Response of methanogen community to elevation of cathode … · 2020. 11. 24. · 4 Running title:...

1

Response of methanogen community to elevation of cathode 1

potentials in the presence of magnetite 2

3

Running title: Electromethanogenesis of paddy soil community 4

5

Kailin Gao, † Xin Wang,

‡ Junjie Huang,

† Xingxuan Xia,

† Yahai Lu

*, † 6

7

†College of Urban and Environmental Sciences, Peking University, Beijing, 100871, 8

China 9

‡College of Environmental Science and Engineering, Nankai University, Tianjin 10

300350, China 11

12

*Corresponding author: 13

Yahai Lu, Peking University, College of Urban and Environmental Sciences, No. 5, 14

Yiheyuan Road, Haidian District, Beijing 100871, China. 15

Phone/fax: 0086 10 62750669 16

E-mail address: [email protected] (Y. Lu) 17

18

Keywords Magnetite; Electromethanogenesis; Methanogens; Paddy soil; 19

Methanospirillum 20

21

preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.24.397190doi: bioRxiv preprint

https://doi.org/10.1101/2020.11.24.397190

2

ABSTRACT 22

Electromethanogenesis refers to the process where methanogens utilize electrons 23

derived from cathodes for the reduction of CO2 to CH4. Setting of low cathode 24

potentials is essential for this process. In this study, we test if magnetite, an iron oxide 25

mineral widespread in environment, can facilitate the adaption of methanogen 26

community to the elevation of cathode potentials in electrochemical reactors. 27

Two-chamber electrochemical reactors were constructed with inoculants obtained 28

from a paddy field soil. We elevated cathode potentials stepwise from the initial -0.6 29

V vs standard hydrogen electrode (SHE) to -0.5 V and then to -0.4 V over the 120 30

days acclimation. Only weak current consumption and CH4 production were observed 31

in the reactors without magnetite. But biocathodes were firmly developed and 32

significant current consumption and CH4 production were recorded in the magnetite 33

reactors. The robustness of electro-activity in the magnetite reactors was not affected 34

with the elevation of cathode potentials from -0.6 V to -0.4 V. But, the current 35

consumption and CH4 production were virtually halted in the reactors without 36

magnetite when cathode potential was elevated to -0.4 V. Methanogens related to 37

Methanospirillum were enriched on cathode surface of the magnetite reactors at -0.4 38

V, while Methanosarcina relatively dominated in the reactors without magnetite. 39

Methanobacterium also increased in the magnetite reactors but stayed off electrodes 40

in the culture medium at -0.4 V. Apparently, magnetite greatly facilitates the 41

development of biocathodes, and it appears that with the aid of magnetite 42


https://doi.org/10.1101/2020.11.24.397190

3

Methanospirillum spp. can adapt to high cathode potentials performing the efficient 43

electromethanogenesis. 44

45

46

IMPORTANCE 47

Converting CO2 to CH4 through bioelectrochemistry is a promising approach for 48

development of green energy biotechnology. This process however requires setting 49

the low cathode potentials, which takes cost. In this study, we test if magnetite, a 50

conductive iron mineral, can facilitate the adaption of methanogens to the elevation of 51

cathode potentials. In the two-chamber reactors constructed using inoculants obtained 52

from a paddy field soil, biocathodes were firmly developed in the presence of 53

magnetite, whereas only weak electro-activity was observed in the reactors without 54

magnetite. The elevation of cathode potentials did not affect the robustness of 55

electro-activity in the magnetite reactors over the 120 days acclimation. 56

Methanospirillum was identified as the key methanogens associated with cathode 57

surface during the operation at relatively high potentials. The findings reported in this 58

study shed a new light on the adaption of methanogen community to the elevated 59

cathode potentials in the presence of magnetite. 60

61


https://doi.org/10.1101/2020.11.24.397190

4

INTRODUCTION 62

Bioelectrochemical technology has been developed rapidly in recent decades with one 63

of attracting applications being the conversion of CO2 to CH4 (1-3). 64

Electromethanogenesis refers to the process where methanogens utilize electrons 65

derived from cathodes for the reduction of CO2 to CH4 (4). Two plausible 66

mechanisms have been proposed for electron transfer from cathodes to methanogens. 67

One is the H2-mediated electron transfer, which assumes that H2 is electrochemically 68

produced either abiotically or biotically that is then used by hydrogenotrophic 69

methanogens for reduction of CO2 to CH4 (5, 6) and the other, though not fully 70

proven, is the direct electron transfer from cathodes to methanogens (7-10). 71

Cathode potential is the critical factor controlling either chemical or biological H2 72

evolution. Theoretically, chemical H2 production from proton reduction can occur at 73

-0.414 V (all potentials reported here are relative to standard hydrogen electrode, 74

SHE), but in practice the cathode potential must be set substantially lower due to the 75

overpotential in electrochemical operation (4, 11-13). Consequently, cathode 76

potentials of -0.5 V to -0.8 V were usually applied in electromethanogenic reactors 77

where H2-mediated electron transfer was assumed as the key process (4, 14-16). H2 78

evolution can occur through different bioelectrochemical mechanisms. For instances, 79

it has been proposed that the high rate of hydrogen-mediated electromethanogenesis 80

in Methanococcus maripaludis was due to the release of hydrogenases from living 81

and dead cells, which attached to cathode surface and catalyzed H2 production (5). In 82


https://doi.org/10.1101/2020.11.24.397190

5

mixed culture electrochemical systems, organisms other than methanogens may 83

produce H2 by taking up cathode electrons directly or indirectly and then channel H2 84

to hydrogenotrophic methanogens for CO2 reduction to CH4 (6). In a defined 85

coculture it was demonstrated that the Fe(0)-corroding sulfate-reducing strain IS4 86

performed direct cathode electron uptake during sulfate reduction with active H2 87

production at -0.4 V and -0.6 V, and H2 was consumed by M. maripaludis for CO2 88

reduction to CH4 (17). 89

Methane production from CO2 reduction can occur at a redox potential of -0.244 V 90

under standard conditions. A few studies have suggested that some methanogens can 91

operate the direct electron uptake from cathodes for catabolic metabolism, thus 92

bypassing the H2-mediated processes. In this case it becomes less critical to apply a 93

low potential as compared to the H2-dependent reactors. For instances, a 94

marine-origin Methanobacterium-like strain IM1, which grew on iron specimen but 95

hardly on H2, could sustain electromethanogenesis at -0.4 V, while the typical 96

hydrogenotrophic M. maripaludis failed the operation at this potential (18). Similarly, 97

Methanosarcina barkeri, a methanogen known to conduct direct interspecies electron 98

transfer (DIET) with Geobacter metallireducens, can perform electromethanogenesis 99

at -0.4 V (9, 10, 19). The hydrogenase-independent electron uptake by the M. barkeri 100

mutant lacking hydrogenases has been observed at a potential of -0.484 V (8). 101

Therefore, while H2-mediated electromethanogenesis generally requires low cathode 102


https://doi.org/10.1101/2020.11.24.397190

6

potentials, H2-independent or DET-associated electromethanogenesis can work at 103

relatively high potentials. 104

The performance of electromethanogenesis could be improved by introducing 105

supplemental materials that are optimized for cathode electron transfer or that can 106

efficiently catalyze H2 evolution at low potentials (20-22). Iron oxide minerals such as 107

magnetite are common in soils (23-27). Recently, it has been demonstrated that the 108

presence of magnetite nanoparticles (MNP) greatly promotes methanogenesis from 109

oxidation of short-chain fatty acids in rice paddy soils and anaerobic sludges, and the 110

stimulatory effect has been attributed to the facilitation of DIET (25, 27-30). 111

Magnetite is a mixed-valent iron oxide mineral containing Fe(II) and Fe(III) in a ratio 112

of 1:2. The edge-sharing of the octahedral Fe(II) and Fe(III) in magnetite facilitates 113

the electron hopping or rapid electron exchange along the octahedral sublattice 114

resulting in high electrical conductivity and redox activity (23, 31). It is tempting to 115

explore the influence of MNP on electromethanogenesis. 116

Previous studies on electromethanogensis often collected inoculants from anaerobic 117

digesters. Anaerobic digesters specialized in industry may limit the diversity of 118

methanogens (1, 14, 15, 21, 22, 32), while natural environments such as paddy soil or 119

wetlands can harbor a wider variety of methanogens (33) and hence offer an 120

opportunity to screen methanogens capable of efficient electromethanogenesis. In the 121

present study, electromethanogenic reactors were constructed using an inoculum 122

obtained from a rice paddy soil. To test the effect of magnetite and explore how 123


https://doi.org/10.1101/2020.11.24.397190

7

methanogens response to varying cathode potentials, the cathode potentials of reactors 124

were elevated stepwise from -0.6 V to -0.5 V and finally to -0.4 V. Over four months 125

of electromethanogenesis acclimation, we continuously monitored the CH4 126

production, the electrochemical performance and the microbial population dynamics. 127

Systematic analysis of the combined data about the adaption of methanogens to the 128

increasing cathode potential with and without magnetite sheds the new light on 129

electro-active methanogens community originated from paddy soil. 130

131

RESULTS 132

Magnetite promoted electromethanogenesis 133

Three batches of reactors were constructed, namely the reactors with MNP (MNP 134

reactors), the reactors without MNP (no-MNP reactors), and the reactors without 135

inoculants but with MNP (the abiotic MNP control). All reactors were operated 136

continuously except two breaks. In the initial phase, cathode potential was set at -0.6 137

V. Neither current consumption nor CH4 production were detected in the abiotic 138

control. For the inoculated reactors, electromethanogenesis initiated after over a 139

month acclimation. Magnetite exerted a strong influence. In the MNP reactors, rapid 140

current consumption was detected at 33 d with concomitant sharp increase of CH4 141

production at 40 d (Fig. 2). The current consumption and CH4 production, however, 142

were much lower in the no-MNP reactors where obvious electromethanogenesis 143

occurred only after 50 d. When the current consumption in the MNP reactors did not 144


https://doi.org/10.1101/2020.11.24.397190

8

increase further, the first break was applied. After the CV tests of cathodes and the 145

exchange of catholytes with fresh culture medium, the circuits were reconnected and 146

cathode potentials were elevated to -0.5 V for the second stage of operation. Methane 147

production and current consumption occurred without delay in the MNP reactors, 148

indicating that microbial populations retained on electrode surfaces (microbes in the 149

catholytes were disposed during medium exchange) were well adapted to the 150

electrochemical environment (Fig. 2). The current consumption and CH4 production 151

also occurred in the no-MNP reactors, but the activity remained much lower as 152

compared with the MNP reactors. The total CH4 accumulation was 22.68 mmol L-1

in 153

the MNP reactors and 7.71 mmol CH4 L-1

in the no-MNP reactors, respectively, over 154

the period at -0.5 V operation (Fig. 2a). When the current consumption stopped 155

increasing again, the second break was implemented. After the CV tests of cathodes 156

and culture medium exchange repeated, the cathode potentials were elevated to -0.4 V 157

for the third stage of operation. The current consumption and CH4 production kept 158

highly active in the MNP reactors. The total CH4 accumulation over 30 days 159

operation actually exceeded those observed in the earlier stages at -0.6 V and -0.5 V. 160

However, CH4 production and current consumption in the no-MNP reactors were 161

virtually halted at -0.4 V (Fig. 2). 162

H2 accumulated to 0.02 mmol L-1

in the abiotic control during the initial operation at 163

-0.6 V. H2 was occasionally detected in the MNP reactors at concentrations close to 164

detection limit (0.01 mmol L-1

). Otherwise H2 concentrations were below the 165


https://doi.org/10.1101/2020.11.24.397190

9

detection limit in most cases, especially during the operations at -0.5 V and -0.4 V. To 166

verify if addition of MNP influenced redox potentials (Eh) in culture medium, we 167

measured Eh under open circuit conditions. The results showed no difference in the 168

presence (-0.340 V) and absence (-0.338 V) of MNP. 169

At each break and at the end of experiment, CV tests were conducted to evaluate the 170

catalytic activity of cathodes. At the break after -0.6 V operation, the no-MNP 171

reactors revealed the lowest redox activity of cathodes, followed by the abiotic 172

reactors while the highest redox activity was recorded for the MNP reactors (Fig. 3a). 173

The activity of the abiotic reactors indicates that addition of MNP improved the 174

conductivity of electrodes and electrolytes compared to the inoculated reactors 175

without MNP. CV tests at the break after -0.5 V operation and at the end of 176

experiment exhibited similar results and confirmed the significantly enhanced 177

catabolic activity of cathodes in the MNP reactors compared with the no-MNP 178

reactors (Fig. 3b, c). The voltammograms showed some distinct redox peaks and 179

inflection points in the current profiles (Fig. 3a-c). For instances, at the break after 180

-0.6 V operation, the current generation in the MNP reactors plateaued in the potential 181

range from -0.23 V to -0.51 V, followed by an increase and then flattened again at 182

-0.6 V. The inflection point occurred at -0.48 V for the MNP reactors at the break 183

after -0.5 V operation and at -0.41 V after -0.4 V operation. These reflection points 184

illustrated the robust development of biocathodes in the MNP reactors. 185

Response of archaeal and bacterial communities 186


https://doi.org/10.1101/2020.11.24.397190

10

Amplicon sequencing was used to analyze community compositions of archaea and 187

bacteria attached on the cathode surfaces and living in the culture mediums (catholyte 188

solution). The sequence summary for all archaea amplicons was given in Table S1. 189

Archaea were composed exclusively of methanogen populations. Methanogens in the 190

original inoculum obtained from rice paddy soil were dominated by Methanospirillum 191

accounting for 90% of total archaeal sequences (Fig. 4a). The rest mainly affiliated to 192

Methanosarcina and Methanoregula. Over the operation of electromethanogenesis, 193

Methanobacterium replaced Methanoregula, arising as the third dominant 194

methanogen (Fig. 4a; Fig. S1). The methanogen compositions in reactors were 195

markedly influenced by the magnetite treatment, the potential elevation and the 196

sample location (cathode surface vs catholyte medium). 197

In the no-MNP reactors, the relative abundance of Methanospirillum and 198

Methanobacterium decreased, while that of Methanosarcina increased from 15% to 199

60% with the elevation of cathode potentials from -0.6 V to -0.4 V (Fig. 4a). There 200

was no significant difference in methanogen composition between the cathode surface 201

and the culture medium (Fig. 4a). The MNP reactors showed a significantly different 202

pattern. After the operation at -0.6 V, Methanospirillum remained dominant while 203

Methanobacterium slightly increased compared with the original inoculum. When 204

cathode potential was shifted to -0.5 V, Methanobacterium greatly increased and 205

surpassed Methanospirillum both on cathode surface and in catholyte medium. 206

Methanosarcina also increased relatively in the culture medium. When the cathode 207


https://doi.org/10.1101/2020.11.24.397190

11

potential was further elevated to -0.4 V, Methanospirillum returned as the most 208

dominant methanogen on cathode surface while Methanobacterium kept dominant 209

only in catholyte medium (Fig. 4a). Apparently, Methanospirillum were selected 210

against Methanobacterium and Methanosarcina on cathode surfaces at -0.4 V (Fig. 211

4a). 212

The shift of methanogen community was also illustrated by the nonmetric 213

multidimensional scale (NMDS) analysis (Fig. 4b). Methanogen communities under 214

electromethanogenesis were all distinct from original inoculum. At the break after 215

-0.6 V operation, communities from all samples were clustered (green oval). At the 216

break after -0.5 V operation, communities from the MNP and no-MNP reactors were 217

separated (two purple ovals), while at the end after -0.4 V operation, a more 218

significant divergence was revealed (three red ovals), with the separations not only 219

depending on magnetite treatments but also on sample sites (cathode surface vs 220

culture medium). 221

The phylogenetic relationship of top 10 archaeal OTUs was depicted in Fig. 5a. 222

Clone_Mspi were closely related to Methanospirillum psychrodurum X-18 and 223

Methanospirillum lacunae Ki8-1. Clone_Mba1 and clone_Mba2 were related to 224

Methanobacterium formicicum MF and Methanobacterium flexile GH. Clone_Msar 225

was related to Methanosarcina horonobensis HB-1 and Methanosarcina mazei DSM 226

2053. Given that Methanospirillum were predominant and selected by cathode surface 227

in the MNP reactors at -0.4 V, we further analyzed the relative abundances of 15 228


https://doi.org/10.1101/2020.11.24.397190

12

OTUs affiliated to Methanospirillum (Fig. 5b). A significant shift was revealed for 229

these Methanospirillum phylotypes. Clone_Mspi2 was dominated in the original 230

inoculum. At the first break after -0.6 V operation, Clone_Mspi1, 4 and 14 increased, 231

while clone_Mspi2 dropped. At the second break after -0.5 V operation, all the 232

Methanospirillum OTUs declined. But at the end after -0.4 V operation, ten other 233

OTUs including clone_Mspi3, 6-13 and 15 were substantially enriched (Fig. 5b). 234

These results implied that clone_Mspi3, 6-13 and 15 instead of the dominant 235

clone_Mspi2 in the original inoculum were enriched during electromethanogenesis at 236

-0.4 V. 237

The sequence summary for the bacteria amplicons was listed in Table S2. Bacteria 238

community consisted mainly of Acetobacterium, Anaerolineaceae, Sulfuricurvum, 239

Geobacter, Bacteroidales, and Desulfovibrio (Fig. S2). Acetobacterium and 240

Anaerolineaceae, the first and second most abundant bacteria across reactors, were 241

more enriched in the MNP reactors compared to the no-MNP reactors (Fig. 6). By 242

comparison, the other four bacteria mentioned above exhibited higher relative 243

abundances in the no-MNP reactors than in the MNP reactors. The relative abundance 244

of Acetobacterium however decreased sharply with the elevation of cathode potentials, 245

from 54% to 10% in the MNP reactors and from 32.5% to 0.06% in the no-MNP 246

reactors, respectively. Though relatively higher abundances of Acetobacterium on 247

cathode surface than in catholyte medium of the MNP reactors at -0.6 V and -0.5 V, 248

this difference was diminished at -0.4 V (Fig. 6). Anaerolineaceae showed the 249


https://doi.org/10.1101/2020.11.24.397190

13

opposite tendency. Their relative abundances were low at -0.6 V and -0.5 V, but 250

markedly increased with the elevation of cathode potentials to -0.4 V where they were 251

the most dominant bacteria both on cathode surface and catholyte medium. 252

Actinobacteria showed low relative abundance across all samples except on cathode 253

surface in the MNP-reactors at -0.4 V where they became the second dominant 254

bacteria after Anaerolineaceae. The NMDS analysis revealed that the samples from 255

the MNP reactors at -0.6 and -0.5 V formed a cluster while those at -0.4 V formed 256

another cluster (Fig. S4). The samples from the no-MNP reactors were separated into 257

three clusters depending on cathode potentials and sample locations. 258

259

DISCUSSION 260

In the present study we demonstrated that electromethanogenesis derived from a 261

paddy soil community was substantially promoted by magnetite during the 120 days 262

operation with the elevation of cathode potentials from -0.6 V to -0.4 V (Fig. 2). 263

Granular active carbon and zero-valent iron have been employed to accelerate the 264

start-up of biocathodes and upgrade electromethanogenesis efficiency (20-22). One 265

such a study however showed that electromethanogenesis was not affected by 266

magnetite amendment in comparison with magnetite-free reactors (20). Thus, the 267

stimulating effect appears dependent on reactor conditions and especially microbial 268

identities in operations (34). Nevertheless, it has been demonstrated that in natural 269

systems the syntrophic oxidation of short chain fatty acids and CH4 production were 270


https://doi.org/10.1101/2020.11.24.397190

14

significantly stimulated by magnetite nanoparticles (25-27, 35). Magnetite was also 271

shown to facilitate syntrophic interaction between a phototrophic bacterium 272

Rhodopseudomonas palustris and an iron-reducing Geobacter sulfurreducens (36, 273

37). More recently, it was demonstrated that magnetite accelerated the aceticlastic 274

methanogenesis by a pure culture Methanosarcina mazei zm-15 and its corresponding 275

environment enrichment (38). The mechanisms are considered to be related to DIET 276

in syntrophic interaction (25) or serving as an environmental battery facilitating 277

electron transfer among microbes (37). In the present experiment, we lifted cathode 278

potentials from the initial -0.6 V to the final -0.4 V over the 120 days acclimation. 279

The electromethanogenesis remained robust in the presence of magnetite whereas the 280

activity was substantially inhibited in the reactors without magnetite. Albeit mixture 281

community in our reactors hindered the elucidation of exact mechanism, we assume 282

that magnetite amendment facilitated the electron transfer from cathodes to 283

methanogens either directly or indirectly. 284

Microbial communities in our reactors were markedly influenced by the magnetite 285

treatment, the potential elevation and the sample location (Fig. 4, Fig. 6 and Fig. 286

S3-S4). In the no-MNP reactors, the relative abundance of Methanosarcina gradually 287

increased with the elevation of cathode potentials (Fig. 4a). Methanosarcina barkeri 288

and Methanosarcina mazei have been demonstrated to be capable of DIET with 289

Geobacter metallireducens or perform electromethanogenesis at -0.4 V (8-10, 19). It 290

is possible that Methanosarcina spp. contributed to electromethanogenesis in the 291


https://doi.org/10.1101/2020.11.24.397190

15

no-MNP reactors albeit their weak electrochemical performance. The relative 292

abundance of Methanosarcina, however, substantially declined in the reactors with 293

magnetite. Apparently, Methanosarcina did not respond to the stimulatory effect of 294

magnetite or were less competitive during the development of biocathodes in the 295

MNP reactors. Methanobacterium on the other hand significantly increased in the 296

reactors with magnetite, especially at -0.5 V, reaching the relative abundance of about 297

50% on cathode surface as well as in catholyte medium. These methanogens however 298

wane from cathode surface when cathode potentials were further elevated to -0.4 V. 299

As the replacement, Methanospirillum were dominated on cathode surface at -0.4 V. 300

The concomitant CH4 production and current consumption indicated that biocathodes 301

were well developed in the MNP reactors (Fig. 2). The facts that the culture medium 302

exchange at two breaks and the elevation of cathode potentials from -0.6 V to -0.4 V 303

did not influence the electrochemical performance indicate that the biocathodes 304

remained robust in the MNP reactors over the 120 days operation. This robustness of 305

biocathode development was further illustrated with the CV tests at two breaks and at 306

the end of experiment (Fig. 3). Accordingly, we assumed that microbial populations 307

associated with cathode surface played the key role for electromethanogenesis in the 308

MNP reactors. Many of previous studies have found that Methanobacterium were 309

enriched in the cathode chambers of electrosynthesis reactors (13-16, 39). We show 310

here that Methanospirillum were dominated on cathode surface at -0.4 V in the 311

presence of magnetite (Fig. 4a). Methanospirillum have been underappreciated in the 312


https://doi.org/10.1101/2020.11.24.397190

16

previous studies on electromethanogenesis. Recently, it has been reported that the 313

archaellum of Methanospirillum hungatei is electrically conductive, making it an 314

excellent candidate for the research of extracellular electron transfer in archaea and 315

the application in electrochemical systems (40). It shall warrant a further investigation 316

for whether the Methanospirillum in our reactors contain the electrically conductive 317

archaellum, which facilitates the extracellular electron transfer in the biocathode 318

ecosystems. 319

For the mixed culture reactors, the accompanying bacteria can play important roles in 320

electromethanogenesis. Electro-acetogenesis refers to biological production of acetate 321

from CO2 with electrons derived from cathodes (41, 42). Acetobacterium was the 322

most dominant bacteria across all samples and were relatively enriched in the MNP 323

reactors (Fig. 6), indicating their possible involvement in electro-acetogenesis. But the 324

relative abundance of these acetogens significantly declined in the reactors at -0.4 V, 325

suggesting they were not tolerant to the elevation of cathode potentials. The role of 326

bacteria in the electromethanogenic reactors can be multifold. Firstly, acetogens 327

produce acetate electrochemically which is then used by aceticlastic methanogens like 328

Methanosarcina in the reactors. Second, some electrically-active bacteria may 329

generate H2 by taking up electrons from electrodes and then channel H2 to 330

hydrogenotrophic methanogens, forming syntrophic interaction (17). Third, it is 331

plausible that DIET is established between the electrically-active bacteria and 332

methanogens without the involvement of intermediate H2 production. Except 333


https://doi.org/10.1101/2020.11.24.397190

17

Acetobacterium, some of potentially electrically-active organisms like Geobacter, 334

Sulfuricuvum and Desulfovibrio were present as major bacteria members (Fig. S2). 335

These organisms however exhibited relatively high abundances only in the no-MNP 336

reactors, and thus did not play a significant role in the MNP reactors. 337

Anaerolineaceae were the second dominant bacteria next to Acetobacterium (Fig. 6). 338

These bacteria were significantly enriched in the MNP reactors at -0.4 V. 339

Anaerolineaceae species have long filamentous structure (43, 44), and hence have an 340

advantage of forming microbial aggregates or biofilms by attaching to electrode 341

surface. Some Anaerolineaceae are known to ferment sugars and grow better in 342

coculture with H2-consuming methanogens (44), implying their syntrophic life style. 343

Therefore, it is plausible that Anaerolineaceae are involved in electromethanogenesis 344

through forming syntrophic associations or improving methanogenic aggregate 345

formation in the present experiment. 346

In summary, this study demonstrates that MNP greatly facilitates the adaption of 347

methanogens in electrochemical reactors with the stepwise elevation of cathode 348

potentials from -0.6 V to -0.4 V. Methanospirillum was identified as the key 349

methanogens associated with cathode surface at -0.4 V. Given that the archaellum of 350

Methanospirillum hungatei is known to be electrically conductive, it is worthwhile to 351

further explore if Methanospirillum can perform direct electron transfer in the 352

electrochemical reactors. Though a set potential of -0.4 V is considered to be 353

sufficiently high to limit electrochemical H2 production under pure culture conditions 354


https://doi.org/10.1101/2020.11.24.397190

18

(1, 8-10, 18), we are not able to dissect if the H2-mediated or H2-independent 355

methanogenesis prevails in our reactors. Further research is necessary to determine if 356

syntrophy is involved in electromethanogenesis, in which some bacteria may fetch 357

electrons from electrodes forming H2 which is then utilized by the hydrogenotrophic 358

Methanospirillum and Methanobacterium. It also remains unclear if the redox-active 359

enzymes including hydrogenases are released from living and dead cells, which are 360

then deposited on electrode surface facilitating H2-mediated processes (5, 45). These 361

open questions await a further research in the future. 362

363

MATERIALS AND METHODS 364

Enrichment preparation for cathode inoculation 365

To focus on methanogen community and exclude carbon sources from soil, 366

methanogenic enrichment from a rice paddy soil was prepared as below. The 367

water-saturated soil samples were collected from a paddy field located in the 368

northeastern China close to Heihe city of Heilongjiang province, China (127.36°E, 369

49.90°N). Four successive transfers of anaerobic incubation were conducted. For the 370

first transfer, fresh soil samples were suspended in autoclaved degassed water at a 371

water to soil ratio of 5:1 (soil mass in dry weight). Aliquots (50 ml) of the 372

homogenized soil slurry were then dispensed into 125 ml sterile serum bottles. 373

Glucose was added into bottles at a final concentration of 5 mM in slurries. 374

Headspace of serum bottles were flushed with N2 thoroughly. All the bottles were 375


https://doi.org/10.1101/2020.11.24.397190

19

sealed with butyl stoppers and aluminum crimp caps, and put in the dark at room 376

temperature (27 ℃). When the rate of daily CH4 production reached to the 377

quasi-steady state, the soil slurries were used as inoculum (5% v/v) for the next three 378

transfers where acetate (10 mM) was spiked into 50 ml HEPES-buffered (30 mM, pH 379

7) culture medium and the headspace of serum bottles were flushed with H2/CO2 380

(80:20; v/v). Both acetate and H2/CO2 served as the carbon and energy source for 381

methanogen enrichment (Fig. 1). The basal medium consisted of MgCl2·6H2O (0.4 382

g/L), CaCl2·2H2O (0.1 g/L), NH4Cl (0.1 g/L), KH2PO4 (0.2 g/L), KCl (0.5 g/L). 383

Supplements of vitamin, and trace element solutions were applied. Resazurin (46) and 384

cysteine were omitted to avoid the possible effect of electron shuttle molecules (47). 385

All the bottles for the enrichment cultivation were sealed and put in the dark at room 386

temperature (27 ℃). When acetate was used up in the medium, the enrichment 387

cultures were used to inoculate (10% v/v) the cathode chambers of bioelectrochemical 388

reactors. Abiotic control reactors were established without inoculation. 389

Setup of bioelectrochemical systems 390

The bioelectrochemical reactors consisted of two chambered borosilicate gastight 391

H-type microbial electrolysis cells, in which the 250 ml anode and cathode chambers 392

were separated by a Nafion 117 proton exchange membrane (surface area 5 cm2, 393

DuPont, Wilmington, DE, USA) (Fig. 1). Prior to use, membranes were successively 394

treated one hour each with H2O2 (3.5 wt.%), distilled water, H2SO4 (5 wt.%), and 395

distilled water. In each chamber, there are an upper and a lower openings, which were 396


https://doi.org/10.1101/2020.11.24.397190

20

sealed with butyl stoppers and aluminum crimp caps. The upper openings were used 397

to collect gas samples (Fig. 1). Carbon fiber brush (volume 20 cm3) acted as the work 398

electrode in the cathode chambers and platinum foil was used as the counter electrode 399

in the anode chambers. An Ag/AgCl reference electrode (+0.2046 V vs SHE, 25℃) 400

was placed in the cathode chamber as a reference electrode. Magnetite nanoparticles 401

(MNP) was synthesized via a conventional aqueous co-precipitation method (48) as 402

described previously (27). 403

Bioelectrochemical measurements and cathode potential elevation 404

Nitrogen was flushed up to one hour to make the anaerobic condition. After 405

autoclaving, the anaerobic basal medium was added into two chambers of reactors. 406

The composition of basal medium was same as the enrichment cultivation but without 407

the addition of acetate. 125 ml medium was dispensed into anode chambers. And 408

112.5 ml of basal medium and 12.5 ml of inoculum were dispensed into cathode 409

chambers to bring the total volume to 125 ml. The abiotic control was prepared with 410

addition of 125 ml same medium in both chambers without inoculation. The 411

headspace of each reactor had a volume of 125 ml and was flushed with N2/CO2 412

(80:20; v/v) thoroughly. CO2 was the sole carbon source in reactors. Reactors were 413

put in the dark at room temperature (27℃). The experiment was divided into three 414

groups. For the first group, MNP was added into the cathode chambers with a final 415

concentration of 10 mM in Fe atom. The second group was prepared similarly but 416

without MNP in chambers. The third group was set up as abiotic control with addition 417


https://doi.org/10.1101/2020.11.24.397190

21

of MNP in cathodes but without inoculation of enrichment. All reactors were 418

connected into the eight channel potentiostat (CHI 1000C, CH Instruments Inc., 419

Shanghai, China) which recorded current every 100 seconds automatically. The 420

reactors were operated in a continuous mode except two breaks for the shifting of 421

cathode potential. The initial cathode potential was set at -0.6 V vs standard hydrogen 422

electrode (SHE, all potentials below, unless specified, were all versus standard 423

hydrogen electrode). When the current density of MNP bioreactors showed no further 424

change or started falling, all the reactors were disconnected from the potentiostat for 425

the first break. The cyclic voltammetry (CV) was conducted immediately with a scan 426

rate of 5 mV/s and scan range of -0.8 V to 0 V vs SHE to characterize biocathodes. 427

Microbial samples were also collected immediately from the cathodes and catholyte 428

medium (see details below). The reactors were then reconnected to the potentiostat 429

with the cathode potential elevated to -0.5 V and operated until the current density of 430

MNP bioreactors started falling again. The second break was then applied for CV 431

measurement and microbial sampling. Finally, the cathode potential was raised to -0.4 432

V for the last round of electrochemical operation. At the end, the last CV tests and 433

microbial sampling were performed again. 434

During each break, the chambers were rinsed thoroughly to remove remaining 435

microbes and magnetite, then replenished with the fresh medium. Cathode chambers 436

were inoculated by 10% (v/v) of the used medium and the remaining used medium 437

was discarded. The electrodes with biofilm remained unchanged. Magnetite 438


https://doi.org/10.1101/2020.11.24.397190

22

nanoparticles (MNP) were re-supplemented in the MNP reactors with the same 439

concentration as before. Microbial sampling and medium refreshing were performed 440

in an anaerobic glove box. There were nine reactors in total at the beginning, i.e. three 441

each for MNP bio-reactors, no-MNP bio-reactors and MNP abiotic reactors. But 442

during the first-round operation at -0.6 V, one of the no-MNP biotic reactors failed to 443

produce CH4 after 50 days and hence this reactor was suspended. Thus, there were 444

eight functioning reactors in operation throughout the experiment. 445

Chemical analyses 446

Methane and hydrogen were monitored throughout experiments. Gas samples (200 447

µL) were regularly taken from the headspace of cathode chambers with a 448

Pressure-Lok precision analytical syringe (Bation Rouge, LA, USA). The 449

concentration of CH4 and H2 were analyzed using GC-7890B gas chromatograph 450

(Agilent Technologies, Santa Clara, CA, USA) equipped with flame ionization 451

detector. The detection limits are 5 Pa for both CH4 and H2. Every time after gas 452

sampling, chambers were shaken vigorously for one minute. 453

Microbial community analysis 454

At the end of CV tests, an aliquot of culture medium was collected, while small pieces 455

of carbon brushes were cut off from electrodes (without influencing the electrode 456

integrity). Carbon brush samples were washed twice with sterile demineralized water 457

to remove the loosely attached microbial cells. Microbial DNA were extracted from 458

both culture medium and carbon brush samples. FastDNATM

SPIN Kit (MP 459


https://doi.org/10.1101/2020.11.24.397190

23

Biomedicals, Irvine, CA, USA) was used for DNA extraction according to the 460

manufacturer’s instructions. Archaeal DNA was amplified using the 16S rRNA gene 461

primers 1106F (TTWAGTCAGGCAACGAGC) and 1378R 462

(TGTGCAAGGAGCAGGGAC) (49). The bacterial DNA was amplified using the 463

16S rRNA gene primers 515F (GTGCCAGCMGCCGCGGTAA) and 806R 464

(GGACTACHVGGGTWTCTAAT). Amplicon sequencing was completed by 465

Novogene (Beijing, China) using Ion S5XL platform. About 80000 reads were 466

obtained for each sample and were clustered into operational taxonomic units (OTUs) 467

with a similarity threshold of 97%. Nonmetric multidimensional scale (NMDS) was 468

conducted using R and vegan community ecology package (50). The closest matching 469

sequences were identified by searching with the BLAST program in the NCBI 470

database (51). Neighbor-joining phylogenetic trees were constructed using MEGA-X 471

and presented with ggtree package in R (52, 53). The nucleotide sequences generated 472

from this study has been deposited in SAR database under the accession numbers of 473

SAMN13136352–SAMN13136413. 474

475

ACKNOWLEDGEMENTS 476

This study was financially supported by the National Natural Science Foundation of 477

China (No. 41630857; 91951206). 478

CONFLICT OF INTEREST 479

The authors declare that they have no conflict of interest. 480


https://doi.org/10.1101/2020.11.24.397190

24

SUPPLEMENTAL MATERIAL 481

Sequence summary for amplicon sequencing of microbes, total abundance of 482

methanogens and bacteria in electromethanogenic reactors, relative abundance of 483

bacterial community, nonmetric multidimensional scale (NMDS) plot of bacteria, 484

CH4 production, current density generation and CV tests in replicate or triplicate 485

reactors. 486

REFERENCES 487

1. Blasco-Gomez R, Batlle-Vilanova P, Villano M, Balaguer MD, Colprim J, 488

Puig S. 2017. On the Edge of Research and Technological Application: A 489

Critical Review of Electromethanogenesis. Int J Mol Sci 490

18.doi:10.3390/ijms18040874 491

2. Holmes DE, Smith JA. 2016. Biologically Produced Methane as a Renewable 492

Energy Source, p 1-61. In Sariaslani S, Gadd GM (ed), Adv Appl Microbiol, 493

Vol 97, vol 97. Elsevier Academic Press Inc, San Diego. 494

3. Logan BE, Rabaey K. 2012. Conversion of Wastes into Bioelectricity and 495

Chemicals by Using Microbial Electrochemical Technologies. Science 496

337:686-690.doi:10.1126/science.1217412 497

4. Cheng SA, Xing DF, Call DF, Logan BE. 2009. Direct Biological Conversion 498

of Electrical Current into Methane by Electromethanogenesis. Environ Sci 499

Technol 43:3953-3958.doi:10.1021/es803531g 500

5. Deutzmann JS, Sahin M, Spormann AM. 2015. Extracellular Enzymes 501


https://doi.org/10.1101/2020.11.24.397190

25

Facilitate Electron Uptake in Biocorrosion and Bioelectrosynthesis. Mbio 502

6.doi:10.1128/mBio.00496-15 503

6. Dykstra CM, Pavlostathis SG. 2017. Methanogenic Biocathode Microbial 504

Community Development and the Role of Bacteria. Environ Sci Technol 505

51:5306-5316.doi:10.1021/acs.est.6b04112 506

7. Lohner ST, Deutzmann JS, Logan BE, Leigh J, Spormann AM. 2014. 507

Hydrogenase-independent uptake and metabolism of electrons by the archaeon 508

Methanococcus maripaludis. ISME J 8:1673-1681.doi:10.1038/ismej.2014.82 509

8. Rowe AR, Xu S, Gardel E, Bose A, Girguis P, Amend JP, El-Naggar MY. 510

2019. Methane-Linked Mechanisms of Electron Uptake from Cathodes by 511

Methanosarcina barkeri. Mbio 10:12.doi:10.1128/mBio.02448-18 512

9. Yee MO, Rotaru A-E. 2020. Extracellular electron uptake in 513

Methanosarcinales is independent of multiheme c-type cytochromes. Sci Rep 514

10:372-372.doi:10.1038/s41598-019-57206-z 515

10. Yee MO, Snoeyenbos-West OL, Thamdrup B, Ottosen LDM, Rotaru A-E. 516

2019. Extracellular Electron Uptake by Two Methanosarcina Species. Front 517

Energy Res 7.doi:10.3389/fenrg.2019.00029 518

11. Batlle-Vilanova P, Puig S, Gonzalez-Olmos R, Vilajeliu-Pons A, Baneras L, 519

Dolors Balaguer M, Colprim J. 2014. Assessment of biotic and abiotic 520

graphite cathodes for hydrogen production in microbial electrolysis cells. Int J 521

Hydrogen Energ 39:1297-1305.doi:10.1016/j.ijhydene.2013.11.017 522


https://doi.org/10.1101/2020.11.24.397190

26

12. Mitov M, Chorbadzhiyska E, Rashkov R, Hubenova Y. 2012. Novel 523

nanostructured electrocatalysts for hydrogen evolution reaction in neutral and 524

weak acidic solutions. Int J Hydrogen Energ 525

37:16522-16526.doi:10.1016/j.ijhydene.2012.02.102 526

13. Villano M, Aulenta F, Ciucci C, Ferri T, Giuliano A, Majone M. 2010. 527

Bioelectrochemical reduction of CO2 to CH4 via direct and indirect 528

extracellular electron transfer by a hydrogenophilic methanogenic culture. 529

Bioresource Technol 101:3085-3090.doi:10.1016/j.biortech.2009.12.077 530

14. Batlle-Vilanova P, Puig S, Gonzalez-Olmos R, Vilajeliu-Pons A, Balaguer 531

MD, Colprim J. 2015. Deciphering the electron transfer mechanisms for 532

biogas upgrading to biomethane within a mixed culture biocathode. Rsc Adv 533

5:52243-52251.doi:10.1039/c5ra09039c 534

15. Saheb-Alam S, Persson F, Wilen BM, Hermansson M, Modin O. 2019. A 535

variety of hydrogenotrophic enrichment cultures catalyse cathodic reactions. 536

Sci Rep 9:13.doi:10.1038/s41598-018-38006-3 537

16. Siegert M, Yates MD, Spormann AM, Logan BE. 2015. Methanobacterium 538

Dominates Biocathodic Archaeal Communities in Methanogenic Microbial 539

Electrolysis Cells. ACS Sustain Chem Eng 540

3:1668-1676.doi:10.1021/acssuschemeng.5b00367 541

17. Deutzmann JS, Spormann AM. 2017. Enhanced microbial electrosynthesis by 542

using defined co-cultures. ISME J 11:704-714.doi:10.1038/ismej.2016.149 543


https://doi.org/10.1101/2020.11.24.397190

27

18. Beese-Vasbender PF, Grote JP, Garrelfs J, Stratmann M, Mayrhofer KJJ. 2015. 544

Selective microbial electrosynthesis of methane by a pure culture of a marine 545

lithoautotrophic archaeon. Bioelectrochemistry 546

102:50-55.doi:10.1016/j.bioelechem.2014.11.004 547

19. Rotaru AE, Shrestha PM, Liu F, Markovaite B, Chen S, Nevin KP, Lovley DR. 548

2014. Direct Interspecies Electron Transfer between Geobacter 549

metallireducens and Methanosarcina barkeri. Appl Environ Microb 550

80:4599-4605.doi:10.1128/aem.00895-14 551

20. Dykstra CM, Paylostathis SG. 2017. Zero-Valent Iron Enhances Biocathodic 552

Carbon Dioxide Reduction to Methane. Environ Sci Technol 553

51:12956-12964.doi:10.1021/acs.est.7b02777 554

21. Kim KR, Kang J, Chae KJ. 2017. Improvement in methanogenesis by 555

incorporating transition metal nanoparticles and granular activated carbon 556

composites in microbial electrolysis cells. Int J Hydrogen Energ 557

42:27623-27629.doi:10.1016/j.ijhydene.2017.06.142 558

22. Liu DD, Roca-Puigros M, Geppert F, Caizan-Juanarena L, Ayudthaya SPN, 559

Buisman C, ter Heijne A. 2018. Granular Carbon-Based Electrodes as 560

Cathodes in Methane-Producing Bioelectrochemical Systems. Front Bioeng 561

Biotech 6:10.doi:10.3389/fbioe.2018.00078 562

23. Cornell RM, Schwertmann U. 2004. Electronic, Electrical and Magnetic 563

Properties and Colour, p 111-137, The Iron Oxides. 564


https://doi.org/10.1101/2020.11.24.397190

28

24. Hazen RM, Papineau D, Leeker WB, Downs RT, Ferry JM, McCoy TJ, 565

Sverjensky DA, Yang H. 2008. Mineral evolution. Am Mineral 566

93:1693-1720.doi:10.2138/am.2008.2955 567

25. Li HJ, Chang JL, Liu PF, Fu L, Ding DW, Lu YH. 2015. Direct interspecies 568

electron transfer accelerates syntrophic oxidation of butyrate in paddy soil 569

enrichments. Environ Microbiol 17:1533-1547.doi:10.1111/1462-2920.12576 570

26. Xia XX, Zhang JC, Song TZ, Lu YH. 2019. Stimulation of 571

Smithella-dominating propionate oxidation in a sediment enrichment by 572

magnetite and carbon nanotubes. Env Microbiol Rep 573

11:236-248.doi:10.1111/1758-2229.12737 574

27. Zhang JC, Lu YH. 2016. Conductive Fe3O4 Nanoparticles Accelerate 575

Syntrophic Methane Production from Butyrate Oxidation in Two Different 576

Lake Sediments. Front in Microbiol 7.doi:10.3389/fmicb.2016.01316 577

28. Kato S, Hashimoto K, Watanabe K. 2012. Methanogenesis facilitated by 578

electric syntrophy via (semi)conductive iron-oxide minerals. Environ 579

Microbiol 14:1646-1654.doi:10.1111/j.1462-2920.2011.02611.x 580

29. Viggi CC, Rossetti S, Fazi S, Paiano P, Majone M, Aulenta F. 2014. Magnetite 581

Particles Triggering a Faster and More Robust Syntrophic Pathway of 582

Methanogenic Propionate Degradation. Environ Sci Technol 583

48:7536-7543.doi:10.1021/es5016789 584

30. Zhuang L, Ma JL, Yu Z, Wang YQ, Tang J. 2018. Magnetite accelerates 585


https://doi.org/10.1101/2020.11.24.397190

29

syntrophic acetate oxidation in methanogenic systems with high ammonia 586

concentrations. Microb Biotechnol 11:710-720.doi:10.1111/1751-7915.13286 587

31. Skomurski FN, Kerisit S, Rosso KM. 2010. Structure, charge distribution, and 588

electron hopping dynamics in magnetite (Fe3O4) (100) surfaces from first 589

principles. Geochim Cosmochim Ac 590

74:4234-4248.doi:10.1016/j.gca.2010.04.063 591

32. Sanchez O, Garrido L, Forn I, Massana R, Ignacio Maldonado M, Mas J. 592

2011. Molecular characterization of activated sludge from a 593

seawater-processing wastewater treatment plant. Microb Biotechnol 594

4:628-642.doi:10.1111/j.1751-7915.2011.00256.x 595

33. Conrad R. 2009. The global methane cycle: recent advances in understanding 596

the microbial processes involved. Env Microbiol Rep 597

1:285-292.doi:10.1111/j.1758-2229.2009.00038.x 598

34. Ren GP, Chen P, Yu J, Liu JB, Ye J, Zhou SG (2019) Recyclable 599

magnetite-enhanced electromethanogenesis for biomethane production from 600

wastewater. Water Res 166. Doi:10.1016/j.watres.2019.115095 601

35. Fu L, Song TZ, Zhang W, Zhang J, Lu YH. 2018. Stimulatory Effect of 602

Magnetite Nanoparticles on a Highly Enriched Butyrate-Oxidizing 603

Consortium. Front Microbiol 9.doi:10.3389/fmicb.2018.01480 604

36. Byrne JM, van der Laan G, Figueroa AI, Qafoku O, Wang C, Pearce CI, 605

Jackson M, Feinberg J, Rosso KM, Kappler A. 2016. Size dependent 606


https://doi.org/10.1101/2020.11.24.397190

30

microbial oxidation and reduction of magnetite nano- and micro-particles. Sci 607

Rep 6.doi:10.1038/srep30969 608

37. Byrne JM, Klueglein N, Pearce C, Rosso KM, Appel E, Kappler A. 2015. 609

Redox cycling of Fe(II) and Fe(III) in magnetite by Fe-metabolizing bacteria. 610

Science 347:1473-1476.doi:10.1126/science.aaa4834 611

38. Wang H, Byrne JM, Liu PF, Liu J, Dong X, Lu YH. 2020. Redox cycling of 612

Fe(II) and Fe(III) in magnetite accelerates aceticlastic methanogenesis by 613

Methanosarcina mazei. Env Microbiol Rep 614

12:97-109.doi:10.1111/1758-2229.12819 615

39. Van Eerten-Jansen MCAA, Veldhoen AB, Plugge CM, Stams AJM, Buisman 616

CJN, Ter Heijne A. 2013. Microbial Community Analysis of a 617

Methane-Producing Biocathode in a Bioelectrochemical System. Archaea 618

2013.doi:10.1155/2013/481784 619

40. Walker DJF, Martz E, Holmes DE, Zhou ZM, Nonnenmann SS, Lovley DR. 620

2019. The Archaellum of Methanospirillum hungatei Is Electrically 621

Conductive. Mbio 10.doi:10.1128/mBio.00579-19 622

41. Nevin KP, Woodard TL, Franks AE, Summers ZM, Lovley DR. 2010. 623

Microbial Electrosynthesis: Feeding Microbes Electricity To Convert Carbon 624

Dioxide and Water to Multicarbon Extracellular Organic Compounds. Mbio 625

1.doi:10.1128/mBio.00103-10 626

42. Marshall CW, Ross DE, Fichot EB, Norman RS, May HD. 2012. 627


https://doi.org/10.1101/2020.11.24.397190

31

Electrosynthesis of Commodity Chemicals by an Autotrophic Microbial 628

Community. Appl Environ Microb 78:8412-8420.doi:10.1128/aem.02401-12 629

43. Yamada T, Sekiguchi Y, Hanada S, Imachi H, Ohashi A, Harada H, Kamagata 630

Y. 2006. Anaerolinea thermolimosa sp nov., Levilinea saccharolytica gen. 631

nov., sp nov and Leptolinea tardivitalis gen. nov., so. nov., novel filamentous 632

anaerobes, and description of the new classes Anaerolineae classis nov and 633

Caldilineae classis nov in the bacterial phylum Chloroflexi. Int J Syst Evol 634

Micr 56:1331-1340.doi:10.1099/ijs.0.64169-0 635

44. Yamada T, Imachi H, Ohashi A, Harada H, Hanada S, Kamagata Y, Sekiguchi 636

Y. 2007. Bellilinea caldifistulae gen. nov., sp nov and Longilinea arvoryzae 637

gen. nov., sp nov., strictly anaerobic, filamentous bacteria of the phylum 638

Chloroflexi isolated from methanogenic propionate-degrading consortia. Int J 639

Syst Evol Micr 57:2299-2306.doi:10.1099/ijs.0.65098-0 640

45. Yates MD, Siegert M, Logan BE. 2014. Hydrogen evolution catalyzed by 641

viable and non-viable cells on biocathodes. Int J Hydrogen Energ 642

39:16841-16851.doi:10.1016/j.ijhydene.2014.08.015 643

46. Lian J, Tian XL, Guo JB, Guo YK, Song YY, Yue L, Wang YJ, Liang XH. 644

2016. Effects of resazurin on perchlorate reduction and bioelectricity 645

generation in microbial fuel cells and its catalysing mechanism. Biochem Eng 646

J 114:167-175.doi:10.1016/j.bej.2016.06.028 647

47. Kaden J, Galushko AS, Schink B. 2002. Cysteine-mediated electron transfer in 648


https://doi.org/10.1101/2020.11.24.397190

32

syntrophic acetate oxidation by cocultures of Geobacter sulfurreducens and 649

Wolinella succinogenes. Arch Microbiol 650

178:53-58.doi:10.1007/s00203-002-0425-3 651

48. Kang YS, Risbud S, Rabolt JF, Stroeve P. 1996. Synthesis and characterization 652

of nanometer-size Fe3O4 and gamma-Fe2O3 particles. Chem Mater 653

8:2209-&.doi:10.1021/cm960157j 654

49. Zhang JW, Tang HY, Zhu JG, Lin XG, Feng YZ. 2016. Divergent responses of 655

methanogenic archaeal communities in two rice cultivars to elevated 656

ground-level O-3. Environ Pollut 657

213:127-134.doi:10.1016/j.envpol.2016.01.062 658

50. Dixon P. 2003. VEGAN, a package of R functions for community ecology. J 659

Veg Sci 14:927-930.doi:10.1658/1100-9233(2003)014[0927:vaporf]2.0.co;2 660

51. Lyu Z, Lu YH. 2018. Metabolic shift at the class level sheds light on 661

adaptation of methanogens to oxidative environments. ISME J 662

12:411-423.doi:10.1038/ismej.2017.173 663

52. Yu GC, Smith DK, Zhu HC, Guan Y, Lam TT-Y. 2017. GGTREE: an R 664

package for visualization and annotation of phylogenetic trees with their 665

covariates and other associated data. Methods Ecol Evol 666

8(1):28-36.doi:10.1111/2041-210x.12628 667

53. Yu GC, Lam TT-Y, Zhu HC, Guan Y. 2018. Two Methods for Mapping and 668

Visualizing Associated Data on Phylogeny Using Ggtree. Mol Biol Evol 669


https://doi.org/10.1101/2020.11.24.397190

33

35(12):3041-3043.doi:10.1093/molbev/msy194 670

671


https://doi.org/10.1101/2020.11.24.397190

34

FIGURE CAPTIONS 672

673

Fig. 1 The schematic figure for enrichment cultivation of methanogens and the set of 674

electrochemical reactors. 675

676


https://doi.org/10.1101/2020.11.24.397190

35

677

Fig. 2 The production of CH4 and current density generation. The initial potential was 678

set at -0.6 V and then was elevated to -0.5 V and -0.4 in two steps (yellow arrows). 679

Data shown is a representative example of replicate or triplicate experiments (n=2 or 680

3), data for replicate or triplicate experiments could be found in Fig. S5 and S6 681

682

0 70 80 90Time (Days)

Cu

rre

nt

de

ns

ity

(A

m)

Abiotic

MNP

0

5

10

15

20

25

30

35

0 10 20 30 40 50 60 70 80 90 100 110 120 130

CH

4 (m

mo

l L

-1)

MNP added

0

5

10

15

20

25

30

35

0 10 20 30 40 50 60 70 80 90 100 110 120 130

CH

4 (m

mo

l L

-1)

MNP added

a

b

-0.6 V -0.5 V -0.4 V

-0.6 V -0.5 V -0.4 V


https://doi.org/10.1101/2020.11.24.397190

36

683

Fig. 3 Cyclic voltammogram determined at the end of electromethanogenic operations 684

under three successively elevated cathodic potentials (a, b, c separately). Red, MNP 685

reactors; black, no-MNP reactors; blue, abiotic control reactors, which contained 686

MNP but without inoculum. L, M, H denote -0.6 V, -0.5 V and -0.4 V; n, magnetite 687

nanoparticles; A, abiotic control. Data shown is a representative example of replicate 688

experiments (n=2 or 3), data for replicate experiments could be found in Fig. S7 689

690

-0.8 -0.6 -0.4 -0.2 0.0

-150

-100

-50

0

50

100

150

200 LnLAnL

Cu

rre

nt

de

ns

ity

(A

m-3)

-0.6 V

E (V vs SHE)

-0.8 -0.6 -0.4 -0.2 0.0-600

-400

-200

0

200

400MnM

-0.5 V

E (V vs SHE)

-0.8 -0.6 -0.4 -0.2 0.0-400

-300

-200

-100

0

100

200 -0.4 V

E (V vs SHE)

HnH

a b c


https://doi.org/10.1101/2020.11.24.397190

37

691

Fig. 4 Composition dynamics of methanogens during the operation of 692

electromethanogenesis. The relative abundance of methanogen community at genera 693

level (a). Nonmetric multidimensional scale (NMDS) plot of methanogen community 694

across samples (b). L, no-MNP biocathode at -0.6 V; nL, MNP biocathode at -0.6 V; 695

cL, no-MNP catholyte at -0.6 V; ncL, MNP catholyte at -0.6 V; M, no-MNP 696

biocathode at -0.5 V; nM, MNP biocathode at -0.5 V; cM, no-MNP catholyte at -0.5 697

V; ncM, MNP catholyte at -0.5 V; H, no-MNP biocathode at -0.4 V; nH, MNP 698

biocathode at -0.4 V; cH, no-MNP catholyte at -0.4 V; ncH, MNP catholyte at -0.4 V. 699

Error bars represent mean values ± one standard deviation, n=2 or 3 700

701

a b

Inocu

lum

L cL M cM H cH nL ncL nM ncM nH ncH

Re

lati

ve

ab

un

da

nc

eMethanospirillum

Methanobacterium

Methanosarcina

Methanoregula

Methanosphaerula

Methanocella

Methanobrevibacter

Methanosaeta

Methanolobus

Methanosphaera

Methanimicrococcus

Others0.0

0.2

0.4

0.6

0.8

1.0

No-MNP reactors MNP reactors

NMDS1

H

cH

Inoculum

nH

nM

M

cM

nL

L

cL

-0.4

V

-0.5

V

-0.6

V

0.4

0.2

0.0

-0.2

ncM

ncH

ncL

NM

DS

2

-0.50 -0.25 0.00 0.25


https://doi.org/10.1101/2020.11.24.397190

38

702

Fig. 5 Neighbor-joining phylogenetic relationship of methanogens (a) and heatmap 703

showing the change of phylotypes within Methanospirillum with the elevation of 704

cathode potential (b). nL, MNP biocathode at -0.6 V; nM, MNP biocathode at -0.5 V; 705

nH, MNP biocathode at -0.4 V 706

707

clone_Mspi1

clone_Mspi2

clone_Mspi3

clone_Mspi4

clone_Mspi5

clone_Mspi6

clone_Mspi7

clone_Mspi8

clone_Mspi9

clone_Mspi10

clone_Mspi11

clone_Mspi12

clone_Mspi13

clone_Mspi14

clone_Mspi15

0

1

2

-2

-1

Inoculu

mnL1

nL2

nL3

nM

1nM

2nM

3nH

1nH

2nH

3

a b

Methanospirillum hungatei JF-1 (NR 074177.1)

Methanospirillum stamsii ps (NR 117705.1)

clone_Msph

Methanosphaerula palustris E1-9c (NR 074167.1)

Methanoregula boonei 6A8 (NR 074180.1) clone_Mre

Methanoregula formicica SMSP (NR 102441.1)

Methanobacterium formicicum MF (NR 115168.1) clone_Mba1

Methanobacterium palustre F (NR 041713.1)

Methanobacterium bryantii MOH (NR 042781.1)

clone_Mba2

Methanobacterium flexile GH (NR 116276.1)

clone_Msar Methanosarcina horonobensis HB-1 JCM 15518 (NR 112648.1)

Methanosarcina mazei DSM 2053 OCM 26 (NR 041956.1)

Methanosarcina acetivorans C2A (NR 074110.1)

Methanosarcina barkeri MS (NR 118371.1) clone_Mce

Methanocella arvoryzae

Methanocella conradii HZ254 (NR 118245.1)

Methanocella paludicola SANAE (NR 074192.1)

0.050

Methanospirillum hungatei JF-1 i (NR 074177.1)


clone_MsarMethanosarcina horonobensis HB-1 JCM 15518 (NR 112648.1)


Methanosarcina acetivorans C2A (NR 074110.1)A

Methanosarcina barkeri MS (NR 118371.1)

Methanobacterium formicicum MF (NR 115168.1)clone_Mba1



clone_Mba2


clone_Mspi

(including 15 clones)

MRE50 (NR074232.1)


https://doi.org/10.1101/2020.11.24.397190

39

708

Fig. 6 Bacterial community composition and relative abundance. Error bars represent 709

mean values ± one standard deviation, n=2 or 3 710

711

712

0.00

0.25

0.50

0.75

1.00

Inoculum L cL M cM H cH nL ncLnM ncM nH ncH

Re

lati

ve

Ab

un

dan

ce

Others

Syntrophobacter

Desulfomicrobium

Verrucomicrobia

Actinobacteria

Gracilibacter

Alphaproteobacteria

Anaerolineaceae

Anaerovorax

Lentimicrobium

Desulfovibrio

Spirochaetes

Bacteroidales

Geobacter

Sulfuricurvum

Gammaproteobacteria

Acetobacterium



https://doi.org/10.1101/2020.11.24.397190

Counter electrodeWork electrode Reference

electrode

Gas

sample

Liquid

sample

Anode

Cathode

!"##$%&'()

*)+,'-.%/0%123

4! (5%67.%7."#-8",.

95',+)"6.

:,.6"6.%/;<%123

=!>?@! (5%67.%7."#-8",.:,.6"6.%/;<%123

=!>?@! (5%67.%7."#-8",.

:,.6"6.%/;<%123=!>?@! (5%67.%7."#-8",.

95',+)"6. 95',+)"6. 95',+)"6.%

67.%,"67'#.%

,7"1A.B-

C5B(,7%67.%1.67"5'D.5-

/E7B..%-+,,.--(F.%6B"5-G.B3

:,6(F"6.%6'6")%1(,B'A(")%",6(F(6$%


https://doi.org/10.1101/2020.11.24.397190

0 70 80 90Time (Days)

Cu

rre

nt

de

ns

ity

(A

m)

Abiotic

MNP

0

5

10

15

20

25

30

35

0 10 20 30 40 50 60 70 80 90 100 110 120 130

CH

4 (m

mo

l L

-1)

MNP added

0

5

10

15

20

25

30

35

0 10 20 30 40 50 60 70 80 90 100 110 120 130

CH

4 (m

mo

l L

-1)

MNP added

a

b

-0.6 V -0.5 V -0.4 V

-0.6 V -0.5 V -0.4 V


https://doi.org/10.1101/2020.11.24.397190

-0.8 -0.6 -0.4 -0.2 0.0

-150

-100

-50

0

50

100

150

200 LnLAnL

Cu

rren

t d

en

sit

y (

A m

-3)

-0.6 V

E (V vs SHE)

-0.8 -0.6 -0.4 -0.2 0.0-600

-400

-200

0

200

400MnM

-0.5 V

E (V vs SHE)

-0.8 -0.6 -0.4 -0.2 0.0-400

-300

-200

-100

0

100

200 -0.4 V

E (V vs SHE)

HnH

a b c


https://doi.org/10.1101/2020.11.24.397190

a b

Inoculu

m L cL M cM H cH nL ncL nM ncM nH ncH

Re

lati

ve

ab

un

da

nc

e

Methanospirillum

Methanobacterium

Methanosarcina

Methanoregula

Methanosphaerula

Methanocella

Methanobrevibacter

Methanosaeta

Methanolobus

Methanosphaera

Methanimicrococcus

Others0.0

0.2

0.4

0.6

0.8

1.0


NMDS1

H

cH

Inoculum

nH

nM

M

cM

nL

L

cL

-0.4

V

-0.5

V

-0.6

V

0.4

0.2

0.0

-0.2

ncM

ncH

ncL

NM

DS

2-0.50 -0.25 0.00 0.25


https://doi.org/10.1101/2020.11.24.397190

clone_Mspi1

clone_Mspi2

clone_Mspi3

clone_Mspi4

clone_Mspi5

clone_Mspi6

clone_Mspi7

clone_Mspi8

clone_Mspi9

clone_Mspi10

clone_Mspi11

clone_Mspi12

clone_Mspi13

clone_Mspi14

clone_Mspi15

0

1

2

-2

-1

Inoculu

mnL1

nL2

nL3

nM

1nM

2nM

3nH

1nH

2nH

3

a b

Methanospirillum hungatei JF-1 (NR 074177.1)


clone_Msph

Methanosphaerula palustris E1-9c (NR 074167.1)

Methanoregula boonei 6A8 (NR 074180.1) clone_Mre

Methanoregula formicica SMSP (NR 102441.1)




clone_Mba2


clone_Msar Methanosarcina horonobensis HB-1 JCM 15518 (NR 112648.1)


Methanosarcina acetivorans C2A (NR 074110.1)

Methanosarcina barkeri MS (NR 118371.1) clone_Mce

Methanocella arvoryzae

Methanocella conradii HZ254 (NR 118245.1)

Methanocella paludicola SANAE (NR 074192.1)

0.050

Methanospirillum hungatei JF-1 ei (NR 074177.1)


clone_MsarMethanosarcina horonobensis HB-1 JCM 15518 (NR 112648.1)


Methanosarcina acetivorans C2A (NR 074110.1) C2A

Methanosarcina barkeri MS (NR 118371.1)




clone_Mba2


clone_Mspi

(including 15 clones)

MRE50 (NR074232.1)


https://doi.org/10.1101/2020.11.24.397190

0.00

0.25

0.50

0.75

1.00

Inoculu

m L cL M cM H cH nL ncLnM ncM nH ncH

Re

lati

ve

Ab

un

da

nc

e

Others

Syntrophobacter

Desulfomicrobium

Verrucomicrobia

Actinobacteria

Gracilibacter

Alphaproteobacteria

Anaerolineaceae

Anaerovorax

Lentimicrobium

Desulfovibrio

Spirochaetes

Bacteroidales

Geobacter

Sulfuricurvum

Gammaproteobacteria

Acetobacterium



https://doi.org/10.1101/2020.11.24.397190

Date post:	21-Jan-2021
Category:	Documents
Upload:	others
View:	1 times
Download:	0 times

Response of methanogen community to elevation of cathode … · 2020. 11. 24. · 4 Running title:...

Documents