of 70%, the average formate production

342

rate maximized at around 60 mg L-1 h-1 (or 1.18 ± 0.08 mg h-1 by one unit of CbFDH). If the

343

huge amount of CO2 (i.e. 1.21 × 104 tons day-1 emitted by wastewater treatment plants in

344

year 2025 as introduced earlier) can be captured in an aqueous system, thousand tons of

345

formate will be produced worldwide every day. The EFC also gains an advantage of energy

346

efficiency over the widely used metal catalysts which require excessive overpotential

347

(usually higher than -1.0 V) to initiate the electron transfer from cathode to CO2 species [28].

348

Consequently, bioelectrochemical CO2 sequestration and/or reduction therein lay a great

349

potential in sustainable development.

350 351

Table 1

352

Performance of the MFC-EFC system in CO2-to-formate conversion powered by

353

wastewater MFC unit (n)

Series 19

Parallel

ACCEPTED MANUSCRIPT n=1

n=2

n=3

n=2

n=3

46.11±5.34

57.12±5.18

59.13±3.85

47.19±7.41

56.01±6.89

0.35±0.04

0.43±0.04

0.45±0.03

0.36±0.06

0.42±0.05

0.92±0.11

1.14±0.10

1.18±0.08

0.94±0.15

1.12±0.14

57.36±7.60

65.58±6.56

69.94±4.34

37.14±3.60

26.72±3.26

13.25±1.74

12.00±1.58

20.04±2.49

33.30±2.55

Enzymatic fuel cell (EFC) Production rate (mg L-1 h-1) Production yield (mg mg CO2-1 h-1) Specific productivity (mg U CbFDH-1 h-1) Faraday efficiency (%)

Microbial fuel cell (MFC) stack Electric quantity (C)

13.71±2.05

354

Note: each data value represents an average at 95% confidence level over an experimental

355

period of 120 min.

356 357

The biodegradable organic pollutants in wastewater hold great promise as a variable

358

electron source for EES of formate. In our optimized hybrid biofuel cells, within a short

359

experimental period of 120 min, up to 2×1020 electrons are derived from the microbial

360

degradation of organic pollutants in low-strength wastewater. That is equivalent to 0.28 mM

361

CO2 reduction or formate generation (via a two-electron transfer reaction) based on an

362

electric quantity of 33 C from a three-MFC stack in parallel (total wastewater volume of 200

363

mL × 3). Domestic wastewater is a typical low-strength wastewater. The average

364

concentration of biodegradable organic matters was only 220 ± 34 mg L-1 (vs. > 10,000 mg

365

L-1 of industrial wastewater) in the water samples from Ulu Pandan Reclamation Plant

366

(Singapore). As for a conventional domestic wastewater treatment plant with a working

367

capacity of 10,000 m3 day-1, more than 120 kg of CO2/formate will be reduced/synthesized 20

ACCEPTED MANUSCRIPT 368

daily. This is commercially viable to meet the great market demand of million tons per year

369

for formic acid (formate) in the production of food additives and preservatives [29]. Thus,

370

domestic wastewater turns out to be a promising candidate to power MFC-MFC systems for

371

CO2 conversion to formate.

372

Furthermore, the real domestic wastewater has several attributes as a potential feedstock

373

for EES of valuable chemicals in a green pathway: Wastewater → CO2 → Formate (this

374

study) →∙Biofuels (future study). There are more biodegradable organic compounds and less

375

toxicant to support the growth of bacteria in MFCs. If the carbonate/bicarbonate-containing

376

effluent from the anode chambers of MFC (final products of organic matter degradation) can

377

be reused in the cathode of EFC in future study, the hybrid system shall be a more attractive

378

technology to make better use of the organic wastes in water rather than merely treating or

379

disposing them. However, proper operation like adjusting the pH of treated wastewater is

380

necessary to ensure an optimal working condition in the EFC. As of such, the constructed

381

MFC-EFC system is energy-and-performance efficient in mitigating the environmental

382

impact of CO2 and securing the molecular value of organic pollutants in wastewater.

383

4. Conclusion

384

The MFC-EFC system has been proven feasible to synthesize valuable chemical of

385

formate from CO2. On an appropriately fabricated enzymatic cathode with CbFDH and

386

PolyNR, sustainable CO2 reduction occurs at lowered overpotentials as low as 0.10 V.

387

Formate can be confirmed as the quantitative product of bioelectrochemical CO2 reduction.

388

Performance of metallurgical MFC stacked in both series and parallel has been investigated

389

to explore the potential application of CO2-to-formate conversion driven by the bioelectric

390

power supplied by water pollutants. Higher yield of formate can be obtained by inputting

391

flux of electrons from more MFC units. The optimal MFC-EFC system demonstrates 21

ACCEPTED MANUSCRIPT 392

competitive advantages in (1) oriented CO2 reduction, (2) efficient yield of formate and (3)

393

sustainable utilization of bioresource from wastewater.

394

Acknowledgements

395

The authors gratefully acknowledge the financial support from the National University of

396

Singapore, National Research Foundation and Economic Development Board (SPORE,

397

COY-15-EWI-RCFSA/N197-1) and Ministry of Education (R-143-000-519-112).

398

Appendices

399

Appendix A E-pH diagram of CO2 reduction by CbFDH

400

The E-pH diagram of CO2 reduction by immobilized CbFDH in aqueous system was

401

plotted based on the electrocatalytic voltammetry. Data have been fitted by using the Nernst

402

equation as follows:

403

E  E 

404

where Eθ = -0.21 V, pKRed = 3.75, pKOx1 = 6.39 and pKOx2 = 10.32 [10].

RT 1  K Ox1 / [ H  ](1  K Ox 2 / [ H  ]) ln 2F (1  K Re d / [ H  ])[ H  ]2

22

(A.1)

ACCEPTED MANUSCRIPT

405 406

Fig. A.1. pH-dependent reduction potential of CO2 in aqueous system. Potential values were

407

measured by the electrocatalytic voltammetry of immobilized CbFDH. The reduction

408

potentials (blue open circles) were recorded using 10 mM NaHCO3 and 10 mM formate in

409

100 mM PBS from pH 5.0 to 8.0 at 0.50 intervals. CV measurement was carried out as

410

indicated in Fig. 2a, but the enzymatic electrode rotated at a fixed rate of 2000 rpm.

411

23

ACCEPTED MANUSCRIPT 412

Appendix B Electro-polymerization of neutral red

413 414

Fig. B.1. Cyclic voltammetry for the electropolymerization of neutral red onto the graphite

415

rod electrode.

416

References

417

[1] E. Dogan, F. Seker, Determinants of CO2 emissions in the European Union: The role of

418

renewable and non-renewable energy, Renew. Energ., 94 (2016) 429-439.

419

[2] M.J. Frank, J.A. Kuipers, W.P. van Swaaij, Diffusion coefficients and viscosities of

420

CO2+H2O, CO2+CH3OH, NH3+H2O, and NH3+CH3OH liquid mixtures, J. Chem. Eng.

421

Data., 41 (1996) 297-302.

422

[3] H.Z. Zhao, Y. Zhang, Y.Y. Chang, Z.S. Li, Conversion of a substrate carbon source to

423

formic acid for carbon dioxide emission reduction utilizing series-stacked microbial fuel

424

cells, J. Power Sources, 217 (2012) 59-64.

24

ACCEPTED MANUSCRIPT 425

[4] G. Mohanakrishna, J.S. Seelam, K. Vanbroekhoven, D. Pant, An enriched electroactive

426

homoacetogenic biocathode for the microbial electrosynthesis of acetate through carbon

427

dioxide reduction, Faraday Discuss., 183 (2015) 445-462.

428

[5] L.J. Zhang, J.Y. Liu, J. Ong, S.F.Y. Li, Specific and sustainable bioelectro-reduction of

429

carbon dioxide to formate on a novel enzymatic cathode, Chemosphere, 162 (2016) 228-

430

234.

431

[6] W. Lv, R. Zhang, P. Gao, L. Lei, Studies on the faradaic efficiency for electrochemical

432

reduction of carbon dioxide to formate on tin electrode, J. Power Sources, 253 (2014)

433

276-281.

434

[7] A. Del Castillo, M. Alvarez-Guerra, J. Solla-Gullon, A. Saez, V. Montiel, A. Irabien,

435

Electrocatalytic reduction of CO2 to formate using particulate Sn electrodes: Effect of

436

metal loading and particle size, Appl. Energ., 157 (2015) 165-173.

437

[8] A. Kumar, S. Ergas, X. Yuan, A. Sahu, Q.O. Zhang, J. Dewulf, F.X. Malcata, H. van

438

Langenhove, Enhanced CO2 fixation and biofuel production via microalgae: recent

439

developments and future directions, Trends Biotechnol., 28 (2010) 371-380.

440

[9] S. Bajracharya, A. ter Heijne, X.D. Benetton, K. Vanbroekhoven, C.J.N. Buisman, D.

441

Strik, D. Pant, Carbon dioxide reduction by mixed and pure cultures in microbial

442

electrosynthesis using an assembly of graphite felt and stainless steel as a cathode,

443

Bioresour. Technol., 195 (2015) 14-24.

444

[10] T. Reda, C.M. Plugge, N.J. Abram, J. Hirst, Reversible interconversion of carbon

445

dioxide and formate by an electroactive enzyme, P. Natl. Acad. Sci. Usa., 105 (2008)

446

10654-10658.

447

[11] H. Hwang, Y.J. Yeon, S. Lee, H. Choe, M.G. Jang, D.H. Cho, S. Park, Y.H. Kim,

448

Electro-biocatalytic production of formate from carbon dioxide using an oxygen-stable

449

whole cell biocatalyst, Bioresour. Technol., 185 (2015) 35-39. 25

ACCEPTED MANUSCRIPT 450

[12] A. Bassegoda, C. Madden, D.W. Wakerley, E. Reisner, J. Hirst, Reversible

451

interconversion of CO2 and formate by a molybdenum-containing formate

452

dehydrogenase, J. Am. Chem. Soc., 136 (2014) 15473-15476.

453

[13] S. Srikanth, M. Maesen, X. Dominguez-Benetton, K. Vanbroekhoven, D. Pant,

454

Enzymatic electrosynthesis of formate through CO2 sequestration/reduction in a

455

bioelectrochemical system (BES), Bioresour. Technol., 165 (2014) 350-354.

456

[14] X. Dominguez-Benetton, S. Srikanth, Y. Satyawali, K. Vanbroekhoven, D. Pant,

457

Enzymatic electrosynthesis: An overview on the progress in enzyme-electrodes for the

458

production of electricity, fuels and chemicals, J. Microb. Biochem. Technol., S6: 007

459

(2013) 1-20.

460

[15] Y. Amao, S. Ikeyama, Discovery of the reduced form of methylviologen activating

461

formate dehydrogenase in the catalytic conversion of carbon dioxide to formic acid,

462

Chem. Lett., 44 (2015) 1182-1184.

463

[16] P.K. Addo, R.L. Arechederra, S.D. Minteer, Evaluating enzyme cascades for

464

methanol/air biofuel cells based on NAD+‐dependent enzymes, Electroanalysis, 22

465

(2010) 807-812.

466

[17] B.E. Logan, B. Hamelers, R. Rozendal, U. Schröder, J. Keller, S. Freguia, P. Aelterman,

467

W. Verstraete, K. Rabaey, Microbial fuel cells: methodology and technology, Environ.

468

Sci. Technol., 40 (2006) 5181-5192.

469

[18] W.Q. Li, S.H. Zhang, G. Chen, Y.M. Hua, Simultaneous electricity generation and

470

pollutant removal in microbial fuel cell with denitrifying biocathode over nitrite, Appl.

471

Energ., 126 (2014) 136-141.

472

[19] B. Virdis, K. Rabaey, R.A. Rozendal, Z.G. Yuan, J. Keller, Simultaneous nitrification,

473

denitrification and carbon removal in microbial fuel cells, Water Res., 44 (2010) 2970-

474

2980. 26

ACCEPTED MANUSCRIPT 475 476

[20] L.J. Zhang, X.J. Yin, S.F.Y. Li, Bio-electrochemical degradation of paracetamol in a microbial fuel cell-Fenton system, Chem. Eng. J., 276 (2015) 185-192.

477

[21] P. Pandey, V.N. Shinde, R.L. Deopurkar, S.P. Kale, S.A. Patil, D. Pant, Recent advances

478

in the use of different substrates in microbial fuel cells toward wastewater treatment and

479

simultaneous energy recovery, Appl. Energy, 168 (2016) 706-723.

480

[22] A.A. Karyakin, O.A. Bobrova, E.E. Karyakina, Electroreduction of NAD+ to

481

enzymatically active NADH at poly (neutral red) modified electrodes, J. Electroanal.

482

Chem., 399 (1995) 179-184.

483 484

[23] S. Meredith, S. Xu, M.T. Meredith, S.D. Minteer, Hydrophobic salt-modified Nafion for enzyme immobilization and stabilization, J. Vis. Exp., (2012).

485

[24] L.J. Zhang, Y. Gao, L.K. Lai, S.F.Y. Li, Whole-cell-based identification of

486

electrochemically active bacteria in microbial fuel cells by matrix-assisted laser

487

desorption/ionization time-of-flight mass spectrometry, Rapid Commun. Mass. Sp, 29

488

(2015) 2211-2218.

489

[25] B.E. Logan, Microbial Fuel Cells, John Wiley & Sons, Inc., Hoboken, New Jersey, 2007.

490

[26] D. Park, J. Zeikus, Utilization of electrically reduced neutral red byActinobacillus

491

succinogenes: physiological function of neutral red in membrane-driven fumarate

492

reduction and energy conservation, J. Bacteriol., 181 (1999) 2403-2410.

493

[29] M.N. Arechederra, P.K. Addo, S.D. Minteer, Poly (neutral red) as a NAD+ reduction

494

catalyst and a NADH oxidation catalyst: towards the development of a rechargeable

495

biobattery. Electrochim. Acta, 56 (2011) 1585-1590.

496

[28] Y. Hori, H. Wakebe, T. Tsukamoto, O. Koga, Electrocatalytic process of co selectivity

497

in electrochemical reduction of CO2 at metal-electrodes in aqueous-media, Electrochim.

498

Acta, 39 (1994) 1833-1839.

27

ACCEPTED MANUSCRIPT 499

[29] A. ElMekawy, H.M. Hegab, G. Mohanakrishna, A.F. Elbaz, M. Bulut, D. Pant,

500

Technological advances in CO2 conversion electro-biorefinery: A step toward

501

commercialization, Biochem. Technol., 215 (2016) 357-370.

28