Microbial Selenate Reduction Driven by a Denitrifying Anaerobic

Advanced Water Management Centre, Faculty of Engineering, Architecture and Information. 3. Technology .... internal diameter). The schematic diagram o...
0 downloads 8 Views 788KB Size
Subscriber access provided by - Access paid by the | UCSB Libraries

Environmental Processes

Microbial Selenate Reduction Driven by a Denitrifying Anaerobic Methane Oxidation Biofilm Jinghuan Luo, Hui Chen, Shihu Hu, Chen Cai, Zhiguo Yuan, and Jianhua Guo Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05046 • Publication Date (Web): 13 Mar 2018 Downloaded from http://pubs.acs.org on March 14, 2018

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

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

Page 1 of 22

Environmental Science & Technology

1

Microbial Selenate Reduction Driven by a Denitrifying Anaerobic Methane Oxidation Biofilm

2

Jing-Huan Luo#, Hui Chen#, Shihu Hu, Chen Cai, Zhiguo Yuan, Jianhua Guo*

3

Advanced Water Management Centre, Faculty of Engineering, Architecture and Information

4

Technology, The University of Queensland, St Lucia, Queensland 4072, Australia.

5

*Corresponding author: Jianhua Guo, Phone: + 61 7 3346 3222; FAX: + 61 7 3365 4726; E-mail:

6

[email protected]

7

# These authors contributed equally to this work.

8

9

ABSTRACT

10 11

Anaerobic oxidation of methane (AOM) plays a crucial role in controlling the flux of methane from

12

anoxic environments. Sulfate-, nitrite-, nitrate-, and iron-dependent methane oxidation processes

13

have been considered to be responsible for the AOM activities in anoxic niches. Here, we report

14

that nitrate-reducing AOM microorganisms, enriched in a membrane biofilm bioreactor (MBfR),

15

are able to couple selenate reduction to AOM. According to ion chromatography (IC), X-ray

16

photoelectron spectroscopy (XPS) and long-term bioreactor performance, we reveal that soluble

17

selenate was reduced to nanoparticle elemental selenium. High-throughput 16S rRNA gene

18

sequencing indicates that Candidatus Methanoperedens and Candidatus Methylomirabilis remained

ACS Paragon Plus Environment

Environmental Science & Technology

19

the only known methane-oxidising microorganisms after nitrate was switched to selenate,

20

suggesting that these organisms could couple anaerobic methane oxidation to selenate reduction.

21

Our findings suggest a possible link between the biogeochemical selenium and methane cycles.

22

INTRODUCTION

23

Methane is a potent greenhouse gas, with a global warming potential 28 times that of carbon

24

dioxide over a 100-year horizon.1 Aerobic or anaerobic methane oxidation processes are the

25

dominant methane sinks to regulate methane concentration in the atmosphere, which is 1.8 ppm

26

currently.2-4 In particular, the anaerobic oxidation of methane (AOM), mainly driven by anaerobic

27

methanotrophic (ANME) archaea, consumes 90% of methane produced in ocean sediments before it

28

enters the atmosphere.5 Sulfate-coupled AOM has been mostly studied, which is mediated by a

29

synergistic consortium between ANME archaea and sulfate-reducing bacteria (SRB),5, 6 or by the

30

ANME alone (sulfate reduced to elemental sulfur by ANME directly).7 More recently, it was found

31

that AOM could also be coupled to denitrification, a process termed as denitrifying anaerobic

32

methane oxidation (DAMO). Two microorganisms have so far been found to be able to mediate

33

these reactions. Candidatus ‘Methanoperedens nitroreducens’, an archaeal DAMO organism

34

affiliated to the ANME-2d cluster, reduces nitrate to nitrite,8 while Candidatus ‘Methylomirabilis

35

oxyfera’, a bacterial DAMO organism affiliated to the NC10 phylum, reduces nitrite to dinitrogen

36

gas,9 both with methane as the electron donor. In addition, ANME archaea have also been reported

37

to be capable of utilizing iron or manganese10-12 and hexavalent chromium13 for anaerobic oxidation

38

of methane, suggesting that AOM can be coupled to a wide variety of electron acceptors. However,

39

to the best of our knowledge, there is no study to investigate whether ANME are able to couple

40

AOM to selenate reduction, which indicates a significant gap of our understanding in the dynamics

41

of anaerobic methane oxidation in nature given the wide distribution of selenium in virtually all

42

materials of the earth's crust.

ACS Paragon Plus Environment

Page 2 of 22

Page 3 of 22

Environmental Science & Technology

43

Selenium (Se), a naturally occurring trace element, can be found in the Earth crust and minerals.14

44

Some anthropogenic activities, such as coal mining and combustion, metal mining and smelting, oil

45

refining and utilization, or agricultural irrigation.15 would increase Se concentrations in aquatic

46

ecosystems. Of considerable biological interest, Se constitutes one kind of necessary micronutrients

47

for human and other animals, but it is a toxic contaminant at excessive concentrations (a maximum

48

contaminant level of 50 µg total Se/L for drinking water was set by US EPA).16, 17 Previous studies

49

reported that selenate could be microbially reduced with acetate18, lactate19 or hydrogen20 as

50

electron donors. During the reduction process, inorganic selenate (SeO42-) and selenite (SeO32-),

51

both quite water-soluble and toxic, can be reduced to much less bioavailable and non-toxic

52

elemental selenium (Se0).21,

53

Enterobacter taylorae and Pelobacter seleniigenes) capable of selenate bio-reduction have been

54

detected or isolated in selenate-contaminated aquifer,23 agricultural drainage water24,

55

diversity of sediment types26, 27 (more details in SI Table S1), suggesting this dissimilatory process

56

be ubiquitous in natural environments. Although selenate could be organotrophic reduced using

57

organic carbon compounds or lithotrophic reduced using hydrogen as electron donors, selenate

58

reduction driven by methane has been largely overlooked so far. Considering that both methane and

59

selenate co-occur in aquatic environments, it is hypothesized that the microbial selenate-dependent

60

AOM process could occur in such environments.

61

Here, we attempted to demonstrate microbial selenate reduction coupled to the anaerobic oxidation

62

of methane. We enriched a culture able to couple selenate reduction to AOM by changing the

63

nitrate-containing feed to a reactor performing DAMO to selenate-containing feed. Ion

64

chromatography (IC), inductively coupled plasma-optical emission spectroscopy (ICP-OES) and X-

65

ray photoelectron spectroscopy (XPS) were employed to monitor the Se transformation. A series of

66

batch tests were also carried out to confirm the link between selenate reduction and AOM.

67

Fluorescence in situ hybridization (FISH) and high-throughput 16S rRNA gene sequencing were

68

carried out to characterize the microbes involved.

22

Diverse microbial species (e.g. Dechloromonas sp., Thauera sp.,

ACS Paragon Plus Environment

25

and a

Environmental Science & Technology

69

MATERIALS AND METHODS

70

Experimental Setup

71

Microbial selenate reduction coupled to anaerobic oxidation of methane was conducted using a

72

laboratory-scale MBfR reactor with a working volume of 800 mL (200 mm in height and 80 mm in

73

internal diameter). The schematic diagram of the MBfR system is similar with our previous studies

74

28, 29

75

diameter and 300 µm outer diameter, Mitsubishi, Japan) were inserted into the reactor evenly. Each

76

bundle was made up by 64 fibers with a length of 300 mm. These 512 fibers gave a membrane

77

surface area of 0.145 m2 and a membrane surface/reactor volume ratio of 181 m2/m3. Each bundle

78

of hollow fibers was bent to be U-shaped and the end was connected to a gas cylinder (95% CH4

79

and 5% CO2, Coregas, Australia), by which the methane can penetrate into the liquid phase from the

80

hollow fiber membrane. The methane pressure in the interior of hollow fibres was controlled using

81

a gas-pressure regulator (150 kPa, Ross Brown, Australia). The maximum CH4 flux was quantified

82

according to the previous method

83

18.8 L CH4/m2/d (12.5 g CH4/m2/d). The bulk medium in the MBfR was completely mixed with a

84

magnetic stirrer (500 rpm, Labtek, Australia) as well as a peristaltic recirculating pump (Masterflex,

85

USA). pH in the reactor was maintained between 7 and 8 by manual injection of 1 M HCl or 1 M

86

NaOH solutions. The reactor was operated in a temperature-controlled lab with temperature

87

maintained at 22±2 oC.

88

MBfR Operation

89

The MBfR was operated for 453 days divided into two phases, namely the nitrate-fed phase for the

90

enrichment of DAMO organisms in the biofilm (Phase I) and selenate-fed phase (Phase II), both

91

under anoxic conditions. For Phase I, a nitrate stock solution (3 M) was dosed weekly to reach 2-11

92

mmol/L nitrate after each injection. For Phase II, selenate was used to replace nitrate, and was

93

added weekly by injection of a concentrated stock solution (0.05 M) to reach an in-reactor selenate

(shown in SI Figure S1). Eight bundles of composite hollow fiber membranes (200 µm inner

30, 31

, and the maximum methane delivery capacity at 150 kPa was

ACS Paragon Plus Environment

Page 4 of 22

Page 5 of 22

Environmental Science & Technology

94

concentration of 20-60 µmol/L after each injection. The stock solutions were prepared with

95

degassed milli-Q water, and stored in sealed nitrogen-atmosphere bottles. In addition, 400 mL fresh

96

mineral medium was used to replace an equivalent volume of liquid in the reactor monthly. The

97

fresh medium consisted of (unit: g/L if not specified)32: KH2PO4 0.038, MgCl2.6H2O 0.008,

98

CaCl2.2H2O 0.015, NH4Cl 0.019, acidic trace elements (including FeSO4 2.085, ZnSO4.7H2O 0.068,

99

CoCl2.6H2O 0.12, MnCl2.4H2O 0.5, CuSO4.5H2O 0.32, NiCl2.6H2O 0.095, H3BO4 0.014, HCl 100

100

mM) 0.5 mL/L, and alkaline trace elements (including NaOH 0.4, NaWO4.2H2O 0.05, NaMoO4

101

0.242, SeO2 0.067) 0.2 mL/L.

102

During the whole experimental period, liquid samples were taken 2-3 times per week from the

103

reactor to determine the concentrations of nitrate, nitrite, selenate and selenite. Nitrate and selenate

104

reduction rates were determined as the slopes of the nitrate and selenate profiles, respectively.

105

Biofilm samples were collected for microbial analysis and XPS analysis in both phases on Day 271

106

and Day 442, respectively (see methods below).

107

Batch Tests

108

Batch tests were undertaken to observe selenate reduction in the presence or absence of methane.

109

Se(VI) reduction in the presence of methane was conducted to determine the stoichiometric electron

110

balance. To monitor the methane consumption, the reactor was disconnected from the gas cylinder

111

to cut off methane supply. Freshly prepared medium was sparged with methane gas (95% methane

112

and 5% carbon dioxide) for 60 min to make sure that the medium was saturated with methane. Then

113

the liquid phase of the MBfR was replaced and filled with this methane saturated liquid medium.

114

Afterwards, 1 mL of stock solution of selenate was injected into the reactor to obtain a

115

concentration of approximately 50.0 µmol/L. During the test for 64 h, liquid samples were taken

116

every 7h to 9h to determine concentrations of selenate and dissolved methane. To measure if

117

selenate would be reduced in the absence of methane, the methane gas cylinder was closed to stop

118

methane supply. Helium gas was flushed into the reactor through the hollow fibers to remove the

ACS Paragon Plus Environment

Environmental Science & Technology

119

residual methane for 30 min. Similarly, 1 mL of stock solution of selenate was injected into the

120

reactor. In addition, an abiotic control was also conducted to test if the selenate reduction is a

121

microbial process. A 200 mL serum bottle was used for the abiotic control experiment, where the

122

fresh medium was flushed with methane for 30 min to deliver dissolved methane. Similarly, 0.25

123

mL of stock solution of selenate was injected into the serum bottle to obtain a concentration of

124

approximately 60.0 µmol/L. The test was run for 48 h for both abiotic and without methane

125

conditions, during which liquid samples were taken every 7h to 11h to determine concentrations of

126

selenate as described below.

127

Chemical Analyses

128

Liquid samples of 2 mL were collected regularly (2-3 times per week) to measure the

129

concentrations of selenium species or nitrate after 0.22 µm-filtration. Selenate and selenite

130

concentrations were determined by ion chromatography (IC), which consisted of an AS-3000

131

Autosampler, a Dionex ICS-2100 equipped with an IonPac AS19 column (4 mm * 250 mm), an

132

ASRS suppressor (4 mm, 75 mA) and an electrochemical conductivity detector (ECD)16. For total

133

dissolved selenium, the sample was centrifuged at 15,000 g for 5 min, and analysed with

134

inductively coupled plasma-optical emission spectroscopy (ICP-OES, Perkin Elmer Optima

135

7300DV) after 10% nitric acid-assisted microwave digestion. The amount of elemental selenium

136

produced was calculated as the decrease of the total dissolved Se concentration. Nitrate

137

concentration was measured using a Lachat QuickChem8000 Flow Injection Analyzer (Lachat

138

Instrument, Milwaukee, WI). An Agilent 7890A gas chromatograph (GC) equipped with a Supelco

139

6 feet * 1/8-in stainless steel packed column (HayeSep Q 80/100) and a flame ionisation detector

140

(FID) was employed to determine the dissolved methane concentration. The pH level in the reactor

141

was monitored using a pH meter (Oakton, Australia).

142

To analyse the valence state of selenium attached to the biofilm, the produced precipitate was

143

collected, freeze-dried and analysed with XPS. The XPS spectra were recorded on a Kratos Axis

ACS Paragon Plus Environment

Page 6 of 22

Page 7 of 22

Environmental Science & Technology

144

Ultra X-ray photoelectron spectrometer (Manchester, UK) using a monochromatized Al Kα X-ray

145

(1486.6 eV) at 150 W.

146

DNA Extraction and 16S rRNA Gene Sequencing

147

Biofilm samples were collected in both phases on Day 271 and Day 442, respectively. These

148

samples were analysed with 16S rRNA gene Illumina sequencing along with a biomass sample

149

from the inoculum. DNA extraction was conducted with the use of the FastDNA SPIN for Soil kit

150

(MP Biomedicals, USA) according to the manufacturer’s instructions. The extracted DNA

151

concentration was quantified with NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE,

152

USA). The 16S rRNA gene was amplified using the universal primer set 926F (5’-

153

AAACTYAAAKGAATTGACGG-3’) and 1392R (5’-ACGGGCGGTGTGTRC-3’). A QIAquick

154

PCR Purification Kit (Qiagen) and a Quant-iT dsDNA HS assay kit (Invitrogen) were employed to

155

purify and quantify the PCR products, respectively. Amplicons were pooled in equimolar

156

concentration and sequenced with an Illumina sequencer based on the standard protocols.

157

Raw sequencing data were quality-filtered and de-multiplexed using Trimmomatic, with poor-

158

quality sequences trimmed and removed. Subsequently, high-quality sequences at 97% similarity

159

were clustered into operational taxonomic units (OTUs) using QIIME with default parameters, and

160

representative OTU sequences were taxonomically BLASTed against Greengenes 16S rRNA

161

database. Finally, an OTU table consisting of the taxonomic classification and OTU representative

162

sequences was output as the main analysis results.

163

FISH

164

Biomass from the inoculum, and biofilm samples collected on Day 271 and Day 442 were harvested,

165

fixed, hybridized, and then visualized as described previously.33 The following probes were used in

166

this study: S-*-Darc-872-a-A-18 (5’-GGCTCCACCCGTTGTAGT-3’) for DAMO archaea, S-*-

167

NC10-1162-a-A-18 (5’-GCCTTCCTCCAGCTTGACGCTG-3’) for DAMO bacteria, S-*-Amx-

ACS Paragon Plus Environment

Environmental Science & Technology

168

820-a-A-18 (5’-AAAACCCCTCTACTTAGTGCCC-3’) for anammox bacteria, and S-DArch-

169

0915-a-A-20 for general archaea, EUBmix for general bacteria.

170

171

RESULTS AND DISCUSSION

172

Enriching DAMO Microorganisms in an MBfR Reactor

173

Methane has a very low solubility in water (22.7 mg/L, under 20 oC and 101.3 kPa) 34, which often

174

limits the DAMO rates in suspended cultures if methane is directly sparged into liquid.35 In order to

175

achieve a higher methane transfer efficiency, a lab-scale membrane biofilm bioreactor (MBfR) was

176

set up for the enrichment of DAMO in biofilm,36,

177

pressurized bubbleless hollow fibre membranes. The methane-based MBfR was seeded with 150

178

mL of inoculum taken from a parent DAMO/anammox reactor fed with nitrate, ammonium and

179

methane.8 At the seeding time, an average nitrate removal rate of 1.43 mmol/L/d was achieved in

180

the parent reactor, where DAMO archaea, DAMO bacteria and anammox bacteria were enriched in

181

the suspended culture (see the detailed microbial communities later). After the inoculation, nitrate

182

was provided in the liquid phase as the sole electron acceptor, and methane supplied through the

183

hollow fibre membrane to enrich DAMO microbes on the outer surface of the membrane.

184

With methane as the sole electron donor available, nitrate was reduced with a fluctuant reduction

185

rate in a range of 0.07-0.66 mmol/L/d, with an average of approximately 0.25 mmol/L/d during the

186

271 days of operation (Figure 1). Simultaneously, a layer of biofilm gradually covered the surface

187

of the hollow fiber membrane. In comparison with the parent reactor with a nitrate removal rate of

188

1.43 mmol/L/d, the lower nitrate reduction rate could have possibly resulted from the washout of

189

anammox bacteria (as will be detailed below) due to the absence of ammonium as a substrate for

190

anammox. It has previously been reported that a partnership with anammox bacteria favours the

37

where methane was delivered from the

ACS Paragon Plus Environment

Page 8 of 22

Page 9 of 22

Environmental Science & Technology

191

growth of archaeal DAMO,8 because anammox bacteria could remove nitrite, which is toxic to

192

archaeal DAMO.38

193

Achieving Microbial Selenate Reduction Coupled to AOM

194

On Day 272, the sole electron acceptor provided to the MBfR was switched from nitrate to selenate,

195

in order to investigate if the microbial community enriched could couple selenate reduction to the

196

anaerobic oxidation of methane. Selenate reduction immediately occurred, and continued in the

197

remaining 182 days of the experimental period (Figure 2). Selenate was reduced with a fluctuant

198

reduction rate of 2.8-12.4 µmol/L/d. Even without any adaptation, the enriched culture reduced

199

selenate from 63.3 µmol/L on Day 272 to 4.5 µmol/L on Day 289. Selenite was not detected in the

200

reactor, suggesting that all the selenate should be converted to elemental Se. This can be supported

201

by attachment of reddish precipitates in the biofilm and the recirculation tubing. In order to

202

investigate the speciation of selenium deposited onto the MBfR biofilm surface after microbial

203

selenate reduction, XPS spectra of the selenate-reducing biofilm and the inoculum were both

204

collected (Figure 3). In comparison with the inoculum, there was a distinct peak appearing around

205

55.4 eV of binding energy (BE) from the selenate-intervened biofilm sample, which could be

206

assigned to elemental Se according to the NIST XPS Database. Furthermore, this peak can be

207

resolved into two sub-peaks, i.e. elemental Se 3d3/2 (BE 56.3eV) and elemental Se 3d5/2 (BE 55.4

208

eV). The XPS spectra indicate that the main selenium component of the precipitate deposited onto

209

the methane-based MBfR biofilm during microbial selenate reduction was elemental Se, suggesting

210

complete selenate bio-reduction to elemental Se without the biological absorption of selenate or the

211

produced selenite and selenide. Similarly, elemental Se was previously shown to be the only

212

product of microbial selenate reduction when acetate or lactate was used as an electron donor.18, 39,

213

40

214

microorganims,41 although the formation of selenide from elemental Se is a slow reaction.42

It is also worth noting that elemental Se was reported to be further reduced to selenide by specific

ACS Paragon Plus Environment

Environmental Science & Technology

215

During the first 30 days after selenate dosing was initiated, the culture underwent an adaptation to

216

selenate loading and exhibited a selenate reduction rate of only about 4 µmol/L/d. Later, the

217

reduction rate rapidly increased to ~12 µmol/L/d, which lasted for more than 40 days, before it

218

gradually declined to the final rate of 7.5 µmol/L/d. Based on the flux calculation (see SI), the

219

required methane flux for selenate reduction (8.2×10-4 g/m2/d) was significantly less than the

220

maximum CH4 flux (12.5 g/m2/d), indicating that the supply of methane was not a rate-limiting step.

221

The rate decline herein could potentially be attributed to an adverse impact caused by the

222

continuous attachment of the produced reddish elemental Se precipitates onto the biofilm, as the

223

precipitates may hinder substrate transfer of selenate to microbes in biofilm, or bind with

224

extracellular polymeric substances on the cytomembrane.

225

To determine whether the selenate reduction was coupled to methane oxidation, a batch test was

226

conducted where the methane delivery was stopped through disconnecting the gas cylinder to the

227

reactor. In the absence of methane, no significant selenate was reduced (SI Figure S2, p >0.05). In

228

contrast, selenate was reduced when methane was available as electron donor. The mole ratio

229

between Se(VI) reduction and methane oxidation was about 0.86 according to the data of Fig. S2a

230

(SI). This measured ratio was comparable to the theoretical value of 1.33 (4SeO42- + 3CH4 + 8H+ →

231

4Se0 + 3CO2 + 10H2O). Moreover, an abiotic experiment further confirmed the lack of selenate

232

reduction in the absence of microbes, indicating that the selenate reduction process was a microbial

233

process. The long-term performance data along with the batch test results provide evidence that

234

selenate reduction might be coupled to AOM. Further isotopic labelling experiments using labelled

235

methane and selenate should be performed to verify this coupling process.

236

Microbial Community Structure and Potential Players in Selenate Bio-reduction

237

Molecular characterization of microbial community in inoculum, nitrate-reducing biofilm and

238

selenate-shaped biofilm were conducted using FISH and 16S rRNA gene amplicon sequencing.

239

Based on FISH results, M. nitroreducens, M. oxyfera and anammox bacteria were the dominant

ACS Paragon Plus Environment

Page 10 of 22

Page 11 of 22

Environmental Science & Technology

240

microorganisms in the inoculum (Figure 4a). The anammox population could hardly be detected in

241

the nitrate-reducing (Day 271, Figure 4b) or selenate-reducing (Day 442, Figure 4c) biofilms. Both

242

M. nitroreducens and M. oxyfera remained as the dominating groups.

243

Analysis of the 16S rRNA gene amplicon sequences (Figure 5) further confirmed that all archaea

244

detected in the inoculum or the biofilms fell within one genus of Candidatus Methanoperedens,

245

which are known archaeal DAMO organisms that perform nitrate-driven anaerobic methane

246

oxidation. Candidatus Methylomirabilis, a known bacterial DAMO organism, was also detected

247

with a high abundance in both inoculum and biofilms. Anammox bacteria were washed out from the

248

reactor, which is consistent with the FISH results. After nitrate was replaced with selenate, both

249

Candidatus Methanoperedens (15.7%) and Candidatus Methylomirabilis (12.5%) were still the

250

dominant microorganisms. Analysis of all 16S rRNA gene sequences further suggests Candidatus

251

Methanoperedens and Candidatus Methylomirabilis were the only known methane-oxidising

252

microbes in the reactor after 442-day operation. Other known selenate-reducing microorganisms

253

such as Sulfurospirillum barnesii 43, Bacillus sp.39 and Stenotrophomonas maltophilia40 (SI Table

254

S1) were not present in the reactor.

255

Compared to the communities in the nitrate-reducing biofilm, the abundance of SHA-31 and

256

Ignavibacterium increased from 4.3% to 11.9% and from 4.9% to 8.8%, respectively, after selenate

257

replaced nitrate as the electron acceptor. The phylogenetic tree of 16S rRNA genes of SHA-31 was

258

established in comparison with phylogenetically close sequences from various environments (as

259

shown in SI Figure S3). None of these phylogenetically related microorganisms have previously

260

been shown to be able to reduce selenate. Similarly, there have been no reports indicating that

261

Ignavibacterium has the ability to reduce selenate.44, 45 These two genera in the reactor are therefore

262

unlikely selenate reducers. Their roles remain to be clarified.

263

Possible Roles of Candidatus Methanoperedens and Candidatus Methylomirabilis

ACS Paragon Plus Environment

Environmental Science & Technology

264

In the present study, we reported that an enriched DAMO culture could couple methane oxidation

265

with selenate reduction. Previous studies suggested that genes encoding heme c-containing proteins

266

(c-type cytochromes) could be a genetic basis for metal reduction, which could shuttle electrons

267

from the cell to soluble or solid electron acceptors.11, 46, 47 Here, we searched the reconstructed

268

genome of Candidatus Methanoperedens and found 28 genes encoding heme c-type cytochromes in

269

the Candidatus Methanoperedens (SI Table S2). The result is consistent with a very recent report

270

about the enriched ANME-2d, which encoded 41 multiheme c-type cytochromes.11 This genomic

271

information indicates that Candidatus Methanoperedens should possess versatile abilities to use a

272

range of electron acceptors according to their availability. Previous studies have demonstrated that

273

anaerobic methanotrophic archaea coupled to sulfate6, nitrite9, nitrate8 and iron/manganese10

274

reduction. In addition, Candidatus Methylomirabilis, which are known DAMO bacteria,9 were

275

detected in the bioreactor community. By searching its genetic information,9 we also found genes

276

encoding heme c-type cytochromes in the reconstructed genome of Candidatus Methylomirbilis,

277

suggesting its potential capability of extracellular electron transfer. However, no studies have been

278

reported that Candidatus Methylomirbilis is able to utilize electron acceptors other than nitrite.

279

Without the involvement of any other known electron acceptors (e.g. sulphate, iron or manganese)

280

for ANME in the reactor, we assumed that Candidatus Methanoperedens or Candidatus

281

Methylomirabilis oxidized methane to generate the electrons for selenate reduction. Similar to

282

nitrate, selenate might serve as the terminal electron acceptor for independent AOM driven by

283

DAMO archaea via a reverse methanogenesis pathway8. In addition, the ANME groups responsible

284

for AOM are commonly reported to assemble with synergistic bacterial partners for sulphate5 or

285

iron/manganese10,

286

organic intermediates (e.g. volatile fatty acids29) under certain conditions, then which might be

287

served as carbon sources for heterotrophically reducing selenate. Future studies are required to

288

investigate whether the DAMO co-cultures independently couple anaerobic methane oxidation with

12

reduction. It is also suspected that DAMO microorganisms might generate

ACS Paragon Plus Environment

Page 12 of 22

Page 13 of 22

Environmental Science & Technology

289

selenate reduction or the enriched DAMO cultures (or other unknown methanotrophs) cooperate

290

with other unidentified selenate reducers.

291

Environmental Implications

292

AOM plays an important role in the global methane cycle, with sulfate-, nitrite/nitrate-, and

293

iron/manganese-dependent AOMs considered to significantly impact the methane flux from anoxic

294

environments.6, 8-10 However, no study has been dedicated to investigating anaerobic oxidation of

295

methane coupled to selenate reduction. Selenium compounds are ubiquitous in the Earth crust and

296

minerals14, and have been discharged into aquatic ecosystems15, inducing biological and ecological

297

effects. Previous studies have reported that selenate could be heterotrophically reduced with various

298

organic compounds as electron donors in selenate-contaminated aquifer23, agricultural drainage

299

water24 and/or wetland sediment48. Herein, for the first time, we demonstrate that the microbial

300

selenate reduction could be coupled to AOM. Considering the wide distribution of methane and the

301

very likely coexistence of methane and selenate in aquatic environments, the selenate-dependent

302

AOM process could play a role in methane regulation on Earth. Our results advance our

303

understanding of the biogeochemical selenium and methane cycles, and of the diversity of

304

microbially mediated selenate reduction in natural environments. Our results may also support the

305

development of alternative technologies for selenate removal from selenate-contaminated aquatic

306

environments such as groundwater.

307

ACKNOWLEDGMENTS

308

This work was financially supported by the Australian Research Council (ARC) through the

309

projects DP170104038 and DE130101401. Jing-Huan Luo would like to acknowledge the China

310

Scholarship Council (CSC) for the scholarship support. Hui Chen would like to acknowledge the

311

support of the International Postgraduate Research Scholarship (IPRS) and The University of

312

Queensland Centennial Scholarship (UQCent).

ACS Paragon Plus Environment

Environmental Science & Technology

313

ASSOCIATED CONTENT

314

Supporting information

315

Additional methods, tables and figures as mentioned in the text. This material is available free of

316

charge via the Internet at http://pubs.acs.org.

ACS Paragon Plus Environment

Page 14 of 22

Page 15 of 22

Environmental Science & Technology

317

REFERENCES

318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369

(1) Pachauri, R. K.; Allen, M. R.; Barros, V. R.; Broome, J.; Cramer, W.; Christ, R.; Church, J. A.; Clarke, L.; Dahe, Q.; Dasgupta, P., Climate change 2014: synthesis report. Contribution of Working Groups I, II and III to the fifth assessment report of the Intergovernmental Panel on Climate Change, IPCC, 2014. (2) Ciais, P.; Sabine, C.; Bala, G.; Bopp, L.; Brovkin, V.; Canadell, J.; Chhabra, A.; DeFries, R.; Galloway, J.; Heimann, M., Carbon and other biogeochemical cycles. In Climate change 2013: the physical science basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change, Cambridge University Press: 2014; pp 465-570. (3) Degelmann, D. M.; Borken, W.; Drake, H. L.; Kolb, S., Different atmospheric methane-oxidizing communities in European beech and Norway spruce soils. Appl. Environ. Microbiol. 2010, 76 (10), 32283235. (4) Maxfield, P. J.; Hornibrook, E. R. C.; Evershed, R. P., Estimating high-affinity methanotrophic bacterial biomass, growth, and turnover in soil by phospholipid fatty acid C-13 labeling. Appl. Environ. Microbiol. 2006, 72 (6), 3901-3907. (5) Knittel, K.; Boetius, A., Anaerobic oxidation of methane: progress with an unknown process. Annu. Rev. Microbiol. 2009, 63, 311-334. (6) Boetius, A.; Ravenschlag, K.; Schubert, C. J.; Rickert, D.; Widdel, F.; Gieseke, A.; Amann, R.; Jorgensen, B. B.; Witte, U.; Pfannkuche, O., A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature 2000, 407 (6804), 623-626. (7) Milucka, J.; Ferdelman, T. G.; Polerecky, L.; Franzke, D.; Wegener, G.; Schmid, M.; Lieberwirth, I.; Wagner, M.; Widdel, F.; Kuypers, M. M. M., Zero-valent sulphur is a key intermediate in marine methane oxidation. Nature 2012, 491 (7425), 541-546. (8) Haroon, M. F.; Hu, S. H.; Shi, Y.; Imelfort, M.; Keller, J.; Hugenholtz, P.; Yuan, Z. G.; Tyson, G. W., Anaerobic oxidation of methane coupled to nitrate reduction in a novel archaeal lineage. Nature 2013, 500 (7464), 567-570. (9) Ettwig, K. F.; Butler, M. K.; Le Paslier, D.; Pelletier, E.; Mangenot, S.; Kuypers, M. M. M.; Schreiber, F.; Dutilh, B. E.; Zedelius, J.; de Beer, D.; Gloerich, J.; Wessels, H. J. C. T.; van Alen, T.; Luesken, F.; Wu, M. L.; van de Pas-Schoonen, K. T.; den Camp, H. J. M. O.; Janssen-Megens, E. M.; Francoijs, K. J.; Stunnenberg, H.; Weissenbach, J.; Jetten, M. S. M.; Strous, M., Nitrite-driven anaerobic methane oxidation by oxygenic bacteria. Nature 2010, 464 (7288), 543-548. (10) Beal, E. J.; House, C. H.; Orphan, V. J., Manganese- and iron-dependent marine methane oxidation. Science 2009, 325 (5937), 184-187. (11) Ettwig, K. F.; Zhu, B. L.; Speth, D.; Keltjens, J. T.; Jetten, M. S. M.; Kartal, B., Archaea catalyze iron-dependent anaerobic oxidation of methane. Proc. Natl. Acad. Sci. U.S.A. 2016, 113 (45), 12792-12796. (12) Fu, L.; Li, S. W.; Ding, Z. W.; Ding, J.; Lu, Y. Z.; Zeng, R. J., Iron reduction in the DAMO/Shewanella oneidensis MR-1 coculture system and the fate of Fe(II). Water Res. 2016, 88, 808-815. (13) Lu, Y.-Z.; Fu, L.; Ding, J.; Ding, Z.-W.; Li, N.; Zeng, R. J., Cr (VI) reduction coupled with anaerobic oxidation of methane in a laboratory reactor. Water Res. 2016, 102, 445-452. (14) Simmons, D. B. D.; Wallschlager, D., A critical review of the biogeochemistry and ecotoxicology of selenium in lotic and lentic environments. Environ. Toxicol. Chem. 2005, 24 (6), 1331-1343. (15) Lemly, A. D., Aquatic selenium pollution is a global environmental safety issue. Ecotox. Environ. Safe. 2004, 59 (1), 44-56. (16) Lenz, M.; Gmerek, A.; Lens, P. N. L., Selenium speciation in anaerobic granular sludge. Int. J. Environ. An. Ch. 2006, 86 (9), 615-627. (17) Vickerman, D. B.; Trumble, J. T.; George, G. N.; Pickering, I. J.; Nichol, H., Selenium biotransformations in an insect ecosystem: Effects of insects on phytoremediation. Environ. Sci. Technol. 2004, 38 (13), 3581-3586. (18) Navarro, R. R.; Aoyagi, T.; Kimura, M.; Koh, H.; Sato, Y.; Kikuchi, Y.; Ogata, A.; Hori, T., Highresolution dynamics of microbial communities during dissimilatory selenate reduction in anoxic soil. Environ. Sci. Technol. 2015, 49 (13), 7684-7691. (19) Lenz, M.; Enright, A. M.; O'Flaherty, V.; van Aelst, A. C.; Lens, P. N. L., Bioaugmentation of UASB reactors with immobilized Sulfurospirillum barnesii for simultaneous selenate and nitrate removal. Appl. Microbiol. Biot. 2009, 83 (2), 377-388.

ACS Paragon Plus Environment

Environmental Science & Technology

370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425

(20) Lai, C. Y.; Yang, X. E.; Tang, Y. N.; Rittmann, B. E.; Zhao, H. P., Nitrate shaped the selenatereducing microbial community in a hydrogen-based biofilm reactor. Environ. Sci. Technol. 2014, 48 (6), 3395-3402. (21) Chung, J.; Nerenberg, R.; Rittmann, B. E., Bioreduction of selenate using a hydrogen-based membrane biofilm reactor. Environ. Sci. Technol. 2006, 40 (5), 1664-1671. (22) Lenz, M.; Van Hullebusch, E. D.; Hommes, G.; Corvini, P. F. X.; Lens, P. N. L., Selenate removal in methanogenic and sulfate-reducing upflow anaerobic sludge bed reactors. Water Res. 2008, 42 (8-9), 21842194. (23) Williams, K. H.; Wilkins, M. J.; N'Guessan, A. L.; Arey, B.; Dodova, E.; Dohnalkova, A.; Holmes, D.; Lovley, D. R.; Long, P. E., Field evidence of selenium bioreduction in a uranium-contaminated aquifer. Env. Microbiol. Rep. 2013, 5 (3), 444-452. (24) Zhang, Y. Q.; Zahir, Z. A.; Frankenberger, W. T., Factors affecting reduction of selenate to elemental selenium in agricultural drainage water by Enterobacter taylorae. J. Agr. Food. Chem. 2003, 51 (24), 70737078. (25) Oremland, R. S.; Steinberg, N. A.; Presser, T. S.; Miller, L. G., In Situ Bacterial Selenate Reduction in the Agricultural Drainage Systems of Western Nevada. Appl. Environ. Microbiol. 1991, 57 (2), 615-617. (26) Narasingarao, P.; Häggblom, M. M., Pelobacter seleniigenes sp. nov., a selenate-respiring bacterium. Int. J. Syst. Evol. Micr. 2007, 57 (9), 1937-1942. (27) Steinberg, N. A.; Oremland, R. S., Dissimilatory Selenate Reduction Potentials in a Diversity of Sediment Types. Appl. Environ. Microbiol. 1990, 56 (11), 3550-3557. (28) Luo, J.-H.; Wu, M.; Yuan, Z.; Guo, J., Biological Bromate Reduction Driven by Methane in a Membrane Biofilm Reactor. Environ. Sci. Technol. Lett. 2017, 4 (12), 562-566. (29) Luo, J.-H.; Chen, H.; Yuan, Z.; Guo, J., Methane-supported nitrate removal from groundwater in a membrane biofilm reactor. Water Res. 2018, 132, 71-78. (30) Ontiveros-Valencia, A.; Penton, C. R.; Krajmalnik-Brown, R.; Rittmann, B. E., Hydrogen-Fed Biofilm Reactors Reducing Selenate and Sulfate: Community Structure and Capture of Elemental Selenium Within the Biofilm. Biotechnol. Bioeng. 2016, 113 (8), 1736-1744. (31) Tang, Y.; Zhao, H.; Marcus, A. K.; Krajmalnik-Brown, R.; Rittmann, B. E., A steady-state biofilm model for simultaneous reduction of nitrate and perchlorate, part 2: parameter optimization and results and discussion. Environ. Sci. Technol. 2012, 46 (3), 1608-15. (32) Ettwig, K. F.; van Alen, T.; van de Pas-Schoonen, K. T.; Jetten, M. S.; Strous, M., Enrichment and molecular detection of denitrifying methanotrophic bacteria of the NC10 phylum. Appl. Environ. Microbiol. 2009, 75 (11), 3656-62. (33) Ettwig, K. F.; Shima, S.; van de Pas-Schoonen, K. T.; Kahnt, J.; Medema, M. H.; op den Camp, H. J. M.; Jetten, M. S. M.; Strous, M., Denitrifying bacteria anaerobically oxidize methane in the absence of Archaea. Environ. Microbiol. 2008, 10 (11), 3164-3173. (34) Mackay, D.; Shiu, W. Y., A critical review of Henry’s law constants for chemicals of environmental interest. J. Phys. Chem. Ref. Data 1981, 10 (4), 1175-1199. (35) Hu, S. H.; Zeng, R. J.; Keller, J.; Lant, P. A.; Yuan, Z. G., Effect of nitrate and nitrite on the selection of microorganisms in the denitrifying anaerobic methane oxidation process. Env. Microbiol. Rep. 2011, 3 (3), 315-319. (36) Shi, Y.; Hu, S. H.; Lou, J. Q.; Lu, P. L.; Keller, J.; Yuan, Z. G., Nitrogen Removal from Wastewater by Coupling Anammox and Methane-Dependent Denitrification in a Membrane Biofilm Reactor. Environ. Sci. Technol. 2013, 47 (20), 11577-11583. (37) Cai, C.; Hu, S. H.; Guo, J. H.; Shi, Y.; Xie, G. J.; Yuan, Z. G., Nitrate reduction by denitrifying anaerobic methane oxidizing microorganisms can reach a practically useful rate. Water Res. 2015, 87, 211217. (38) Hu, S. H.; Zeng, R. J.; Haroon, M. F.; Keller, J.; Lant, P. A.; Tyson, G. W.; Yuan, Z. G., A laboratory investigation of interactions between denitrifying anaerobic methane oxidation (DAMO) and anammox processes in anoxic environments. Sci. Rep. 2015, 5 (8706), 1-9. (39) Fujita, M.; Ike, M.; Kashiwa, M.; Hashimoto, R.; Soda, S., Laboratory-scale continuous reactor for soluble selenium removal using selenate-reducing bacterium, Bacillus sp SF-1. Biotechnol. Bioeng. 2002, 80 (7), 755-761. (40) Dungan, R. S.; Yates, S. R.; Frankenberger, W. T., Transformations of selenate and selenite by Stenotrophomonas maltophilia isolated from a seleniferous agricultural drainage pond sediment. Environ. Microbiol. 2003, 5 (4), 287-295.

ACS Paragon Plus Environment

Page 16 of 22

Page 17 of 22

426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448

Environmental Science & Technology

(41) Herbel, M. J.; Blum, J. S.; Oremland, R. S.; Borglin, S. E., Reduction of Elemental Selenium to Selenide: Experiments with Anoxic Sediments and Bacteria that Respire Se-Oxyanions. Geomicrobiol. J. 2003, 20 (6), 587-602. (42) Zehr, J. P.; Oremland, R. S., Reduction of Selenate to Selenide by Sulfate-Respiring Bacteria: Experiments with Cell Suspensions and Estuarine Sediments. Appl. Environ. Microbiol. 1987, 53 (6), 13651369. (43) Oremland, R. S.; Blum, J. S.; Culbertson, C. W.; Visscher, P. T.; Miller, L. G.; Dowdle, P.; Strohmaier, F. E., Isolation, Growth, and Metabolism of an Obligately Anaerobic, Selenate-Respiring Bacterium, Strain SES-. Appl. Environ. Microbiol. 1994, 60 (8), 3011-3019. (44) Iino, T.; Mori, K.; Uchino, Y.; Nakagawa, T.; Harayama, S.; Suzuki, K., Ignavibacterium album gen. nov., sp nov., a moderately thermophilic anaerobic bacterium isolated from microbial mats at a terrestrial hot spring and proposal of Ignavibacteria classis nov., for a novel lineage at the periphery of green sulfur bacteria. Int. J. Syst. Evol. Micr. 2010, 60, 1376-1382. (45) Liu, Z. F.; Frigaard, N. U.; Vogl, K.; Iino, T.; Ohkuma, M.; Overmann, J.; Bryant, D. A., Complete genome of Ignavibacterium album, a metabolically versatile, flagellated, facultative anaerobe from the phylum Chlorobi. Front. Microbiol. 2012, 3, 185. (46) Shi, L.; Squier, T. C.; Zachara, J. M.; Fredrickson, J. K., Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes. Mol. Microbiol. 2007, 65 (1), 1220. (47) Weber, K. A.; Achenbach, L. A.; Coates, J. D., Microorganisms pumping iron: anaerobic microbial iron oxidation and reduction. Nat. Rev. Microbiol. 2006, 4 (10), 752-764. (48) Narasingarao, P.; Haggblom, M. M., Identification of anaerobic selenate-respiring bacteria from aquatic sediments. Appl. Environ. Microbiol. 2007, 73 (11), 3519-3527.

449

ACS Paragon Plus Environment

Nitrate removal rate (mmol/L/d)

Nitrate/nitrite concentration (mmol/L)

Environmental Science & Technology

Page 18 of 22

12 Nitrate Nitrite

a

9 6 3 0 0.8 0.6

b

0.4 0.2 0.0 0

40

80

120

160

200

240

280

Time (d)

Figure 1. (a) Nitrate and nitrite concentrations, and (b) Nitrate reduction rate in Phase I. Nitrate was periodically fed to the reactor resulting in sudden increases in its concentration. The nitrate reduction rate was calculated as the slope of the concentration profile following each pulse-feed of nitrate.

ACS Paragon Plus Environment

Environmental Science & Technology

Selenate reduction rate ( µmol/L/d)

Selenate/selenite/Se concentration ( µmol/L)

Page 19 of 22

64 Selenate Selenite Elemental Se

a

48 32 16 0 16

b

12 8 4 0 280

300

320

340

360

380

400

420

440

460

Time (d) Figure 2. (a) Selenate, selenite and elemental Se concentrations, and (b) Selenate reduction rate in Phase II. Selenate was periodically fed to the reactor resulting in sudden increases in its concentration. The selenate reduction rate was calculated as the slope of the concentration profile following each pulse-feed of selenate.

ACS Paragon Plus Environment

Environmental Science & Technology

Page 20 of 22

Relative intensity (count)

800

Inoculum

700

Peak sum Elemental Se 3d3/2

600

Elemental Se 3d5/2 500

Selenate-reducing biofilm 400 60

58

56

54

52

50

Binding energy (eV)

Figure 3. Se XPS high-resolution spectra of the selenate-reducing biofilm and the inoculum. Compared to inoculum, the selenate-reducing biofilm exhibits a distinct peak sum of elemental Se (BE 55.4 eV), which can be further resolved into elemental Se 3d3/2 (BE 56.3eV) and elemental Se 3d5/2 (BE 55.4 eV).

ACS Paragon Plus Environment

Page 21 of 22

Environmental Science & Technology

Figure 4. FISH micrographs of the (a) inoculum, (b) nitrate-reducing biofilm (Day 271) and (c) selenate-reducing biofilm (Day 442) showing predominant M. nitroreducens (green) in glomerate clusters, M. oxyfera (red) and anammox bacteria (blue) in scattered clusters. Specific probes for hybridization: Cy3 ARC872 for M. nitroreducens, Cy5 NC1162 for M. oxyfera, and FITC AMX820 for anammox bacteria.

ACS Paragon Plus Environment

Environmental Science & Technology

Figure 5. Relative genus-level abundance of dominating organisms in the microbial community in the nitrate and selenate bio-reduction phases compared to the inoculum. The relative abundance is defined as a percentage in total effective microbial sequences in a sample. Genera with an abundance of >=1% in at least one sample are presented.

ACS Paragon Plus Environment

Page 22 of 22