Changes in Glucose Fermentation Pathways as a Response to the

Subscriber access provided by University of Florida | Smathers Libraries

Article

Changes in glucose fermentation pathways as a response to the free ammonia concentration in microbial electrolysis cells Mohamed Mahmoud, César Iván Torres, and Bruce E. Rittmann Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05620 • Publication Date (Web): 17 Oct 2017 Downloaded from http://pubs.acs.org on October 19, 2017

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 free 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 accessible to all readers and 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.

Environmental Science & Technology 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 43

1 2 3 4 5

Environmental Science & Technology

Changes in glucose fermentation pathways as a response to the free ammonia concentration in microbial electrolysis cells Mohamed Mahmoud § ¥ £ *, César I. Torres § Φ, and Bruce E. Rittmann § ¥

6 7

§

8

Tyler Road, Tempe, Arizona, USA.

9

¥

Biodesign Swette Center for Environmental Biotechnology, Arizona State University, 727

School of Sustainable Engineering and the Built Environment, Arizona State University,

10

Tempe, Arizona, USA.

11

Φ

12

Tempe, Arizona, USA.

13

£

14

Cairo 12311, Egypt

School for Engineering of Matter, Transport and Energy, Arizona State University,

Water Pollution Research Department, National Research Centre, 33 El-Buhouth St., Dokki,

15 16

* Corresponding author: Mohamed Mahmoud

17

Email: [email protected] & [email protected]; tel.: +1 480 727 7596; fax: +1 480 727

18

0889.

1 ACS Paragon Plus Environment


19

Abstract

20 21

When a mixed-culture microbial electrolysis cell (MEC) is fed with a fermentable substrate, such

22

as glucose, a significant fraction of the substrate’s electrons ends up as methane (CH4) through

23

hydrogenotrophic methanogenesis, an outcome that is undesired. Here, we show that free

24

ammonia-nitrogen (FAN, which is NH3) altered the glucose fermentation pathways in batch

25

MECs, minimizing the production of H2, the “fuel” for hydrogenotrophic methanogens.

26

Consequently, the Coulombic efficiency (CE) increased: 57% for 0.02 g FAN/L fed-MEC,

27

compared to 76% for 0.18 fed-MECs and 62% for 0.37 g FAN/L fed-MECs. Increasing FAN

28

concentration was associated with the accumulation of higher organic acids (e.g., lactate, iso-

29

butyrate, and propionate), which was accompanied by increasing relative abundances of

30

phylotypes that are most closely related to anode respiration (Geobacteraceae), lactic-acid

31

production (Lactobacillales), and syntrophic acetate oxidation (Clostridiaceae). Thus, the

32

microbial community established syntrophic relationships among glucose fermenters, acetogens,

33

and anode-respiring bacteria (ARB). The archaeal population of the MEC fed 0.02 g FAN/L was

34

dominated by Methanobacterium, but 0.18 and 0.37 g FAN/L led to Methanobrevibacter

35

becoming the most abundant species. Our results provide insight into a way to decrease CH4

36

production and increase CE using FAN to control the fermentation step, instead of inhibiting

37

methanogens using expensive or toxic chemical inhibitors, such as 2-bromoethane sulfonic acid.

38 39

Keywords: Microbial electrolysis Cells, Free ammonia-nitrogen, Syntrophy, Anode-respiring

40

bacteria, Fermenters.


Page 2 of 43

Page 3 of 43

41


TOC Art

42 43

44 45



46

Introduction

47

Microbial electrolysis cells (MECs) represent one of the newest environmental

48

biotechnologies for wastewater treatment coupled to the production of renewable energy in the

49

form of electrical power, hydrogen gas (H2), or valuable chemicals.1 Since the organic

50

compounds – the “fuel” for the MECs – are complex in nature, their biodegradation requires

51

cooperation among different trophic groups: fermenters, homoacetogens, and anode-respiring

52

bacteria (ARB), as well as competition with methanogens.2,3 Generally, an MEC’s microbial

53

community has a high complexity in terms of community structure and diversity, but key

54

microbial populations have been identified.4,5

55

The defining group in an MEC is the ARB, which perform a unique type of respiration.

56

Most respiring anaerobes transfer electrons intracellularly to a terminal electron acceptor, such as

57

sulfate, nitrate, or carbon dioxide.6,7 ARB oxidize organic matter internally, but transfer the

58

resulting electrons outside their membrane to a solid electron acceptor, the anode.2,5 Another

59

feature of majority of ARB in mixed community is that their electron donors are limited to only a

60

few simple substrates, such as acetate and H2.8–10 Thus, fermentation is necessary to generate the

61

mixture of simple fermentation products that the ARB can utilize directly.

62

In environments lacking electron acceptors, fermentable organic compounds, such as

63

glucose, are transformed into a variety of organic acids and H2. Acetate is the most prevalent

64

organic acid, and Table 1 presents a number of reactions in which acetate and H2 are formed or

65

consumed. H2-consumers include hydrogenotrophic methanogens, which use H2 as the main

66

electron donor and produce methane (reaction 3 in Table 1).7,11–13 Homoacetogens also scavenge

67

H2 to yield acetate. Homoacetogenesis and hydrogenotrophic methanogenesis have very low

68

energy yields.14 H2 must be kept at low level to allow fermentation to be thermodynamically


Page 4 of 43

Page 5 of 43


69

possible.3,11,15 When H2 builds up, the fermentation stoichiometry changes such that higher

70

organic acids, such as lactate, butyrate, propionate, and ethanol, are produced rather than acetate

71

and H2.3,15

72

When the MEC’s anode is the only respiratory electron acceptor, ARB can out-compete

73

methanogens for acetate, due to their thermodynamic and kinetic advantages over the acetate-

74

consuming methanogens (reaction 5 in Table 1); hence, acetoclastic methanogens cannot

75

compete with acetate-oxidizing ARB, although H2-oxidizing methanogens compete well with

76

H2-oxidizing ARB.8 However, ARB do not have similar advantages for H2 consumption, and the

77

electrons in H2 often are channeled to methane (CH4) via hydrogenotrophic methanogens, which

78

are able to grow in suspension, as well as in the anode’s biofilm.9,16 Thus, one of the challenges

79

for MEC scale-up and commercialization is to minimize production of CH4 from the H2

80

generated via fermentation in the presence of hydrogenotrophic methanogens.

81 82

Table 1.

83 84

Previous researchers proposed different strategies to inhibit methanogenesis in

85

laboratory-scale MECs, including thermal treatment, periodic exposure to oxygen (O2), pH

86

excursions, alamethicin exposure, and use of chemical inhibitors such as 2-bromoethanesulfonate

87

(BES).8,17, 18 Among the proposed strategies, chemical inhibitors seem to be the most effective

88

approach, due to their selectivity for inhibiting the activity of methyl coenzyme M reductase A

89

(mcrA) gene in acetoclastic and hydrogenotrophic methanogens. For example, Parameswaran et

90

al.8 inhibited methanogens in an ethanol-fed MEC with 50 mM BES, which boosted the

91

Coulombic efficiency (CE) by ~40%, as electrons from H2 were rerouted to anode respiration



92

instead of to CH4 production. Although BES is an effective way to inhibit methanogens,8,9 it is

93

not practical for field applications due to the continuous need to supply the inhibitor, which is

94

environmentally toxic and expensive.

95

We recently revealed that ARB are tolerant to relatively-high free-ammonia nitrogen

96

(FAN) concentrations, up to at least 200 mg/L; however, high total-ammonia nitrogen (TAN)

97

concentrations (i.e., > 2.2 g/L) might cause inhibition (or lowering) of the anode respiration

98

rate.19 The relatively high threshold for FAN inhibition by ARB may be a tool for managing

99

microbial communities to favor ARB and high electron recovery in MECs fed with fermentable

100

substrates, as long as TAN is not too high. Therefore, we hypothesize that high-enough FAN

101

can promote the desired syntrophy in MECs fed with fermentable substrate by inhibiting

102

hydrogenotrophic methanogens. Suppressing H2-oxidizing methanogens provides ideal

103

conditions for homoacetogens to oxidize H2 and grow, leading to the generation of acetate, the

104

ideal substrate for ARB. 8,9 This hypothesis requires that a FAN concentration high enough to

105

suppress methanogens not have a strong inhibitory effect on ARB and fermenters.14,20 This

106

strategy is highly relevant to MECs treating real wastewater that are characterized by high FAN

107

concentrations, including landfill leachate and animal wastewater.21,22

108

A high FAN concentration also might promote a new pathway for acetate consumption to

109

produce CH4 at very low H2 partial pressure: syntrophic acetate oxidation (SAO) (alternatively

110

known as reverse acetogenesis), which has been explored in anaerobic digesters.23,25 SAO is a

111

two-step process in which acetate is utilized by syntrophic acetate-oxidizing bacteria (SAOB)

112

(reaction 7 in Table 1) with the generation of reducing equivalents, often in the form of H2. This

113

step is a highly energy-demanding reaction that requires syntrophy with H2-consuming bacteria

114

(e.g., hydrogenotrophic methanogens or hydrogenotrophic ARB) to maintain a very low level of


Page 6 of 43

Page 7 of 43


115

H2 (reaction 3 or reaction 6 in Table 1, respectively) in order to make the overall reaction

116

thermodynamically favorable.11,24 In the absence of other electron acceptors, such as nitrate and

117

sulfate, the produced H2 is most likely routed to CH4 through hydrogenotrophic methanogenesis,

118

since ARB are known of being relatively poor H2-consumers.8,25 So far, nothing is known about

119

the SAO process in MECs fed with high FAN concentration.

120

Our overarching goal is to investigate the effect of different FAN levels on the

121

interactions among ARB and other members of the communities, particularly the fermenters and

122

methanogens. We document the microbial interactions through a combination of chemical,

123

electrochemical, and genomic tools. In order to study electron-flow and synergies in an MEC’s

124

anode, we use a fixed concentration of glucose (5 mM) as the sole electron-donor substrate, and

125

we vary the influent FAN concentration (i.e., 0.02, 0.18, and 0.37 g FAN/L) going into the anode

126

during batch and semi-continuous (hydraulic retention time of 2 days) MECs. Key is that we

127

establish electron-equivalent mass balances. We also characterize the relative abundance and

128

composition of bacteria and Archaea by Illumina sequencing, and we track homoacetogens and

129

methanogens by targeting the formyltetrahydrofolate synthetase (FTHFS) gene – a conserved

130

gene involved in their CO2 fixation pathway – and mcrA gene, respectively, by quantitative real-

131

time PCR (qPCR).

132



133

Materials and methods

134 135 136

MEC design and operation For our experiments to understand the fundamentals of how the microbial community

137

changes in response to high FAN concentration, we used half-cell MECs with its anode potential

138

controlled using a potentiostat. Each MEC held ~ 320 mL in each chamber and had a square

139

graphite anode having a total surface area of ~ 11.6 cm2, and a 0.8-cm OD graphite rod as

140

cathode. This setup excluded any interference from potential losses at the cathode or due to a

141

large distance between the anode and the cathode; fixing the anode potential allowed us to focus

142

only on the anodic reactions. In order to ensure that the ARB were not limited by a low anode

143

potential, we poised the anode at a fixed potential of – 0.3 V vs. Ag/AgCl (or ~ – 0.04 V vs.

144

standard hydrogen electrode (SHE) in our media) using a VMP3 digital potentiostat (Bio-Logic

145

USA, Knoxville, TN) by placing an Ag/AgCl reference electrode (BASI Electrochemistry, west

146

Lafayette, IN) about 0.5 cm away from the anode. We separated the anode chamber from the

147

cathode chamber with an anion exchange membrane (AMI 7001, Membranes International, Glen

148

Rock, NJ). The pH of the cathode chamber was adjusted to 12 by addition of 10 N NaOH to

149

facilitate the transport of hydroxide ions (OH–) from the cathode compartment to the anode

150

compartment through the anion-exchange membrane.

151

We inoculated the MEC’s anode chamber with 2 mL of biofilm inoculum from a

152

previously operated MEC fed with 5 mM glucose as the sole electron donor. After sparging the

153

MEC with ultra-high purity N2 gas (≥99.9%) for ~ 45 min, we fed the MEC with autoclaved (for

154

90 min at 121°C) glucose medium (initial pH of 8.1) containing: 5 mM glucose (or 38.4 me–eq),

155

100 mM phosphate buffer (KH2PO4/Na2HPO4), 2.1 g NaHCO3, and 10 mL of trace minerals as


Page 8 of 43

Page 9 of 43


156

outlined in Parameswaran et al.8 We added different amounts of ammonium chloride as the sole

157

N-source, giving FAN (and TAN) concentrations of 0.02 g FAN/L (0.2 g TAN/L), 0.18 g FAN/L

158

(2 g TAN/L), and 0.37 g FAN/L (4 g TAN/L). We eliminated the possibility of ARB biofilm

159

needing to fix atmospheric N2 by using an ammonium concentration more than sufficient for

160

growth in the control experiments (0.2 g N/L or 14 mM-N). Prior to the MEC’s electron balance

161

experiments, we acclimated the inoculum by continuously feeding the developed biofilm with 5

162

mM glucose medium as the sole electron donor at relatively short hydraulic retention time (i.e.,

163

24 h), followed by performing 2 consecutive batch cycles to select for a microbial community

164

that was efficient at consuming glucose and producing electrical current. We controlled the

165

temperature at 30°C in a temperature-controlled room, and the liquid in both chambers was

166

mixed using a magnetic stirrer at 220 rpm. We performed all batch MEC experiments in

167

duplicate.

168

In order to confirm and expand the results from the batch MEC experiments, we

169

evaluated MEC performance with semi-continuous operation. We operated two independent

170

MECs in parallel and fed in a semi-continuous mode once every 2 days. The MECs were

171

operated at a fixed temperature of 30°C and fed with a 5-mM glucose medium having the same

172

composition as mentioned in the previous paragraph. The liquid in both chambers was mixed

173

using a magnetic stirrer at 220 rpm. We tested the MECs with different FAN concentrations

174

achieved with different combinations of TAN concentration and pH: (1) 0.003 g FAN/L (pH = 7

175

and TAN = 0.2 g/L), (2) 0.02 g FAN/L (pH = 8.1 and TAN = 0.2 g/L), 0.18 g FAN/L (pH = 8.1

176

and 2 g/L), and (4) 0.37 g FAN/L (pH = 8.1 and TAN = 4 g/L).

177



178 179

Chemical analyses We measured TAN, in duplicate, using HACH kits (HACH, Ames, IA) after filtration

180

through a 0.22-µm membrane filter (PVDF GD/X, Whatman, GE Healthcare, Ann Arbor, MI).

181

We quantified liquid samples for organic fermentation products and glucose using high-

182

performance liquid chromatography (HPLC; Model LC-20AT, Shimadzu, Columbia, MD) with

183

an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA) after filtration through a

184

0.22-µm membrane filter according the method described in Mahmoud et al.21 Briefly, we used

185

2.5 mM sulfuric acid and 18-MΩ reverse-osmosis water as eluents for determining organic

186

fermentation products and glucose, respectively, at a constant flow rate of 0.6 mL/min. We

187

developed a five-point calibration curve for every set of analyses, performed duplicate assays,

188

and report the average concentrations.

189

We measured the volume of gas produced with a friction-free glass syringe of 10- or 25-

190

mL volume (Popper & Sons, Inc., New Hyde Park, NY, USA). We estimated gas percentages of

191

H2, CH4, and CO2 in samples taken with a gas-tight syringe (SGE 500 µL, Switzerland) using a

192

gas chromatograph (GC 2010, Shimadzu Corporation, Columbia, MD) equipped with a thermal

193

conductivity detector and a packed column (CarboxenTM 1010 PLOT Capillary Column,

194

Supleco, Inc.). Helium was the carrier gas at a constant flow rate of 10 mL/min and pressure of

195

42.3 kPa. Temperature conditions for column, injection, and detector were 80, 150, and 220 °C,

196

respectively. We employed analytical grade H2, CH4, and CO2 gases for standard curves, carried

197

out gas analyses in duplicate, and averaged the two values.

198


Page 10 of 43

Page 11 of 43

199 200


Microbial community analyses DNA extraction. At the end of each batch experiment, we harvested the entire biofilm

201

biomass from each MEC anode by scraping it off with a sterilized pipette tip and suspending the

202

biomass sample in a sterile centrifuge tube containing DNA-free water. We then centrifuged the

203

contents at 10,000g (Eppendorf Centrifuge 5414 D, USA) for 10 min to concentrate the biomass,

204

which we stored at −20 °C prior to DNA extraction. We also centrifuged the entire liquid of the

205

MEC chamber to concentrate the suspended phase for extraction. We extracted the total

206

genomic DNA using the MOBIO Powersoil DNA extraction kit according to manufacturer’s

207

instructions, and determined the quality and quantity of the extracted DNA using a nanodrop

208

spectrophotometer (ND 1000, Thermo Scientific) by measuring absorbance at 260 and 280 nm.

209

Quantitative real-time PCR (qPCR). We evaluated the presence and abundance of

210

methanogenic Archaea and homoacetogens by targeting the mcrA and FTHFS genes,

211

respectively, using qPCR. We carried out all PCR reactions in optically clear tubes with caps in

212

an Eppendorf Realplex 4S realcycler with a 20 µL total reaction volume. We performed all

213

qPCR reactions in triplicate along with a six-point standard curve by following modified assays

214

for FTHFS gene9 and mcrA gene.26 We performed negative-control assays by using DNA-free

215

water instead of DNA templates. We reported the results as the number of gene copies per

216

reactor, after calculating the number of 16S rRNA genes per the entire biofilm or suspended

217

phase of each MEC.

218

Illumina sequencing. We amplified the extracted DNA using 16S rRNA gene-targeting

219

forward and reverse fusion primers at MR DNA laboratory (www.mrdnalab.com, Shallowater,

220

TX, USA). We performed bacterial and archaeal sequence using the bar-coded primer set 515F

221

(5′–GTGCCAGCMGCCGCGGTAA–3′)/806R (5′–GGACTACHVGGGTWTCTAAT–3′)27 and



222

349F (5′–GYGCASCAGKCGMGAAW–3′)/806R (5′–GGACTACVSGGGTATCTAAT–3′),28

223

respectively, in a single-step 28 cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen,

224

USA) with the following conditions: 94°C for 3 min, followed by 28 cycles of 94°C for 30 s,

225

53°C for 40 s, and 72°C for 1 min, after which a final elongation step at 72°C was for 5 min. In

226

order to determine the amplification success and the relative intensity of bands, PCR products

227

were checked in 2% agarose gel.29 Then, the amplified products were pooled in equal

228

proportions based on their molecular weight and DNA concentrations, purified using calibrated

229

Ampure XP beads, and used to prepare DNA libraries following the Illumina TruSeq DNA

230

library preparation protocol. Sequencing was performed on a MiSeq Illumina sequencer

231

(Illumina Inc., USA) following the manufacturer’s guidelines.

232

Bioinformatics analysis. We analyzed the sequences data using QIIME software package

233

version 1.9.130 after trimming off low-quality bases and discarding sequences shorter than 25 bp,

234

longer than 450 bp, or labeled as chimeric sequences. We performed taxonomic classification at

235

3% sequence divergence (97% sequence similarity) and assigned taxonomy to operational

236

taxonomic units (OTUs) by using the Ribosomal Database Project (RDP) classifier with a 50%

237

confidence threshold.31 We performed rarefaction on the OTU table at a depth of 100 sequences

238

in 10 replicates, and we analyzed the rarefaction measures with the same sequence numbers per

239

sample (i.e., 223,000 sequences per sample for bacteria population) with a python script in

240

QIIME software.31 We performed alpha- and beta-diversity calculations, including richness of

241

each samples with Chao1 index,32 the diversity with Shannon and Phylogenetic Distance Whole

242

Tree metrics,33 the evenness with the equitability coefficient on a normalized scale from zero, in

243

which community is perfectly even, to 1, in which community has one dominant OTU and many

244

singlets,29 and principal-coordinates analysis (PCoA) using python script in QIIME software.


Page 12 of 43

Page 13 of 43


245

Sequence data sets are available at NCBI/Sequence Read Archive (SRA) under study with

246

BioProject accession number PRJNA343831.

247 248 249

Electron balance and electron flow into various sinks We established electron balances by estimating the electron equivalents of experimentally

250

measured glucose and fermentation products with the following equivalences:12 24 me– eq per

251

mmole glucose, 8 me– eq per mmole acetate, 14 me– eq per mmole propionate, 20 me– eq per

252

mmole butyrate, 12 me– eq per mmole lactate, 25 me– eq per mmole valerate, 0.32 me– eq per mL

253

CH4 (@30°C), and 0.08 me– eq per mL H2 (@30°C).

254

First, we established electron flow from glucose when hydrogenotrophic methanogens

255

are not suppressed or are completely suppressed. We assumed stoichiometric fermentation of

256

glucose (24 me– eq) into 2 moles of acetate (16 me– eq) and 4 moles of H2 (8 me– eq). Acetate is

257

efficiently consumed by acetate-consuming ARB to generate electric current, while H2 is

258

consumed by hydrogenotrophic methanogens that have thermodynamic, kinetic, and metabolic

259

advantages over H2-consuimng ARB and homoacetogens.8 In parallel, a fraction of electrons is

260

utilized by different microbial groups in the MEC’s anode to synthesize new microbial cells. We

261

quantify the fraction of electrons used for biomass synthesis based on fs° (me– eq biomass per

262

me– eq substrate) values of 0.18 for fermenters,12 0.08 for hydrogen-consuming methanogens,12

263

~0.02 for homoacetogens, 34,35 and ~ 0.09 for ARB.19 Supplementary Figure S1 shows the

264

different paths for electron flow from glucose when hydrogenotrophic methanogens are not

265

suppressed (Figure S1A) or are completely suppressed (Figure S1B). The resulting CE for the

266

electron flows in Figure S1A (when methanogenesis is not suppressed) is 50%, with 25% of the



267

electrons routed to CH4 gas and 25% to biomass. CE increases to 76% when methanogenesis is

268

completely inhibited, with comparable electrons routed to biomass (24%) (Figure S1B).


Page 14 of 43

Page 15 of 43

269


Results and discussion

270 271

Framework for explaining electrons distribution for batch MECs

272

Figure 1 presents the electron distribution from glucose into the possible electron sinks

273

over the course of the experiments with different FAN concentrations. The inoculum had been

274

pre-acclimated to glucose, and then we performed an electron balance on batch experiments with

275

glucose as the sole electron donor. With the lowest initial FAN (0.02 g FAN/L), current

276

production increased significantly during the first 2 days (Figure 1A). Simultaneously, acetate

277

production increased to ~ 32% of the total electrons, and glucose decreased below the detection

278

limit. Methane slowly accumulated within 2 days of operation and reached a plateau after day 6,

279

when methane generation accounted for ~ 15% of the total electrons supplied from glucose.

280

15% methane is less than the estimate in Figure 1A (25%), probably due to either H2

281

consumption by either ARB or homo-acetogenesis. The total electrons contained in other

282

fermentation products were small: ~ 3.3 % for propionate and ~ 3.1% for butyrate within the

283

first 4 days of operation. Propionate and butyrate were completely consumed at day 17 and day

284

8, respectively.

285

We detected no H2 in the headspace gas of the MEC’s anode. Thus, H2 produced during

286

glucose fermentation was quickly channeled to methane by H2-consuming methanogens, to

287

current by ARB, or to acetate and then current by homoacetogens and ARB.13,24 The Coulombic

288

recovery at the end of batch cycle (~ 57%) was slightly greater than the estimated value in Figure

289

S1, which suggests that a modest flow of electron where routed through H2 to the anode.

290 291

Figure 1B shows the distribution of electrons from glucose during 20 days of batch operation with 0.18 g FAN/L, where methane generation was partly inhibited. Compared to the



292

MEC fed with 0.02 g FAN/L (Figure 1A), current slowly increased during the first 2 days and

293

was associated with complete glucose fermentation. However, lactate started to increase,

294

becoming the largest electron sink (~ 30%) at day 2, followed by acetate (~ 13%), propionate (~

295

5%), and iso-butyrate (~ 2%). Since lactate fermentation is more likely than direct anode

296

respiration, it has to be fermented first before it can be utilized by ARB. Lactate fermentation

297

proceeded rapidly, and the current increased to become the largest electron sink after day 6, at

298

which time methane accounted for ~ 9% of the total electrons supplied from glucose. Although

299

we did not detect any H2 in the headspace gas, formate was detected in low concentration (0.67

300

mM), accounting for nearly 2% of glucose’s total electrons. These results support that that 0.18

301

g FAN/L delayed lactate fermentation, rather than glucose fermentation, and significantly

302

inhibited methanogenesis. A key finding is that CE was ~ 76%, which illustrates the benefit to

303

inhibiting methanogenesis.

304

Further increasing the initial FAN concentration to 0.37 g FAN/L (Figure 1C) had a more

305

pronounced effect on fermentation, although the impact on methanogenesis was not much

306

greater than 0.18 g FAN/L. Similar to the lower FAN concentration, the rate of glucose

307

consumption did not change; however, 0.37 g FAN-N/L altered the fermentation kinetics and

308

pathway. During the first 4 days, lactate, iso-butyrate, propionate, butyrate, and acetate

309

accumulated, collectively accounting for ~ 25% of glucose’s electrons. After 4 days,

310

fermentation proceeded rapidly with concurrent increases in electric current, which became the

311

largest electron sink by the end of the batch cycle (~ 62%), suggesting that this high FAN

312

concentration might partially inhibit ARB. However, methanogenesis was not completely

313

inhibited, as it accounted for ~ 6% of glucose’s electrons.


Page 16 of 43

Page 17 of 43

314


As a result of organic-acids accumulation, the pH at the end of all batch cycles decreased

315

from their initial pH value (8.1) to a narrow range of 6.85−7.10. Since the pH values were not

316

much different from each other, variations in pH should have had a minimal effect on

317

fermentation pathway and kinetics.36

318 319

Figure 1.

320 321 322

Electron balances for batch MECs Figure S2 presents the electron balances at the end of batch-cycle operation: 57±2.9%,

323

76±3.5%, and 62±2.7% of the total electrons from glucose ended up as electric current for MECs

324

fed with 0.02, 0.18, and 0.37 g FAN/L, respectively. Theoretically, ~ 25% (or 9.7 me– eq) of

325

glucose’s electrons (i.e., 38.4 me– eq) should have been ended up as methane, if all the generated

326

H2 were utilized by H2-consuming methanogens (Figure S1A). However, we detected as

327

methane only about 15%, 9%, and 6%, respectively, of the original electrons.

328

Electrons ending up as organic acids at the end of the experiment were by far the highest

329

for the MEC fed with 0.37 g FAN/L: ~ 16% of the influent electrons, versus ~ 0.2% and ~ 2%

330

for MECs fed 0.02 and 0.18 g FAN-N/L, respectively. Approximately 28%, 13%, and 16% of

331

the electron from glucose were unaccounted by directly measured components. Unidentified

332

components include biomass, soluble microbial products, and fermentation products not

333

measured by HPLC. Figure S1 shows an estimate of 25 and 24% of electrons ending up in

334

biomass when methanogenesis is active versus completely inhibited.



335 336

Semi-continuous glucose fermentation in MECs To examine whether high FAN concentration affected fermentation, methanogenesis, or

337

both, we performed MEC experiments using semi-continuous operation by replacing the feed

338

medium (i.e., 5 mM glucose) once every two days. Figure 2 summarizes the results for both

339

MECs, which showed similar trends. The current density was stable over the influent FAN

340

range of 0.003 to 0.18 g FAN-N/L (Figure 2A), but a further increase of influent FAN to 0.37 g

341

N/L led to a gradual decrease in the current density after 2 semi-continuous cycles. This pattern

342

suggests that higher FAN slowed acetate oxidation by ARB, since acetate accumulated at the end

343

of semi-continuous cycle (Figure 2B).

344

Similar to the batch MECs, glucose was completely fermented for all FAN conditions,

345

confirming that high FAN concentration did not affect the first fermentation step. As the FAN

346

concentration increased, more electrons ended up in organic acids, and this was associated with

347

17 – 29% decreases in methane yield. Perhaps high FAN altered the fermenters’ intracellular pH

348

homeostasis, leading them to divert electrons away from H2 production and toward formation of

349

more-reduced organic products.37 A shift from H2 to organic acids is consistent with high FAN

350

maintaining a low H2 concentration in the MEC, a change that should be beneficial for

351

syntrophic interactions not involving hydrogenotrophic methanogens.

352 353

Figure 2.

354

355

Distribution of bacterial population in batch MECs

356

Figure 3 and Figure S3 report the sequence analyses of the V4 region in the bacterial 16S

357

rRNA gene in the batch-MEC samples. The majority of bacterial 16S rRNA genes in all samples 18 ACS Paragon Plus Environment

Page 18 of 43

Page 19 of 43


358

belonged to 3 phyla: Proteobacteria, Firmicutes, and Bacteroidetes (Figure 3A), which is

359

consistent with previous studies.9,38,39 Several members of phyla Bacteroidetes and Firmicutes

360

are known to ferment sugar, whereas many members of Proteobacteria are known to perform

361

anode respiration.40,41 The lower abundance of Proteobacteria in the suspended-phase (SP)

362

samples agrees with the fact that most of ARB can only use the anode surface as terminal

363

electron acceptor.

364

Among the Proteobacteria, Deltaproteobacteria were the largest sub-group. The

365

predominant genus among Deltaproteobacteria was Geobacter, and their relative abundance

366

increased with higher FAN. Whereas biofilm (Bf) samples from 0.02 g FAN/L had ~ 2.6% of

367

the total genus sequences in Geobacter, samples from 0.18 and 0.37 g FAN/L had 34.5% and

368

62% of the total genus sequences, respectively (Figure 3B). The high abundance of

369

Geobacteraceae in 0.18 and 0.37 g FAN/L Bf samples agrees with their higher CEs compared to

370

the control MEC, and it confirms that the relatively high FAN concentration gave the ARB an

371

ecological advantage that enhance electron flow towards anode respiration versus

372

methanogenesis. Although we observed high relative abundance of Geobacteraceae for the MEC

373

fed with 0.37 g FAN/L, its CE was lower when compared to MEC fed with 0.18 g FAN/L (p-

374

value < 0.05; pairwise t-test). This probably was due to partial inhibition to the biofilm’s ARB at

375

higher FAN. Partial inhibition of acetate uptake by ARB in the biofilm anode made the

376

microbial community less functionally effective for anode respiration, even though the relative

377

abundance of Geobacteraceae in anode’s biofilm remained high.

378 379

Figure 3



380

The relative abundance of Firmicutes was similar in all samples except for 0.37 g FAN/L

381

Bf, but their compositions varied significantly. Figure S4 shows community breakdown at the

382

family level within the phylum Firmicutes. An increase in FAN concentration led to the

383

emergence of Clostridiaceae and Ruminococcaceae families, which belong to the order

384

Clostridiales. Compensating losses occurred for the family Tissierellaceae. Recently, Müller et

385

al. 20 revealed that several members of the Clostridia class, mainly belonging to the orders

386

Clostridiales, can perform syntrophic acetate oxidation (SAO) (See Table 1), which was the

387

predominant pathway for methane production with elevated ammonia levels.20,42

388

We also detected an increase in the relative abundance of Lactobacillales, which include

389

genera Lactobacillus and Enterococcus, at the highest FAN conditions (0.18 and 0.37 g FAN/L).

390

Lactobacillales are reported to play a major role in lactic-acid production in fermentation,43 and

391

this is consistent with the observation of lactate accumulation during glucose fermentation in

392

MECs fed with 0.18 and 0.37 g FAN/L (Figure 1).

393

Supplementary Table S1 and Figure S5A show all the values for the bacterial community

394

diversity metrics for Bf and SP samples. The Chao1, Shannon, and PD values reflect that the Bf

395

samples developed more diverse bacterial community than the corresponding SP samples. Also,

396

the bacterial diversity of Bf and SP samples from 0.18 g FAN/L-fed MEC, which achieved the

397

highest CE (Figure 1), was greater than from the other two MECs. Consistent with the diversity

398

results and based on the equitability coefficient, the evenness increased as FAN concentration

399

increased from 0.02 to 0.18 g FAN/L, while the 0.37 g FAN/L-fed MEC was least even (Figure

400

S5B). Previous studies revealed that microbial communities with greater evenness are often

401

associated with more robustness and functional stability compared to less even microbial

402

communities.29,44


Page 20 of 43

Page 21 of 43


The results of weighted PCoA analysis, Figure 4, show that principal components (PC) 1

403 404

and 2 explained 33.7% and 29.8% of the total bacterial community variations, respectively.

405

Increasing FAN concentration correlated with the PC2 vector, whereas the type of sample (Bf

406

versus SP) correlated with the PC1 vector. The trend along PC2 was associated with the emergence of the orders

407 408

Desulfuromonadales, Lactobacillales, and Synergistales at elevated FAN concentration; to

409

compensate, the relative abundances of other orders, such as Clostridiales and Bacteroidales,

410

decreased in response in change in FAN concentration (Figure 4). Several members of

411

Desulfuromonadales, a sub-group of Deltaproteobacteria, and Lactobacillales are well-known

412

ARB and lactic acid producers, respectively. Although Synergistales, a sub-group of phylum

413

Synergistetes, have been detected in anaerobic digesters treating different wastewater, their

414

functions are still mysterious.45,46 One study suggested that members of this order are potential

415

propionate consumers,47 but another study suggested that they might be SAOB.48 The

416

emergence of those orders, coupled with the decreases of Clostridiales and Bacteroidales, well-

417

known acetic acid-producing fermenters, supports that higher FAN altered glucose fermentation

418

towards the production of higher organic acids (e.g., lactate) and away from H2, rather than the

419

formation of 2 moles of acetate and 4 moles of H2 per mole of glucose, as illustrated in Figure

420

S1.

421

The trend along PC1 was associated with the emergence of the orders Bacteroidales and

422

Lactobacillales, which play an important role in fermentation of complex substrates.

423

Compensating for these increases, the relative abundance of Desulfuromonadales decreased

424

along PC1 vector. These results confirm that most of fermentation occurred in the suspension

425

phase, since we operated MECs in batch mode; ARB dominated the MECs’ biofilm.



426

Figure 4.

427 428

429

Distribution of Archaea population in batch MECs Figure 5 summarizes the sequencing results for Archaea. Hydrogenotrophic

430

methanogens dominated all Bf and SP Archaea communities, regardless the FAN concentration,

431

which is consistent with previous work.9,40 The percentage of Archaea relative to bacteria, based

432

on the prokaryotic library results, decreased as FAN increased, supporting that the methanogens

433

were partially inhibited by higher FAN (Figure S6). For example, the percentage of Archaea

434

relative to Bacteria in Bf samples was about 10.8%, 3.8%, and 4.0% for 0.02 g FAN/L, 0.18 g

435

FAN/L, and 0.37 g FAN/L, respectively. The percentage of Archaea to bacteria was higher for

436

all SP samples than for Bf samples, confirming that the methanogens were predominantly in the

437

suspended phase of MECs operated in batch mode. This trend is reinforced by the qPCR results

438

targeting the mcrA gene (Figure 5A) and by the decrease in CH4 production in the MECs’

439

headspace fed with 0.18 g FAN/L and 0.37 g FAN/L (i.e., 9% and 6% of the original electrons,

440

respectively) compared to control MEC (i.e., 15% of the original electrons) (Figure 1). The

441

mcrA gene copies in the MECs’ anodes fed with high FAN concentrations (0.18 g FAN/L, and

442

0.37 g FAN/L) were statically significant lower (p-value < 0.05; pairwise t-test) compared to

443

control experiment (0.02 g FAN/L). The most likely reason for increasing the mcrA gene copies

444

per reactor volume in the SP sample is either biomass detachment from the biofilm into the

445

supernatant or DNA residual from the inoculum.

446

For all experimental runs, we did not detect any acetoclastic methanogens in all Bf and

447

SP samples, which were dominated by hydrogenotrophic methanogens (i.e., Methanobacterium

448

and Methanobrevibacter) (Figure 5B). For the 0.02 g FAN/L bf, Methanobacterium was


Page 22 of 43

Page 23 of 43


449

dominant (~83% of sequence), while Methanobrevibacter (74% for 0.18 g FAN/L biofilm and

450

91% for 0.37 g FAN/L biofilm) and Methanobacterium (16% for 0.18 g FAN/L biofilm and 11%

451

for 0.37 g FAN/L biofilm) shared the archaeal community for higher FAN concentrations.

452

Several members of both genera are reported to be resistant to ammonia inhibition up to 400 mM

453

(i.e., 5.6 g TAN/L or ~ 0.04 g FAN/L at pH 6.8).49

454

The higher relative abundance of Methanobrevibacter with higher FAN suggests that

455

they contained the strains more resistant to FAN inhibition. Following the FAN increase,

456

Methanobacterium started to outcompete Methanobrevibacter (Figure 5B). A new uncultured

457

Archaea, classified as a candidate genus vadinCA11 (Thermoplasmata sp.), emerged as FAN

458

decreased. This genus was detected in an anaerobic fluidized bed fed with wine-distillation

459

waste,51 and it is potentially halophilic.52 The results suggest that the community shifted toward

460

methanogens tolerant to FAN.

461 462

Figure 5.

463 464 465

High FAN did not affect homo-acetogens Previous work revealed that suppressing methanogenesis was the main reason that high

466

CEs were achieved in MECs.9 Suppressing H2-oxidizing methanogens provides ideal conditions

467

for homoacetogens to oxidize H2 and grow, leading to the generation of acetate, the ideal

468

substrate for ARB. 8,9 If this scenario were true, we should have observed a larger population of

469

homoacetogens with higher FAN concentration. To test this hypothesis, we tracked the relative

470

abundance of homoacetogens by targeting the FTHFS gene using qPCR; the results are in Figure

471

S7. FTHFS gene copies for all Bf and SP samples were comparable, regardless of the FAN



472

concentration. The qPCR data also are consistent with bacterial 16S rRNA gene sequence data,

473

where we detected genus Treponema, a well-known homoacetogenic Spirochaetes (Figure 3B),

474

but at a very low relative abundance (< 1% of total sequences) for all samples. Thus,

475

homoacetogens were consistently present, and changes in their numbers likely were not

476

responsible for enhancing CE.

477 478 479

New insights into metabolic flexibility in MECs Our results provide insight for possible way to decrease CH4 production and increase CE

480

by controlling the fermentation step using FAN, instead of inhibiting methanogens using

481

expensive or toxic chemical inhibitors. Altering fermentation led to less production of H2, which

482

is the “fuel” for methanogens, resulting in less CH4 production and higher CE, even though

483

methanogenesis was not completely suppressed. The lowering of H2 generation was

484

accompanied by more production of short-chain organic acids. However, relatively high FAN

485

(0.37 g FAN/L in my study) might promote SAOB, which are competitors for acetoclastic ARB,

486

introducing the possibility of a new pathway for acetate consumption in MECs fed with

487

fermentable substrates; it is a pathway to be avoided.

488

SAO might be the reason for the lower CE we observed with the MEC fed with 0.37 g

489

FAN/L, compared to MEC fed with 0.18 g FAN/L. Although acetate-based anode respiration is

490

thermodynamically favorable compared to SAO (See Table 1), the half-saturation constant (Ks)

491

for SAOB ranges from 4.7 to 13 mM,48,53 values ~2.5- to 7-fold higher than the maximum Ks

492

reported for mixed-culture ARB (1.86 mM).54 Despite the higher Ks value for acetate, the SAOB

493

may be more tolerant to high FAN, which would give them a kinetic advantage compared to

494

ARB, since ARB often are susceptible to a high FAN concentration. This observation coincides


Page 24 of 43

Page 25 of 43


495

with emergence of Clostridiaceae and Ruminococcaceae families, which are potential SAOB.

496

Further investigation of SAOB in MECs is warranted.

497

An important implication of this study is that methanogenesis does not need to be

498

completely suppressed to achieve high current production and CE from MECs fed with

499

fermentable substrates. Using FAN to suppress methanogenesis is a realistic option for scaling-

500

up MECs during the biodegradation of fermentable substrates, particularly when the feed stream

501

has a high nitrogen content, such as animal manures and landfill leachate.

502 503

Acknowledgements

504 505

We thank Dr. Juan Maldonado, Dr. Zehra Esra Ilhan, and Dr. Prathap Parameswaran for helpful

506

discussion and guidance. This study was partially supported by the endowment fund from Mr.

507

Brian Swette to the Swette Center for Environmental Biotechnology and Graduate and

508

Professional Student Association at Arizona State University. Mohamed Mahmoud was

509

supported by an Egyptian government fellowship (GM 0931).

510

511

Conflicts of interest

512 513

The authors declare that there is no conflict of interest regarding the publication of this paper.

514 515 516 517



518

Supporting Information

519 520

Available in the Supporting Information are: Figure S1-S8: Electron flows from glucose into

521

different electron sinks in an MEC’s anode, Electron balance of MECs at the end of batch-cycle

522

operation, bacterial community distribution at the class level, composition of phylum Firmicutes

523

at the family level, bacterial community diversity and richness results, archaea-to-bacteria ratio,

524

FTHFS gene copies per reactor determined by qPCR at the end of batch cycles; and Table S1:

525

Chao1, Shannon, and PD indices for bacterial sequences.

526


Page 26 of 43

Page 27 of 43

527


References

528 529

1. Torres, C. I. On the importance of identifying, characterizing, and predicting fundamental

530

phenomena towards microbial electrochemistry applications. Curr. Opin. Biotechnol. 2014, 27,

531

107–114.

532 533

2. Lovley, D. R. The microbe electric: conversion of organic matter to electricity. Curr. Opin.

534

Biotechnol. 2008, 19, 564–571.

535 536

3. Angenent, L. T.; Karim, K.; Al-Dahhan, M. H.; Wrenn, B. A.; Domíguez-Espinosa, R.

537

Production of bioenergy and biochemicals from industrial and agricultural wastewater. Trends

538

Biotechnol. 2004, 22, 477–485.

539 540

4. Kiely, P. D.; Regan, J. M.; Logan, B. E. The electric picnic: synergistic requirements for

541

exoelectrogenic microbial communities. Curr. Opin. Biotechnol. 2011, 22, 378–385.

542 543

5. Logan, B. E.; Regan, J. M. Electricity-producing bacterial communities in microbial fuel cells.

544

Trends Microbiol. 2006, 14, 512–518.

545 546

6. Lovley, D. R.; Coates, J. D. Novel forms of anaerobic respiration of environmental relevance.

547

Opin. Microbiol. 2000, 3, 252–256.

548



549

7. Thauer, R. K.; Jungermann, K.; Decker, K. Energy conservation in chemotrophic anaerobic

550

bacteria. Bacteriol. Rev. 1977, 41, 100–180.

551 552

8. Parameswaran, P.; Torres, C. I.; Lee, H. S.; Krajmalnik-Brown, R.; Rittmann, B. E.

553

Syntrophic interactions among anode respiring bacteria (ARB) and Non-ARB in a biofilm anode:

554

electron balances. Biotechnol. Bioeng. 2009, 103, 513–523.

555 556

9. Parameswaran, P.; Zhang, H.; Torres, C. I.; Rittmann, B. E.; Krajmalnik-Brown, R. Microbial

557

community structure in a biofilm anode fed with a fermentable substrate: the significance of

558

hydrogen scavengers. Biotechnol. Bioeng. 2010, 105, 69–78.

559 560

10. Freguia, S.; Rabaey, K.; Yuan, Z.; Keller, J. Syntrophic processes drive the conversion of

561

glucose in microbial fuel cell anodes. Environ. Sci. Technol. 2008, 42, 7937–7943.

562 563

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

564

and archaea. Nat. Rev. Microbiol. 2009, 7, 568–577.

565 566

12. Rittmann, B. E.; McCarty, P. L. Environmental biotechnology: Principles and applications;

567

McGraw-Hill: New York, 2001.

568 569

13. Zinder, S. H. Physiological ecology of methanogens. In: Ferry JG, editor. Methanogenesis—

570

Ecology, physiology, biochemistry & genetics. New York, NY: Chapman & Hall. 1993, pp 128–

571

206.


Page 28 of 43

Page 29 of 43


572

14. Schuchmann, K.; Müller, V. Autotrophy at the thermodynamic limit of life: a model for

573

energy conservation in acetogenic bacteria. Nat. Rev. Microbiol. 2014, 12, 809–821.

574 575

15. McInerney, M. J.; Struchtemeyer, C. G.; Sieber, J.; Mouttaki, H.; Stams, A. J.; Schink, B.;

576

Rohlin, L.; Gunsalus, R. P. Physiology, ecology, phylogeny, and genomics of microorganisms

577

capable of syntrophic metabolism. Ann. New York Acad. Sci. 2008, 1125, 58–72.

578 579

16. Ishii, S.; Shimoyama, T.; Hotta, Y.; Watanabe, K. Characterization of a filamentous biofilm

580

community established in a cellulose-fed microbial fuel cell. BMC Microbiol. 2008, 8, 1–12.

581 582

17. Zhu, X.; Siegert, M.; Yates, M. D.; Logan, B. E. Alamethicin suppresses methanogenesis and

583

promotes acetogenesis in bioelectrochemical systems. Appl. Environ. Microbiol. 2015, 81, 3863–

584

3868.

585 586

18. Chae, K. J.; Choi, M. J.; Kim, K. Y.; Ajayi, F. F.; Park, W.; Kim, C. W.; Kim, I. S.

587

Methanogenesis control by employing various environmental stress conditions in two-chambered

588

microbial fuel cells. Bioresour. Technol. 2010, 101, 5350–5357.

589 590

19. Mahmoud, M.; Parameswaran, P.; Torres, C. I.; Rittmann, B. E. Electrochemical techniques

591

reveal that total ammonium stress increases electron flow to anode respiration in mixed-species

592

bacterial anode biofilms. Biotechnol. Bioeng. 2017 (In press; DOI: 10.1002/bit.26246).

593



594

20. Müller, B.; Sun, L.; Westerholm, M.; Schnürer, A. Bacterial community composition and fhs

595

profiles of low- and high-ammonia biogas digesters reveal novel syntrophic acetate-oxidising

596

bacteria. Biotechnol. Biofuels. 2016, 9, 48.

597 598

21. Mahmoud, M.; Parameswaran, P.; Torres, C. I.; Rittmann, B. E. Fermentation pre-treatment

599

of landfill leachate for enhanced electron recovery in a microbial electrolysis cell (MEC).

600

Bioresour. Technol. 2014, 151, 151–158.

601 602

22. Yenigün, O.; Demirel, B. Ammonia inhibition in anaerobic digestion: A review. Process

603

Biochem. 2013, 48, 901–911.

604 605

23. Zinder, S. H.; Koch, M. Non-aceticlastic methanogenesis from acetate: acetate oxidation by a

606

thermophilic syntrophic coculture. Arch. Microbiol. 1984, 138, 263–272.

607 608

24. Shimada T.; Morgenroth, E.; Tandukar, M.; Pavlostathis, S. G.; Smith, A.; Raskin, L.; Kilian,

609

R. E. Syntrophic acetate oxidation in two-phase (acid-methane) anaerobic digesters. Water Sci.

610

Technol. 2011, 64, 1812–1820.

611 612

25. Lee, H. S.; Torres, C. I.; Parameswaran, P.; Rittmann, B. E. Fate of H2 in an upflow single-

613

chamber microbial electrolysis cell using a metal-catalyst-free cathode. Environ. Sci. Technol.

614

2009, 43, 7971–7976.

615


Page 30 of 43

Page 31 of 43


616

26. Steinberg, L. M.; Regan, J. M. mcrA-targeted real-time quantitative PCR method to examine

617

methanogen communities. Appl. Environ. Microbiol. 2009, 75, 4435–4442.

618 619

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

620

Owens, S. M.; Betley, J.; Fraser, L.; Bauer, M.; Gormley, N.; Gilbert, J. A.; Smith, G.; Knight,

621

R. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq

622

platforms. ISME J. 2012, 6, 1621–1624.

623 624

28. Takai, K.; Horikoshi, K. Rapid detection and quantification of members of the archaeal

625

community by quantitative PCR using fluorogenic probes. Appl. Environ. Microbiol. 2000, 66,

626

5066–5072.

627 628

29. Werner, J. J.; Knights, D.; Garcia, M. L.; Scalfone, N. B.; Smith, S.; Yarasheski, K.;

629

Cummings, T. A.; Beers, A. R.; Knight, R.; Angenent, L. T. Bacterial community structures are

630

unique and resilient in full-scale bioenergy systems. Proc. Natl. Acad. Sci. U.S.A. 2011, 108,

631

4158–4163.

632 633

30. Caporaso, J. G.; Kuczynski, J.; Stombaugh, J.; Bittinger, K.; Bushman, F. D.; Costello, E. K.;

634

Fierer, N.; Pena, A. G.; Goodrich, J. K.; Gordon, J. I.; Huttley, G. A.; Kelley, S. T.; Knight, D.;

635

Koenig, J. E.; Ley, R. E.; Lozupone, C. A.; McDonald, D.; Muegge, B. D.; Pirrung, M.; Reeder,

636

J.; Sevinsky, J. R.; Tumbaugh, P. J.; Walters, W. A.; Widmann, J.; Yatsunenko, T.; Zaneveld, J.;

637

Knight, R. QIIME allows analysis of high-throughput community sequencing data. Nature

638

Methods 2010, 7, 335–336.



639

31. Cole, J. R.; Wang, Q.; Cardenas, E.; Fish, J.; Chai, B.; Farris, R. J.; Kulam-Syed-Mohideen,

640

A. S.; McGarrell, D. M.; Marsh, T.; Garrity, G. M.; Tiedje, J. M. The Ribosomal Database

641

Project: Improved alignments and new tools for rRNA analysis. Nucl. Acids Res. 2009, 37,

642

D141–D145.

643 644

32. Chao, A. Estimating the population-size for capture recapture data with unequal catchability.

645

Biometrics 1987, 43, 783–791.

646 647

33. Faith, D. P. Conservation evaluation and phylogenetic diversity. Biol. Cons. 1992, 61, 1–10.

648 649

34. Ni, B. J.; Liu, H.; Nie, Y. Q.; Zeng, R. J.; Du, G. C.; Chen, J.; Yu, H. Q. Coupling glucose

650

fermentation and homoacetogenesis for elevated acetate production: Experimental and

651

mathematical approaches. Biotechnol. Bioeng. 2011, 108, 345–353.

652 653

35. Graber, J. R.; Breznak, J. A. Physiology and nutrition of Treponema primitia, an H2/CO2-

654

acetogenic spirochete from termite hindguts. Appl. Environ. Microbiol. 2004, 70, 1307–1314.

655 656

36. Lee, H. S.; Salerno, M. B.; Rittmann, B. E. Thermodynamic evaluation on H2 production in

657

glucose fermentation. Environ. Sci. Technol. 2008, 42, 2401–2407.

658 659

37. González-Cabaleiro, R.; Lema, J. M.; Rodríguez, J. Metabolic energy-based modelling

660

explains product yielding in anaerobic mixed culture fermentations. PLoS ONE 2015, 10,

661

e0126739.


Page 32 of 43

Page 33 of 43


662

38. Mahmoud, M.; Parameswaran, P.; Torres, C. I.; Rittmann, B. E. Relieving the fermentation

663

inhibition enables high electron recovery from landfill leachate in a microbial electrolysis cell.

664

RSC Advances 2016, 6, 6658 – 6664.

665 666

39. Sanchez-Herrera, D.; Pacheco-Catalan, D.; Valdez-Ojeda, R.; Canto-Canche, B.;

667

Dominguez-Benetton, X.; Domínguez-Maldonado, J.; Alzate-Gaviria, L. Characterization of

668

anode and anolyte community growth and the impact of impedance in a microbial fuel cell. BMC

669

Biotechnol. 2014, 14, 102.

670 671

40. Siegert, M.; Li, X. F.; Yates, M. D.; Logan, B. E. The presence of hydrogenotrophic

672

methanogens in the inoculum improves methane gas production in microbial electrolysis cells.

673

Front. Microbiol. 2015, 5, 778.

674 675

41. Rismani-Yazdi, H.; Carver, S. M.; Christy, A. D.; Yu, Z.; Bibby, K.; Peccia, J.; Tuovinen, O.

676

H. Suppression of methanogenesis in cellulose-fed microbial fuel cells in relation to

677

performance, metabolite formation, and microbial population. Bioresour. Technol. 2013, 129,

678

281–288.

679 680

42. Westerholm, M.; Levén, L.; Schnürer, A. Bioaugmentation of syntrophic acetate-oxidizing

681

culture in biogas reactors exposed to increasing levels of ammonia. Appl. Environ. Microbiol.

682

2012, 78, 7619–7625.

683



684

43. De Vrieze, J.; Saunders, A. M.; He, Y.; Fang, J.; Nielsen, P. H.; Verstraete, W.; Boon, N.

685

Ammonia and temperature determine potential clustering in the anaerobic digestion microbiome.

686

Water Res. 2015, 75, 312–323.

687 688

44. Wittebolle, L.; Marzorati, M.; Clement, L.; Balloi, A.; Daffonchio, D.; Heylen, K.; De Vos,

689

P.; Verstraete, W.; Boon, N. Initial community evenness favours functionality under selective

690

stress. Nature 2009, 458, 623–626.

691 692

45. Zamanzadeh, M.; Hagen, L. H.; Svensson, K.; Linjordet, R.; Horn, S. J. Anaerobic digestion

693

of food waste – Effect of recirculation and temperature on performance and microbiology. Water

694

Res. 2016, 96, 246–254.

695 696

46. Delbès, C.; Moletta, R.; Godon, J. J. Bacterial and archaeal 16S rDNA and 16S rRNA

697

dynamics during an acetate crisis in an anaerobic digestor ecosystem. FEMS Microbiol. Ecol.

698

2001, 35, 19–26.

699 700

47. Hagen, L. H.; Vivekanand, V.; Linjordet, R.; Pope, P. B.; Eijsink, V. G. H.; Horn, S. J.

701

Microbial community structure and dynamics during co-digestion of whey permeate and cow

702

manure in continuous stirred tank reactor systems. Bioresour. Technol. 2014, 171, 350–359.

703 704

48. Ito, T.; Yoshiguchi, K.; Ariesyady, H. D.; Okabe, S. Identification of a novel acetate-utilizing

705

bacterium belonging to Synergistes group 4 in anaerobic digester sludge. ISME J. 2011, 5, 1844–

706

1856.


Page 34 of 43

Page 35 of 43


707

49. Sprott, G. D.; Patel, G. B. Ammonia toxicity in pure cultures of methanogenic bacteria. Syst.

708

Appl. Microbiol. 1986, 7, 358–363.

709 710

51. Godon, J. J.; Zumstein, E.; Dabert, P.; Habouzit, F.; Moletta, R. Molecular microbial

711

diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. Appl.

712

Environ. Microbiol. 1997, 63, 2802–2813.

713 714

52. Durbin, A. M.; Teske, A. Archaea in organic-lean and organic-rich marine subsurface

715

sediments: an environmental gradient reflected in distinct phylogenetic lineages. Front.

716

Microbiol. 2012, 3, 168.

717 718

53. Rivera-Salvador, V.; López-Cruz, I. L.; Espinosa-Solares, T.; Aranda-Barradas, J. S.; Huber,

719

D. H.; Sharma, D.; Toledo, J. U. Application of Anaerobic Digestion Model No. 1 to describe the

720

syntrophic acetate oxidation of poultry litter in thermophilic anaerobic digestion. Bioresour.

721

Technol. 2014, 167, 495–502.

722

723

54. Lee, H. S.; Torres, C. I.; Rittmann, B.E. Effects of substrate diffusion and anode potential on

724

kinetic parameters for anode-respiring bacteria. Environ. Sci. Technol. 2009, 43, 7571–7577.



725

List of Tables

726 727

Table 1. Overview of reactions involving acetate and H2


Page 36 of 43

Page 37 of 43

728


Table 1

729

Process (1) Glucose fermentation: C6H12O6 + 2H2O → 2 CH3COO− + 4H2 + 2CO2 + 2H+

∆G°rxn (kJ/e– eq) a – 8.59

(2) Acetoclastic methanogenesis: CH3COO− + H2O → CH4 + HCO3−

– 3.88

(3) Hydrogenotrophic methanogenesis: HCO3− + 4H2 + H+ → CH4 + 3H2O

−16.38

(4) Homoacetogenesis: 2 CO2 + 4H2 → CH3COO− + H+ + 2H2O

− 11.88

(5) Acetoclastic anode respiration b: CH3COO− + 2H2O → 2CO2 + 7H+ + 8e−

− 19.30

(6) Hydrogenotrophic anode respiration b: 2H2 → 2H+ + 2e−

− 9.65

(7) Acetate oxidation to H2: CH3COO− + 4H2O → 2HCO3− + 4H2 + H+

+13.10

730

a

We calculated ∆G°rxn at standard conditions (i.e., 298 K, 1 atm for gases, pH 7, and 1 M for

731

soluble reactants) based on the thermodynamic data provided in Thauer et al.11; b Near-optimum

732

anode potential = +0.2 V versus SHE.15

733



734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756

Figures caption Figure 1. Electron distributions in MECs fed with 5-mM glucose at an initial pH of 8.1: (A) 0.02 g FAN/L or 0.2 g TAN/L (final pH = 6.85), (B) 0.18 g FAN/L or 2 g TAN/L (final pH = 7.10), and (C) 0.37 g FAN-N/L or 4 g TAN/L (final pH = 6.89). Figure 2. Performance of semi-continuous MECs fed with different FAN concentrations: (A) Current generation profile of duplicate MECs versus time. (B) Electron balance of MECs at the end of semi-continuous cycle operation in panel A. Figure 3. Bacterial community sequencing results. (A) bacterial community distribution at the phylum level. Phyla with less than 1% of total sequences are grouped as “others”. (B) bacterial community distribution at the genus level. Genera with less than 1% of total sequences are grouped as “others”. Figure 4. Weighted Unifrac analysis shows that the relative abundance of order-level phylotypes on the Principal Coordinates. FAN concentration determined the main phylotypes that drove the community structures in MECs. Figure 5. Archaeal community analyses. (A) mcrA gene copies per reactor determined by qPCR at the end of batch cycles. All error bars show standard deviations of triplicate measurements. (B) Archaeal community distribution at the genus level. Genera with less than 1% of total sequences are grouped as “others”.


Page 38 of 43

Page 39 of 43

757 758 759


Figure 1



760 761

762 763 764 765

Figure 2


Page 40 of 43

Page 41 of 43


766 767

768 769 770

Figure 3



771 772 773 774

Figure 4


Page 42 of 43

Page 43 of 43


775

776 777 778 779 780

Figure 5

781


Changes in Glucose Fermentation Pathways as a Response to the

Recommend Documents