Biochar Modulates Methanogenesis through Electron Syntrophy of

Subscriber access provided by University of Sunderland

Characterization of Natural and Affected Environments

Biochar modulates methanogenesis through electron syntrophy of microorganisms with ethanol as a substrate Hai-Yan Yuan, Long-Jun Ding, Eric Fru Zama, Pan-pan Liu, Wael N. Hozzein, and Yong-Guan Zhu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b04121 • Publication Date (Web): 10 Oct 2018 Downloaded from http://pubs.acs.org on October 14, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41

Environmental Science & Technology

1

Biochar Modulates Methanogenesis through Electron Syntrophy of Microorganisms

2

with Ethanol as a Substrate

3

Hai-Yan Yuan†,‡, Long-Jun Ding†, Eric Fru Zama§, Pan-Pan Liu||, Wael N. Hozzein ⊥

4

and Yong-Guan Zhu *,†,§

5

† State

6

Sciences, Chinese Academy of Sciences, Beijing 100085, People’s Republic of China.

7

‡ University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China.

8

§

9

Academy of Sciences, Xiamen 361021, People’s Republic of China.

Key Lab of Urban and Regional Ecology, Research Center for Eco-Environmental

Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese

10

|| State

11

Environment, Tsinghua University, Beijing 100084, People’s Republic of China.

12

⊥

13

University, Riyadh 11451, Kingdom of Saudi Arabia.

Key Joint Laboratory of Environment Simulation and Pollution Control, School of

Bioproducts Research Chair, Zoology Department, College of Science, King Saud

ACS Paragon Plus Environment


15

TOC/Abstract art

16


Page 2 of 41

Page 3 of 41


18

ABSTRACT

19

Biochar has the potential to influence methanogenesis which is a key component of global

20

carbon cycling. However, the mechanisms governing biochar's influence on

21

methanogenesis is not well understood, especially its effects on interspecies relationships

22

between methanogens and anaerobic bacteria (e.g. Geobacteraceae). To understand how

23

different types of biochar influence methanogenesis, biochars derived from rice straw (RB),

24

wood chips (WB) and manure (MB) were added to the methanogenic enrichment culture

25

system of a paddy soil. Compared to the non-biochar control (NB), RB and MB additions

26

accelerated methanogenesis remarkably, showing 10.7 and 12.3-folds higher methane

27

production rate, respectively; while WB had little effect on methanogenesis. Using fourier

28

transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and

29

electrochemical methods, RB and MB also had higher redox-active properties or charging

30

and discharging capacities than WB, and the functional groups, mainly quinones, on the

31

biochar surface played an important role in facilitating methanogenesis. Quantitative

32

polymerase chain reaction (qPCR) result demonstrated that electronic syntrophy did exist

33

between methanogens and Geobacteraceae. RB and MB stimulate methanogenesis by

34

facilitating direct interspecies electron transfer between methanogens and Geobacteraceae.

35

Our findings contribute to a better understanding of the effects of biochars from different

36

feedstocks on methanogenesis and provide new evidence to the mechanisms of stimulating

37

methanogenesis via biochar.

38

INTRODUCTION ACS Paragon Plus Environment


Page 4 of 41

39

Methanogenesis is a key component of global carbon cycling and widely distributed

40

in both natural environments and engineered systems1-4. Methanogenesis includes

41

hydrogenotrophic

42

electromethanogenesis. Electromethanogenesis is a novel manner of methane production5,

43

6.

44

production of methane7. However, high H2 concentration could inhibit syntrophic oxidation

45

of organic matters and suppress growth of syntrophic microorganisms, which makes

46

interspecies electron transfer be the rate-limiting step of methanogenesis8, 9. But electron

47

transfer between different species is not limited to H2 or formate as electron carriers5.

48

Different species often engage in direct interspecies electron transfer (DIET)10. DIET could

49

transfer electrons more efficiently than H2 or formate as electron carriers11.

methanogenesis,

acetoclastic

methanogenesis

and

H2 or formate, as electron carriers, could facilitate electron exchange, leading to the

50

Recent studies have found that some materials with good conductivity such as

51

granular activated carbon and magnetite could accelerate DIET11, 12. Biochar, as a carbon-

52

rich product of incomplete combustion of biomass with a limited oxygen supply13-15, has

53

been found to be redox active. Oxidized biochar could function as electron acceptors to

54

enable organic matter degradation and the reduced biochar could serve as electron donors

55

for nitrate16, magnetite reduction17 or for bacteria18. It has been suggested that biochar

56

serving as an electron shuttle could accelerate DIET between Geobacter metallireducens

57

and Methanosarcina in the co-culture system18. During anaerobic digestion process,

58

biochar could also facilitate CH4 emissions through facilitating electron transfer processes

59

directly by functioning as electron shuttle19. Yet little is known about how biochar


Page 5 of 41


60

functioning as an electron shuttle influences microbial methanogenesis in soils. Biochar

61

has organic functional groups such as quinone and hydroquinone on its surface, which lead

62

to its redox active properties20. However, the knowledge of how the redox properties from

63

different types of biochars (i.e., biochars produced from different feedstocks) influence

64

methanogenesis is rather limited. Considering that feedstock has been recognized as a

65

determinant of properties of biochar21, 22, we thus hypothesize that biochars derived from

66

different feedstocks have remarkably different redox properties and exert distinct effects

67

on methanogenesis.

68

Therefore, in this study, biochars derived from rice straw (RB), manure (MB) and

69

wood chips (WB) were employed to investigate their effects on methanogenesis. Biochars

70

from rice straw and manure are widely applied to agricultural ecosystems, because they

71

can increase crop yield, sequester carbon and immobilize contaminants such as lead and

72

atrazine23, 24. Biochar from wood chips is a major component of natural organic matter

73

worldwide, due to frequent wildfires25. We aimed at (i) investigating the influence of three

74

types of biochars from rice straw, wood chips and manure on methanogens from paddy

75

soils via anaerobic incubation, (ii) revealing the underlying mechanisms regulating

76

biochars' effects on methanogenesis by qualitative and quantitative analyses of the

77

chemical composition of biochars using X-ray photoelectron spectroscopy (XPS) and

78

fourier transform infrared spectroscopy (FTIR), and quantifying the changes in the

79

charging and discharging capacities of biochars using electrochemical analyses, and (iii)



80

exploring the electronic syntrophy between Geobacteraceae and methanogens via

81

incubation with or without biochar.

82

MATERIALS AND METHODS

83

Characterization of Biochars

84

Three types of biochars (RB, WB and MB) were made from rice straw, poplar wood

85

chips and cow manure biomasses, separately. Each type of biomass was pyrolyzed at 600ºC

86

for 2 h in a closed container under oxygen-limited conditions. Then biochars were grinded

87

to small particle sizes and passed through 2 mm sieve. A pH meter was used to measure

88

the pH of biochar at 1:15 biochar:water ratio (w/v). Brunauer-Emmett-Teller (BET) surface

89

area of RB, WB and MB was determined with an ASAP 2020 HD88 instrument

90

(Micromeritics, USA). Electrical conductivity (EC) of the three biochars was measured

91

with a conductivity meter26. The total C and H of the biochars were determined with an

92

elemental analyzer (Vario EL III, Elementar, Germany). Before application, RB, WB and

93

MB were washed in order to remove dissolved organic matter. The washed biochars were

94

made by leaching pyrolized biochars with deionized water, and then dried in the oven.

95

Qualitative and quantitative information of the chemical composition of biochars was

96

obtained with X-ray photoelectron spectroscopy (XPS) (ESCALAB 250 Xi- ThermoFisher,

97

UK). Fourier transform infrared spectroscopy (FTIR) (Thermo Scientific Nicolet 8700)

98

was used to determine organic functional groups such as hydroxyls and carbonyls on the

99

surface of biochars. Biochar was prepared in pellets of fused KBr within the 4000-400 cm-1

100

regions27. Scanning electron microscopy (SEM) (Hitachi S4800＋EDS, Hitachi Limited,


Page 6 of 41

Page 7 of 41


101

Japan), operated at 20 kV with an energy X-ray dispersive (EDX) detector was used to

102

observe the microstructure and element distribution of biochars.

103

Three biochar powder were immobilized on graphite rod electrodes for

104

electrochemical tests of surface quinone groups. 8 mg biochar solids and 40 μL of nafion

105

were dispersed in 1 mL of a mixture of ethanol and water (1:4) with a vortex. Then, 15 μL

106

of the resulting dispersion was dropped onto the surface of the working electrodes and

107

dried at room temperature. Afterwards, cyclic voltammetry was performed in a three-

108

electrode configured cell with a potentiostat (CHI1040B, Chenhua, China). Graphite rods

109

and Ag/AgCl were used as counter and reference electrodes, respectively. 0.1 M KCl was

110

used as the supporting electrolyte. Before measurements, N2 was used to purge the oxygen

111

from solution. Reduction and oxidation currents were designed to a negative and positive

112

value, respectively. The scan direction for the cyclic voltammogram was changed from

113

reduction to oxidation and the scan rate was 50 mV s-1.

114

Enrichment Cultures of Methanogenic Microbial Communities

115

Methanogenic microbial communities were enriched in serum bottles (125 ml) filled with

116

50 ml of anaerobic medium. Ethanol is one of the most abundant low molecular metabolite

117

during organic matter degradation28. Consequently, 30 mM of ethanol was supplemented

118

in serum bottles as organic carbon substrate. The anaerobic medium was comprised of

119

NaHCO3 (5 mM L-1), MgCl2 (0.5 mM L-1), CaCl2 (0.5 mM L-1), KH2PO4 (1 mM L-1),

120

NH4Cl (10 mM L-1), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), 0.05%

121

Bacto yeast extract, 1 ml L-1 trace element solution and 1 ml L-1 Se/W solution29. 100 mg



122

(wet weight) of paddy soil was inoculated as a source of microorganisms. The paddy soil

123

was sampled at Hailun (47o35'N, 126o53'E), Heilongjiang Province, China in October 2014.

124

The soil was air-dried and sieved (2mm mesh size), then stored at 4oC before use. The

125

physicochemical properties of the inoculated paddy soils are shown in Table S1. In order

126

to test the effect of biochar amendments, 1 g of RB, WB, or MB was added to the

127

enrichment culture medium. Control cultures (NB) were not amended with biochars.

128

Moreover, in order to investigate the electronic syntrophy between Geobacteraceae and

129

methanogens in the enrichment culture systems, a methanogenesis inhibitor (2-

130

bromoethane sulfonate (BES)) was added to the enrichment culture systems. Meanwhile,

131

control enrichment culture systems were not amended with BES (Non-BES). Consequently,

132

the crossed design resulted in eight treatments: (1) control with no additions, (2) control

133

with BES, (3) with addition of RB, (4) with addition of RB and BES, (5) with addition of

134

WB, (6) with addition of WB and BES, (7) with addition of MB and (8) with addition of

135

MB and BES. The treatments of the experiment were shown in Fig. S1. Each treatment has

136

three replicates.

137

BES could inhibit the growth of methanogens, but could not inhibit the growth of

138

Geobacteraceae directly30. If electronic syntrophy exists between Geobacteraceae and

139

methanogens, the growth of Geobacteraceae would also be suppressed when that of

140

methanogens was inhibited by BES. Conversely, the growth of Geobacteraceae would not

141

be influenced in the enrichment culture systems without BES. Consequently, different

142

treatments with or without BES were carried out to demonstrate the electronic syntrophy


Page 8 of 41

Page 9 of 41

143


between Geobacteraceae and methanogens.

144

The headspace of all the cultures were flushed with N2/CO2 [80:20 (v/v)] and then

145

incubated at 30°C without shaking. Methane concentrations in the enrichment cultures

146

were measured with gas chromatography (Agilent Technologies, CA, USA).

147

Analyses of Archaeal and Bacterial Community from the Enrichment Cultures Using

148

Illumina MiSeq Sequencing

149

After 42 days, all the enrichment samples were collected by centrifugation. Microbial

150

DNA from each enrichment culture system was extracted with FAST DNA Spin Kit (MP

151

Biomedicals) as described previously31. NanoDrop spectrophotometer (ND-1000, USA)

152

was used to quantify DNA of the extracts. The V3-V4 region of the bacterial 16S rRNA

153

gene was amplified by Polymerase Chain reaction (PCR) using a primer set: 338f (5'-

154

ACTCCTACGGGAGGCAGCAG-3') and 806r (5'- GGACTACHVGGGTWTCTAAT-3')

155

which contained 8-bp barcodes for sample identification. The V4-V5 region of the archaeal

156

16S rRNA gene was amplified via PCR with a primer set of 524f (5'-

157

TGYCAGCCGCCGCGGTAA-3') and 958r (5'-YCCGGCGTTGAVTCCAATT-3') which

158

also contained 8-bp barcodes for sample identification. The details about PCR reaction

159

mixture and thermal programs for 16S rRNA genes of bacteria and archaea are shown on

160

Table S2. The obtained amplicons from the PCR reactions were pooled in equimolar and

161

paired-end sequenced (2 × 250) on an Illumina MiSeq platform according to the standard

162

protocols of the Majorbio Bioinformatics Institute (Shanghai, China).



163

Raw sequencing data were demultiplexed and quality-filtered using Quantitative

164

Insights Into Microbial Ecology (QIIME) (version 1.17)32. Briefly, low-quality reads

165

which contained primer mismatches and incorrect barcodes were removed, and ambiguous

166

reads were also removed. Then, the primers and barcodes were trimmed off. The raw pair-

167

end reads were assembled to generate clean joined reads. The clean joined reads were

168

clustered into operational taxonomic units (OTUs) at a 97% similarity level. The

169

representative read which was the most abundant sequence within each OTU was classified

170

with the ribosome database project (RDP) classifier (http://rdp.cme.msu.edu/) using

171

confidence threshold of 80%33.

172

Quantitative PCR of 16S rRNA Genes of Geobacteraceae and McrA

173

Quantification of 16S rRNA genes of Geobacteraceae was performed by quantitative

174

PCR (qPCR) with the primer set Geo494F/Geo825R34. Methyl coenzyme M reductase

175

(mcrA) gene was also analyzed by qPCR with the primer set ME1/ME235. Tables S3-S4

176

show the details of qPCR primers, reaction mixtures and thermal programs. Standard

177

plasmids which carried the corresponding genes were obtained through cloning the genes

178

from samples. For a standard curve, a 10-fold serial dilution of the standard plasmid DNA

179

was used and it covered seven orders of magnitude from 102 to 108 copies of template per

180

test. Fig. S2 shows the Standard curves for qPCR. qPCR was performed in triplicate and

181

the final Geobacteraceae bacterial 16S rRNA and mcrA genes were obtained by calibrating

182

against total DNA concentrations extracted and the volume of anaerobic medium.

183

Statistical Analysis


Page 10 of 41

Page 11 of 41


184

QIIME (http://qiime.sourceforge.net/), R (http://www.r-project.org/), and Origin 8.5

185

(OriginPro 8.5.lnk) were used to conduct the statistical analyses. Additionally, SPSS (SPSS

186

statistics 17.0) software was employed to carry out one-way analysis of variance (ANOVA).

187

Linear regression analysis was carried out to demonstrate the syntrophy between

188

Geobacteraceae and methanogens. In order to compare with the effects of three different

189

biochars on methanogenesis, the suppression ratio of the Geobacteraceae growth in the

190

enrichment cultures with BES was calculated. Analysis of similarity (ANOSIM) was

191

conducted to determine significance among groups (i.e. biochar type) with R package

192

"vegan". To find the important factors which correlated with archaeal and bacterial

193

community, redundancy analysis (RDA) was carried out using envfit function of R package

194

"vegan".

195

Accession Number of Nucleotide Sequences

196

The MiSeq sequencing data of this study has been deposited in National Center for

197

Biotechnology Information (NCBI) Sequence Read Achives (SRA) database under

198

bioproject number (PRJNA393217) and accession number (SRP111434).

199

Results

200

Methane Production during Enrichment of Methanogenic Communities

201

The initial rate and long-term extent of methane production were both enhanced with

202

the addition of RB and MB. The addition of MB stimulated methanogenesis to a greater

203

extent compared with RB addition (Fig. 1a and b). However, the rate and extent of

204

methanogenesis was lower in presence of WB. More specifically, in the presence of MB



Page 12 of 41

205

there was 34.64 ± 3.04 (mean ± standard deviation, n = 3) mM cumulative methane

206

produced at day 42 (Fig. 1a). Theoretically, there will be 30 mM cumulative methane

207

produced when the acetate from the conversion of ethanol (30 mM added) was completely

208

utilized (equations (1) and (2)). This suggested that the electrons produced from the process

209

of the conversion of ethanol to acetate were utilized to produce methane according to the

210

equations (3), (4) and (5)36. In case of RB, there was 14.58 ± 9.12 (mean ± standard

211

deviation, n = 3) mM cumulative methane produced at day 42, which was lower than that

212

of MB. For WB and NB treatments, 0.08 ± 0.06 (mean ± standard deviation, n = 3) and

213

1.08 ± 1.30 (mean ± standard deviation, n = 3) mM cumulative methane was produced at

214

day 42, respectively, which were significantly lower than that of MB and RB (P < 0.05).

215

Moreover, the enrichment culture with the addition of RB and MB showed 10.7 and 12.3-

216

folds higher methane production rate compared with the NB treatment, while in the

217

presence of WB methanogenesis remained unchanged (Fig. 1b).

218

2CH3CH2OH + 2H2O→ 2CH3COOH + 8H + + 8e -

(1)

219

2CH3COOH → 2CH4 + 2CO2

(2)

220

CO2 + 8e ― + 8H + → CH4 + 2H2O

(3)

221

2CH3CH2OH → 3CH4 + CO2

(4)

222

CH3CH2OH + H2O→CH3COOH + 4H + + 4𝑒 ―

(5)

223

The Physicochemical, Morphological and Electrochemical Properties of Biochars

224

Biochar varied considerably in physicochemical properties including pH, BET surface

225

area, EC, C (%) and H (%) depending on the feedstock (Fig. S3 and Table 1). Biochar


Page 13 of 41


226

morphological properties varied considerably across three types of precursor materials.

227

SEM images showed RB with a rough, elongated appearance and visible porosity (Fig. 2a).

228

WB had a smooth appearance with porous structure, while MB contained rough, irregular

229

structure with visible porosity (Fig. 2b and c). EDX analysis provided information on the

230

localized elemental contents (Fig. S3). C and O were the dominant elements present in all

231

biochars (75.5-89.2% and 1.53-16.7%, respectively). It was noticed that the amount of O

232

in WB (1.53%) was lower than that in RB (10.51%) and MB (16.68%) (Fig. S3). Different

233

biomass materials have various contents of lignocelluloses, which can be degraded

234

differently in response to pyrolysis temperature37. Compared with rice straw and manure,

235

wood chip contains more lignocelluloses, which might be condensed into graphite sheets

236

with more aromatic carbon than oxygen during pyrolysis.

237

Different biomass feedstocks caused remarkable differences in the chemical structure

238

of biochars such as organic functional groups (Fig. 3a and Fig. 4). Reversible charging and

239

discharging current peaks demonstrated that functional groups on the surface of biochars

240

can act as both electron acceptors and donators (Fig. 3b). RB and MB produced larger

241

current (12.0 mA and 11.5 mA) compared with WB (3.3 mA) and the control (0.3 mA),

242

which indicated that the capacitance behavior of biochar might be a significant reason for

243

increasing the interspecies electron transfer. FTIR analysis indicated broad peaks between

244

1500 and 1000 cm-1 which corresponded to C=O or C=C and C-O bonds. These bonds

245

stretched in quinones and hydroquinones across three types of biochars38 and these bonds

246

were prominent on MB based on the intensity of the peak (Fig. 3a). FTIR and



247

electrochemical results were supported by XPS analysis which could determine the

248

bonding energies of carbon on the biochar surface. The wide scan XPS spectra (Fig. 4a, c

249

and e) showed that the O/C atomic ratios of RB, WB and MB are 0.27, 0.11 and 0.91,

250

respectively, indicating that WB is more reduced than RB and MB. The C1s XPS spectra

251

results exhibited the same oxygen functional groups across all the biochars. The peaks in

252

RB with binding energies at about 284.4, 286.2 and 293.6 eV, are attributed to the C-

253

C/C=C, C=O and O-C=O groups, respectively27 (Fig. 4b). Additionally, the peaks in WB

254

with binding energies at about 284.4, 285.5 and 289.8 eV, are attributed to the C-C/C=C,

255

C=O and O-C=O groups, respectively27 (Fig. 4d). The peaks in MB with binding energies

256

at about 284.4, 285.8 and 293.3 eV, are also attributed to the C-C/C=C, C=O and O-C=O

257

groups, respectively27 (Fig. 4f). The comparison of the three spectra shows that the

258

intensities of the peaks associated with oxygen groups in WB are slightly weaker than in

259

the other two types of biochars (i.e. RB and MB) indicating that the oxygen functional

260

groups of WB distributed in the inner sheet were difficult to reduce39.

261

Quantification of Geobacteraceae and McrA in the Enrichment Cultures

262

In this study, to address the electronic syntrophy between methanogens and

263

Geobacteraceae in the enrichment cultures, qPCR was used to quantify Geobacteraceae

264

populations in the presence and absence of a methanogenesis inhibitor, BES. We have

265

found that the number of 16S rRNA genes copies of Geobacteraceae was reduced in the

266

treatments of RB and MB with BES compared with those without BES (Fig. 5a and b).

267

Additionally, linear regression analysis between the abundance of mcrA and


Page 14 of 41

Page 15 of 41


268

Geobacteraceae with or without BES showed that the abundance of mcrA was positively

269

correlated with the abundance of Geobacteraceae (P < 0.001, R2 = 0.689) (Fig. 5c). These

270

indicated that electronic syntrophy did exist between Geobacteraceae and methanogens in

271

the RB and MB treatments and BES could indirectly suppress the growth of

272

Geobacteraceae via directly inhibiting the growth of methanogens due to DIET. While in

273

the treatment of WB and NB with BES, though BES inhibited the growth of methanogens,

274

it did not influence the number of 16S rRNA genes copies of Geobacteraceae compared

275

with those without BES (Fig. 5a and b). Therefore, electronic syntrophy did not exist in the

276

WB and NB treatments.

277

Effects of Biochars on Bacterial and Archaeal Community Composition

278

Illumina-based 16S rRNA metagenomic profiling was used to investigate the

279

influences of biochar amendment on bacterial and archaeal community composition. After

280

processing and quality filtering, 18346 and 28168 high-quality sequences were obtained in

281

each sample for bacteria and archaea, respectively. ANOSIM results indicated that there

282

were remarkable differences in the bacterial (P < 0.01) and archaeal (P < 0.01) community

283

composition among the treatments.

284

For bacteria, at phylum level, compared with the NB, the major bacterial communities

285

were Proteobacteria, Actinobacteria, Chlorobi and Bacteroidetes, which were enriched in

286

all the biochar supplemented enrichment culture (Fig. S5). RB and MB amendments

287

increased the relative abundance of Proteobacteria to 10.09% and 0.71%, respectively,

288

compared to 0.15% in the NB treatment. For MB, the relative abundances of Chlorobi and



289

Bacteroidetes were 1.70% and 0.88%, respectively, while they were not detected in NB.

290

The relative abundance of Actinobacteria increased from 0.02% for NB to 0.86% for MB

291

(Fig. S5). In RB treatment, the relative abundances of Bacteroidetes and Chlorobi were

292

4.91% and 1.67%, respectively, while they were not detected in NB (Fig. S5). Meanwhile,

293

RB amendment increased the relative abundance of Actinobacteria from 0.02% in NB to

294

1.49% (Fig. S5). Five phyla, including Proteobacteria, Actinobacteria, Chlorobi,

295

Bacterioidetes and Firmicutes were also detected in WB treatment. At genus level, in the

296

case of MB, Clostridium genus within Firmicute phylum was significantly enriched (Fig.

297

7a) (P < 0.05), while its relative abundances were significantly decreased from 17.14% in

298

NB to 7.04% and 15.53% in RB and WB treatments (P < 0.05; P < 0.05), suggesting that

299

MB provided a favorable environment for the growth of Clostridum, compared with the

300

RB and WB40. Previous studies have shown that Clostridium was capable of

301

electrosynthesis41 and it was able to reduce insoluble Fe(III)42. This suggested that

302

Clostridium could donate electrons to the biochar which might be utilized by methanogenes.

303

In addition to Clostridium, the relative abundance of Thermincola was increased from 0.01%

304

for NB to 9.31, 23.26 and 0.36% for RB, WB and MB amendments, respectively. Previous

305

studies have showed that Thermincola could transfer electrons to the anode of microbial

306

fuel cells43. However, whether Thermincola contribute to DIET warrants further

307

investigation. Geobacter are considered to be extracellular respiring bacteria and are

308

widely distributed in paddy soils44, 45. For RB, the relative abundance of Geobacter was

309

0.13%, while it was not detected in NB.


Page 16 of 41

Page 17 of 41


310

The major archaeal communities were Methanobacterium and Methanosarcina at

311

genus level, which had been detected as major methanogenic archaea in paddy soils46 and

312

anaerobic digesters47 (Fig. 7b and S6). Methanobacterium, the major genus within

313

Euryarchaeota phylum, was enriched in the MB treatment. The relative abundance of

314

Methanobacterium increased from 49.89 for NB to 71.08% in the MB supplemented

315

enrichment cultures. While RB amendments increased the abundance of Methanosarcina,

316

the major genus within Euryarchaeota phylum, from 2.06% for NB to 95.86% in the RB

317

supplemented enrichment cultures.

318

Based on envfit function, O/C ratio of biochar, maximum rate of methane production

319

and capacitive current of biochar were identified as variables which significantly shaped

320

bacterial and archaeal community in the enrichment culture systems. These three factors

321

explained 93.6% and 86.1% of the variance of bacterial and archaeal community,

322

respectively (Fig. 6a and b).

323

Discussion

324

Previous study reported that Geobacter metallireducens could proceed DIET48. More

325

specifically, electrons released from ethanol could be directly transferred from Geobacter

326

metallireducens to the methanogens such as Methanosarcina barkerior36. Biochar could

327

accelerate DIET between Geobacter metallireducens and Geobacter sulfurreducens or

328

Methanosarcina barkeri18. Moreover, biochar could function as electron shuttle between

329

Fe(III) minerals and bacteria, which influenced soil biogeochemistry17. In this study, linear

330

regression analysis suggested that Geobacteraceae and methanogens (here represented by



331

mcrA) from paddy soils established syntrophic relationship. This result, together with the

332

remarkable inhibition of the growth of Geobacteraceae in the enrichment cultures with

333

BES in the RB and MB treatments, suggested that RB and MB facilitated electronic

334

syntrophy between Geobacteraceae and methanogens. Otherwise, WB did not influence

335

the electronic syntrophy between Geobacteraceae and methanogens. It is generally known

336

that material's conductivity had an effect on the DIET process existing in natural

337

environments49, 50. However, in this study, conductivity of biochars did not promote DIET.

338

As shown in Table 1, there was no significant difference between the conductivity of WB

339

and that of MB, while the maximum methane production rate of MB exhibited 211 times

340

higher than that of WB, which indicated that conductivity of biochars did not play a key

341

role in the facilitating methanogenesis. Likewise, there was no remarkable difference

342

between the BET surface area of WB and that of MB, indicating that BET surface area of

343

the biochars is not the key factor for promoting the methanogenesis. Biochar is a complex

344

material and besides conductivity51, other properties such as redox properties may play

345

important roles in the facilitating methanogenesis in the enrichment culture systems.

346

Previous studies reported that humic substances and activated carbon had the capacity

347

of accepting and donating electrons52, 53. Biochar has quinone and hydroquinone moieties

348

which make biochar redox active (i.e., be capable of donating and accepting electrons)17,

349

54.

350

with WB. These moieties may participate in the DIET. The charging and discharging cycles

351

of the functional groups (e.g., quinone and hydroquinone pairs) on biochar surface have

RB and MB have higher content of the quinone and hydroquinone moieties compared


Page 18 of 41

Page 19 of 41


352

been shown to accept and donate electrons reversibly55, and played an important role in the

353

electron transfer process56. Sun et al. demonstrated that biochar with O/C ratio > 0.09 could

354

be more likely to transfer electrons through charging and discharging cycles of the

355

functional groups on the biochar surface rather than through direct electron transfer by

356

conductivity51, 56. In our study, the O/C ratio of RB and MB were much higher than 0.09

357

(Fig. 4a, c and e), which indicated that the charging and discharging cycles of the functional

358

groups on the biochar surface played an important role in the electron transfer process.

359

A recent study has shown that Methanobacterium could not accept electrons from

360

syntrophic bacterium directly, and only accept electrons from syntrophic microbes via

361

formate which could serve as electron donor36. However, our study suggested that

362

Methanobacterium could engage in microbial electron uptake from MB directly. We then

363

deduced that Methanobacterium could accept electrons from syntrophic bacterium such as

364

Geobacteraceae directly. Meanwhile MB could facilitate the electron exchange between

365

those two kinds of microbes.

366

Methanosarcina has been reported to display high respiratory versatility and could

367

utilize various substrates for methanogenesis, including H2, CO2, ethanol, and acetate30.

368

Under syntrophic methanogenic conditions, where H2 partial pressures are low,

369

Methanosarcina mainly use acetate as a methanogenic substrate rather than H2 or CO2.

370

Methanosarcina were syntrophic partners of Geobacteraceae57 which has been reported to

371

exchange electrons with Methanosarcina and biochar could increase the direct transfer of

372

electrons between Geobacteraceae and Methanosarcina18. In addition, interspecies



373

electron transfer in co-culture of Methanosarcina and Geobacteraceae with ethanol as

374

electron donor was in accordance with our observation that Methanosarcina could utilize

375

the electrons from the conversion of ethanol to acetate (equation (3)).Consequently,

376

biochar facilitated methanogenesis by increasing the direct electron transfer between

377

Methanosarcina and Geobacteraceae from paddy soils, which challenges the present

378

understanding that biochar might decrease methane gas emission58. Considering that

379

biochar is widely distributed in various environments such as paddy soils, river sediments

380

and anaerobic digesters, the influence of different types of biochars on the global carbon

381

cycling should be further investigated.

382

Overall, this study indicated that the effect of biochar on methanogenesis is highly

383

dependent on its redox-active properties or charging and discharging capacities. Biochar

384

derived from rice straw and manure, which had higher redox-active nature than biochar

385

from wood chips, might facilitate the electronic syntrophy between Geobacteraceae and

386

methanogenes with ethanol as substrate and thus increased methane production. This study

387

highlights the influences of biochar from different feedstocks on methanogenesis and

388

corresponding effects on greenhouse gas (e.g., methane) emission. Compared with

389

biochars from wood chips, biochar from rice straw and manure increased methane

390

production. So the application of biochar from manure and rice straw appears to be a good

391

strategy to promote the methane production rates in anaerobic digestions. Additionally,

392

compared with biochars from rice straw and manure, biochar from wood chips had little

393

effect on methane production. We therefore recommend applying biochar from wood chips


Page 20 of 41

Page 21 of 41


394

in paddy soils for sustainable nutrient management. Our results contribute to a better

395

understanding of the effects of biochars from different feedstocks on methanogenesis and

396

provide new evidence to the mechanisms of facilitating methanogenesis by biochar.

397

ASSOCIATED CONTENT

398

Supporting Information

399

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

400

Table captions

401

Table S1 The physicochemical properties of inoculated paddy soil from Hailun, China

402

Table S2 PCR reaction mixture and thermal profiles for 16S rRNA genes of bacteria and

403

archaea

404

Table S3 Primers used for quantitative PCR

405

Table S4 Quantitative PCR reaction mixture and thermal profiles for 16S rRNA genes of

406

Geobacteraceae and mcrA

407 408

Figure captions

409

Figure S1 The treatments of the experiment design.

410

Figure S2 The standard curves of PCR (a-16S rRNA genes of Geobacteraceae, b-mcrA)

411

Figure S3 Cluster and Non-metric multidimensional scaling (NMDS) analysis based on the

412

physicochemical properties of the three kinds of biochars (A-Cluster analysis; B-NMDS

413

analysis).

414

Figure S4 Scanning electron microscopy images with the energy dispersive X-ray analysis

415

of the three kinds of biochars (a-RB, b-WB, c-MB).



416

Figure S5 Bacterial communities at phylum level in the enrichment cultures with ethanol

417

as substrate and with or without amendments of biochar.

418

Figure S6 Archaeal communities at phylum level in the enrichment cultures with ethanol

419

as substrate and with or without amendments of biochar.

420 421

AUTHOR INFORMATION

422

Corresponding Author*Address

423

State Key Lab of Urban and Regional Ecology, Research Center for Eco-Environmental

424

Sciences, Chinese Academy of Sciences, Beijing 100085, China. Phone: +86-10-62-

425

62936940; fax: +86-10-62936940; e-mail: [email protected]

426

Notes

427

The authors declare no competing financial interest.

428

ACKNOWLEDGMENTS

429

This work was supported by the National Natural Science Foundation of China, Grant Nos.

430

41430858 and 41601242, and the Strategic Priority Research Program of Chinese

431

Academy of Sciences (XDB15020402). The authors would like to thank Twasol Research

432

Excellence Program (TRE Program) at King Saud University, Riyadh, Saudi Arabia for

433

support.


Page 22 of 41

Page 23 of 41


REFERENCES

435 436

1.

Liu, W.; Cai, W.; Guo, Z.; Wang, L.; Yang, C.; Varrone, C.; Wang, A., Microbial

437

electrolysis contribution to anaerobic digestion of waste activated sludge, leading to

438

accelerated methane production. Renew Energ 2016, 91, 334-339.

439

2.

440

Biocathodic methanogenic community in an integrated anaerobic digestion and microbial

441

electrolysis system for enhancement of methane production from waste sludge. ACS

442

Sustain Chem Eng 2016, 4, (9), 4913-4921.

443

3.

444

methanogenic archaeal communities in two types of paddy soil amended with different

445

amounts of rice straw. FEMS Microbiol Ecol 2014, 88, (2), 372-385.

446

4.

447

components of municipal solid waste by short-term pre-aeration and its enhancement on

448

anaerobic degradation in simulated landfill bioreactors. Bioresource Technol 2016, 216,

449

250-259.

450

5.

451

interspecies electron transfer. Front Microbiol 2015, 5,1-8.

452

6.

453

accelerating electromethanogenesis on an hour-long timescale in wetland soil. Environ Sci:

454

Nano 2018, 5,436-445.

455

7.

Cai, W.; Liu, W.; Yang, C.; Wang, L.; Liang, B.; Thangavel, S.; Guo, Z.; Wang, A.,

Bao, Q.-L.; Xiao, K.-Q.; Chen, Z.; Yao, H.-Y.; Zhu, Y.-G., Methane production and

Ni, Z.; Liu, J.; Girotto, F.; Cossu, R.; Qi, G., Targeted modification of organic

Shrestha, P. M.; Rotaru, A.-E., Plugging in or going wireless: strategies for

Xiao, L.; Liu, F.; Liu, J.; Li, J.; Zhang, Y.; Yu, J.; Wang, O., Nano-Fe3O4 particles

Stams, A. J.; Plugge, C. M., Electron transfer in syntrophic communities of anaerobic



456

bacteria and archaea. Nature Rev Microbiol 2009, 7, (8), 568-577.

457

8.

458

interspecies hydrogen transfer between Pelotomaculum thermopropionicum and

459

Methanothermobacter thermautotrophicus. Appl Environ Microbiol 2005, 71, (12), 7838-

460

7845.

461

9.

462

ethanol to perform direct interspecies electron transfer for syntrophic metabolism of

463

propionate and butyrate. Water Res 2016, 102, 475-484.

464

10. Liu, F.; Rotaru, A. E.; Shrestha, P. M.; Malvankar, N. S.; Nevin, K. P.; Lovley, D. R.,

465

Magnetite compensates for the lack of a pilin‐associated c‐type cytochrome in extracellular

466

electron exchange. Environ Microbiol 2015, 17, (3), 648-655.

467

11. Cruz Viggi, C.; Rossetti, S.; Fazi, S.; Paiano, P.; Majone, M.; Aulenta, F., Magnetite

468

particles triggering a faster and more robust syntrophic pathway of methanogenic

469

propionate degradation. Environ Science Technol 2014, 48, (13), 7536-7543.

470

12. Zhang, S.; Chang, J.; Lin, C.; Pan, Y.; Cui, K.; Zhang, X.; Liang, P.; Huang, X.,

471

Enhancement of methanogenesis via direct interspecies electron transfer between

472

Geobacteraceae and Methanosaetaceae conducted by granular activated carbon.

473

Bioresour Technol 2017, 245, 132-137.

474

13. Jeffery, S.; Verheijen, F. G.; Van Der Velde, M.; Bastos, A. C., A quantitative review

475

of the effects of biochar application to soils on crop productivity using meta-analysis. Agr

476

Ecosyst Environ 2011, 144, (1), 175-187.

Ishii, S. i.; Kosaka, T.; Hori, K.; Hotta, Y.; Watanabe, K., Coaggregation facilitates

Zhao, Z.; Zhang, Y.; Yu, Q.; Dang, Y.; Li, Y.; Quan, X., Communities stimulated with


Page 24 of 41

Page 25 of 41


477

14. Spokas, K. A., Review of the stability of biochar in soils: predictability of O: C molar

478

ratios. Carbon Manag 2010, 1, (2), 289-303.

479

15. Steinbeiss, S.; Gleixner, G.; Antonietti, M., Effect of biochar amendment on soil

480

carbon balance and soil microbial activity. Soil Biol Biochem 2009, 41, (6), 1301-1310.

481

16. Saquing, J. M.; Yu, Y.-H.; Chiu, P. C., Wood-derived black carbon (biochar) as a

482

microbial electron donor and acceptor. Environ Science Technol Let 2016, 3, (2), 62-66.

483

17. Kappler, A.; Wuestner, M. L.; Ruecker, A.; Harter, J.; Halama, M.; Behrens, S.,

484

Biochar as an electron shuttle between bacteria and Fe (III) minerals. Environ Science

485

Technol Let 2014, 1, (8), 339-344.

486

18. Chen, S.; Rotaru, A.-E.; Shrestha, P. M.; Malvankar, N. S.; Liu, F.; Fan, W.; Nevin,

487

K. P.; Lovley, D. R., Promoting interspecies electron transfer with biochar. Sci Rep 2014,

488

4, 1-7.

489

19. Zhao, Z.; Zhang, Y.; Holmes, D. E.; Dang, Y.; Woodard, T. L.; Nevin, K. P.; Lovley,

490

D. R., Potential enhancement of direct interspecies electron transfer for syntrophic

491

metabolism of propionate and butyrate with biochar in up-flow anaerobic sludge blanket

492

reactors. Bioresource Technol 2016, 209, 148-156.

493

20. Kemper, J. M.; Ammar, E.; Mitch, W. A., Abiotic Degradation of hexahydro-1,3,5-

494

trinitro-1,3,5-triazine in the presence of hydrogen sulfide and black carbon. Environ

495

Science Technol 2008, 42, (6), 2118-2123.

496

21. Ahmad, M.; Lee, S. S.; Dou, X.; Mohan, D.; Sung, J.-K.; Yang, J. E.; Ok, Y. S., Effects

497

of pyrolysis temperature on soybean stover-and peanut shell-derived biochar properties and



Page 26 of 41

498

TCE adsorption in water. Bioresource Technol 2012, 118, 536-544.

499

22. Oleszczuk, P.; Jośko, I.; Kuśmierz, M., Biochar properties regarding to contaminants

500

content and ecotoxicological assessment. J Hazard Mater 2013, 260, 375-382.

501

23. Zhang, A.; Bian, R.; Pan, G.; Cui, L.; Hussain, Q.; Li, L.; Zheng, J.; Zheng, J.; Zhang,

502

X.; Han, X.; Yu, X., Effects of biochar amendment on soil quality, crop yield and

503

greenhouse gas emission in a Chinese rice paddy: A field study of 2 consecutive rice

504

growing cycles. Field Crop Res 2012, 127, 153-160.

505

24. Cao, X.; Ma, L.; Liang, Y.; Gao, B.; Harris, W., Simultaneous immobilization of lead

506

and atrazine in contaminated soils using dairy-manure biochar. Environ Sci Technol 2011,

507

45, (11), 4884-9.

508

25. Santin, C.; Doerr, S. H.; Merino, A.; Bucheli, T. D.; Bryant, R.; Ascough, P.; Gao, X.;

509

Masiello, C. A., Carbon sequestration potential and physicochemical properties differ

510

between wildfire charcoals and slow-pyrolysis biochars. Sci Rep 2017, 7, (1), 11233.

511

26. Li, X.; Shen, Q.; Zhang, D.; Mei, X.; Ran, W.; Xu, Y.; Yu, G., Functional groups

512

determine biochar properties (pH and EC) as studied by two-dimensional

513

correlation spectroscopy. PloS one 2013, 8, (6), e65949.

514

27. Zama, E. F.; Zhu, Y.-G.; Reid, B. J.; Sun, G.-X., The role of biochar properties in

515

influencing the sorption and desorption of Pb(II), Cd(II) and As(III) in aqueous solution. J

516

Clean Product 2017, 148, 127-136.

517

28. Kirchman, D. L.; Hanson, T. E., Cottrell, M. T.; Hamdan, L. J., Metagenomic analysis

518

of organic matter degradation in methane-rich Arctic. Limnol Oceanogr 2014, 59, (2), 548-


(13)C

NMR

Page 27 of 41


519

559.

520

29. Lovley, D. R.; Phillips, E. J., Novel mode of microbial energy metabolism: organic

521

carbon oxidation coupled to dissimilatory reduction of iron or manganese. Appl Environ

522

Microb 1988, 54, (6), 1472-1480.

523

30. Kato, S.; Hashimoto, K.; Watanabe, K., Methanogenesis facilitated by electric

524

syntrophy via (semi) conductive iron‐oxide minerals. Environ Microbiol 2012, 14, (7),

525

1646-1654.

526

31. Zhou, G.-W.; Yang, X.-R.; Li, H.; Marshall, C. W.; Zheng, B.-X.; Yan, Y.; Su, J.-Q.;

527

Zhu, Y.-G., Electron shuttles enhance anaerobic ammonium oxidation coupled to iron (III)

528

reduction. Environ Sci Technol 2016, 50, (17), 9298-9307.

529

32. Caporaso, J. G.; Lauber, C. L.; Walters, W. A.; Berg-Lyons, D.; Huntley, J.; Fierer,

530

N.; Owens, S. M.; Betley, J.; Fraser, L.; Bauer, M., Ultra-high-throughput microbial

531

community analysis on the Illumina HiSeq and MiSeq platforms. The ISME J 2012, 6, (8),

532

1621-1624.

533

33. Ding, L.-J.; Su, J.-Q.; Li, H.; Zhu, Y.-G.; Cao, Z.-H., Bacterial succession along a

534

long-term chronosequence of paddy soil in the Yangtze River Delta, China. Soil Biol

535

Biochem 2017, 104, 59-67.

536

34. Holmes, D. E.; Finneran, K. T.; O'neil, R. A.; Lovley, D. R., Enrichment of members

537

of the family Geobacteraceae associated with stimulation of dissimilatory metal reduction

538

in uranium-contaminated aquifer sediments. Appl Environ Microbiol 2002, 68, (5), 2300-

539

2306.



540

35. Kim, S.-Y.; Lee, S.-H.; Freeman, C.; Fenner, N.; Kang, H., Comparative analysis of

541

soil microbial communities and their responses to the short-term drought in bog, fen, and

542

riparian wetlands. Soil Biol Biochem 2008, 40, (11), 2874-2880.

543

36. Rotaru, A.-E.; Shrestha, P. M.; Liu, F.; Shrestha, M.; Shrestha, D.; Embree, M.;

544

Zengler, K.; Wardman, C.; Nevin, K. P.; Lovley, D. R., A new model for electron flow

545

during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the

546

reduction of carbon dioxide to methane. Energ Environ Sci 2014, 7, (1), 408-415.

547

37. Kan, T.; Strezov, V.; Evans, T. J., Lignocellulosic biomass pyrolysis: A review of

548

product properties and effects of pyrolysis parameters. Renew Sust Energ Rev 2016, 57,

549

1126-1140.

550

38. Trigo, C.; Cox, L.; Spokas, K., Influence of pyrolysis temperature and hardwood

551

species on resulting biochar properties and their effect on azimsulfuron sorption as

552

compared to other sorbents. Sci Total Environ 2016, 566-567, 1454-1464.

553

39. Shi, J.-L.; Du, W.-C.; Yin, Y.-X.; Guo, Y.-G.; Wan, L.-J., Hydrothermal reduction of

554

three-dimensional graphene oxide for binder-free flexible supercapacitors. J Mater Chem

555

A 2014, 2, (28), 10830-10834.

556

40. Zhao, Z.; Zhang, Y.; Quan, X.; Zhao, H., Evaluation on direct interspecies electron

557

transfer in anaerobic sludge digestion of microbial electrolysis cell. Bioresour Technol

558

2016, 200, 235-44.

559

41. Nevin, K. P.; Hensley, S. A.; Franks, A. E.; Summers, Z. M.; Ou, J.; Woodard, T. L.;

560

Snoeyenbos-West, O. L.; Lovley, D. R., Electrosynthesis of organic compounds from


Page 28 of 41

Page 29 of 41


561

carbon dioxide is catalyzed by a diversity of acetogenic microorganisms. Appl Environ

562

Microbiol 2011, 77, (9), 2882-6.

563

42. Park, H. S.; Kim, B. H.; Kim, H. S.; Kim, H. J.; Kim, G. T.; Kim, M.; Chang, I. S.;

564

Park, Y. K.; Chang, H. I., A novel electrochemically active and Fe(III)-reducing bacterium

565

phylogenetically related to Clostridium butyricum isolated from a microbial fuel cell.

566

Anaerobe 2001, 7, (6), 297-306.

567

43. Byrne-Bailey, K. G.; Wrighton, K. C.; Melnyk, R. A.; Agbo, P.; Hazen, T. C.; Coates,

568

J. D., Complete genome sequence of the electricity-producing “Thermincola potens” strain

569

JR. J Bacteriol 2010, 192, (15), 4078-4079.

570

44. Yuan, H.-Y.; Ding, L.-J.; Wang, N.; Chen, S.-C.; Deng, Y.; Li, X.-M.; Zhu, Y.-G.,

571

Geographic distance and amorphous iron affect the abundance and distribution of

572

Geobacteraceae in paddy soils in China. J Soil Sediment 2016, 16, (12), 2657-2665.

573

45. Lovley, D. R., Dissimilatory Fe (III) and Mn (IV) reduction. Microbiol Rev 1991, 55,

574

(2), 259-287.

575

46. Grobkopf, R.; Janssen, P. H.; Liesack, W., Diversity and structure of the methanogenic

576

community in anoxic rice paddy soil microcosms as examined by cultivation and direct

577

16S rRNA gene sequence retrieval. Appl Environ Microbiol 1998, 64, (3), 960-969.

578

47. Leclerc, M.; Delgenes, J. P.; Godon, J. J., Diversity of the archaeal community in 44

579

anaerobic digesters as determined by single strand conformation polymorphism analysis

580

and 16S rDNA sequencing. Environ Microbiol 2004, 6, (8), 809-19.

581

48. Lovley, D. R.; Giovannoni, S. J.; White, D. C.; Champine, J. E.; Phillips, E. J. P.;



582

Gorby, Y. A.; Goodwin, S., Geobacter metallireducens gen. nov. sp. nov., a microorganism

583

capable of coupling the complete oxidation of organic compounds to the reduction of iron

584

and other metals. Arch Microbiol 1993, 159, (4), 336-344.

585

49. Li, H.; Chang, J.; Liu, P.; Fu, L.; Ding, D.; Lu, Y., Direct interspecies electron transfer

586

accelerates syntrophic oxidation of butyrate in paddy soil enrichments. Environ Microbiol

587

2015, 17, (5), 1533-1547.

588

50. Kato, S.; Nakamura, R.; Kai, F.; Watanabe, K.; Hashimoto, K., Respiratory

589

interactions of soil bacteria with (semi) conductive iron‐oxide minerals. Environ Microbiol

590

2010, 12, (12), 3114-3123.

591

51. Zhang, P.; Zheng, S.; Liu, J.; Wang, B.; Liu, F.; Feng, Y., Surface properties of

592

activated sludge-derived biochar determine the facilitating effects on Geobacter co-

593

cultures. Water Res 2018, 142, 441-451.

594

52. Roden, E. E.; Kappler, A.; Bauer, I.; Jiang, J.; Paul, A.; Stoesser, R.; Konishi, H.; Xu,

595

H. F., Extracellular electron transfer through microbial reduction of solid-phase humic

596

substances. Nat Geosci 2010, 3, (6), 417-421.

597

53. Van der Zee, F. P.; Bisschops, I. A. E.; Lettinga, G.; Field, J. A., Activated carbon as

598

an electron acceptor and redox mediator during the anaerobic biotransformation of azo

599

dyes. Environ Sci Technol 2003, 37, (2), 402-408.

600

54. Tong, H.; Hu, M.; Li, F. B.; Liu, C. S.; Chen, M. J., Biochar enhances the microbial

601

and chemical transformation of pentachlorophenol in paddy soil. Soil Biol Biochem 2014,

602

70, 142-150.


Page 30 of 41

Page 31 of 41


603

55. Klüpfel, L.; Keiluweit, M.; Kleber, M.; Sander, M., Redox properties of plant biomass-

604

derived black carbon (biochar). Environ Sci Technol 2014, 48, (10), 5601-5611.

605

56. Sun, T.; Levin, B. D.; Guzman, J. J.; Enders, A.; Muller, D. A.; Angenent, L. T.;

606

Lehmann, J., Rapid electron transfer by the carbon matrix in natural pyrogenic carbon. Nat

607

Commun 2017, 8, 14873.

608

57. Xu, J.; Zhuang, L.; Yang, G.; Yuan, Y.; Zhou, S., Extracellular quinones affecting

609

methane production and methanogenic community in paddy soil. Microb Ecol 2013, 66,

610

(4), 950-60.

611

58. Woolf, D.; Amonette, J. E.; Street-Perrott, F. A.; Lehmann, J.; Joseph, S., Sustainable

612

biochar

613

DOI:10.1038/ncomms1053.

to

mitigate

global

climate

change.

Nature


commun

2010,

1:56,


615

Table 1 Physical and Chemical Properties of Biochars Original

Pyrolysis temperature

Particle size

feedstock

(ºC)

(mm)

RB

rice straw

600 for 2h

≤2

10.40±0.02a

38.8±0.99a

28.53±0.86a 56.42±0.09b 2.10±0.02b

WB

wood chips

600 for 2h

≤2

9.60±0.03c

14.75±0.95b

2.25±0.06b

83.31±0.15a 2.89±0.03a

MB

manure

600 for 2h

≤2

10.24±0.06b

16.91±0.73b

2.77±0.07b

17.09±0.62c 0.30±0.00c

Biochar

616 617

Page 32 of 41

pH

Different superscript letters indicate significant differences ( P< 0.05). Mean ± SD


BET surface area

EC

(m2/g)

(µS/cm)

C (%)

H (%)

Page 33 of 41


618

Figure captions

619

Figure 1. Effects of biochar on methanogenic activities of paddy soil microbial

620

communities. (a) time-courses of methane concentrations in the enrichment cultures with

621

ethanol as a substrate in the absence or presence of biochar; (b) the maximum

622

methanogenic rates estimated from curves in (a). Methane concentrations are expressed

623

as mM by assuming that methane was present in the aqueous phase. Different letters

624

above the bar represent a significant (P < 0.05) difference in the maximum methanogenic

625

rates among different treatments. Data are presented as the means of three independent

626

experiments, and error bars represent standard deviations

627

Figure 2. Scanning electron microscopy images of the three used biochars. (a) (Rice

628

Straw Biochar) RB, (b) (Wood Chips Biochar) WB, (c) (Manure Biochar) MB

629

Figure 3. (a) Stacked fourier transform infrared spectroscopy (FTIR) spectra of (Rice Straw

630

Biochar) RB, (Wood Chips Biochar) WB and (Manure Biochar) MB; (b) charging and

631

discharging cycles. Cyclic voltammograms of immobilized surface functional groups at a

632

graphite working electrode. Scan rate = 50 mV s-1

633

Figure 4. X-ray photoelectron microscopy (XPS) analysis. (a) XPS spectra of (Rice Straw

634

Biochar) RB; (b) C1s spectrum of RB; (c) XPS spectra of (Wood Chips Biochar) WB; (d)

635

C1s spectrum of WB; (e) XPS spectra of (Manure Biochar) MB; (f) C1s spectrum of MB

636

Figure 5. (a) Copy numbers of 16S rRNA genes of Geobacteraceae in enrichment

637

cultures with ethanol as substrate. Error bars indicate the standard deviation of three

638

replicates; (b) Suppression ratio of the growth of Geobacteraceae in the enrichment

639

cultures with BES compared without BES; (c) Relationship between gene copies of mcrA

640

per mL cells and 16S rRNA gene copies of Geobacteraceae per mL cells



641

Figure 6. Redundancy analysis (RDA) compares microbial community composition from

642

enrichment culture systems with different types of biochars and variables, including O/C,

643

maximum rate of methane production (Max. rate) and capacitive current. (a) bacteria; (b)

644

archaea.

645

Figure 7. Relative abundances of the 10 and 6 most abundant taxa at genus level based on

646

16S rRNA gene sequence data in the enrichment cultures with or without amendments of

647

biochar; (a) bacteria; (b) archaea. The relative abundance is expressed as the percentage of

648

the targeted sequences to the total high-quality sequences of each sample.

649


Page 34 of 41

Page 35 of 41


261x232mm (300 x 300 DPI)



174x360mm (300 x 300 DPI)


Page 36 of 41

Page 37 of 41


38x17mm (600 x 600 DPI)



121x173mm (600 x 600 DPI)


Page 38 of 41

Page 39 of 41


76x68mm (600 x 600 DPI)



177x371mm (600 x 600 DPI)


Page 40 of 41

Page 41 of 41


100x119mm (600 x 600 DPI)


Biochar Modulates Methanogenesis through Electron Syntrophy of

Recommend Documents