Microbial Selenate Reduction Driven by a Denitrifying Anaerobic

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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

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Environmental Science & Technology

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Microbial Selenate Reduction Driven by a Denitrifying Anaerobic Methane Oxidation Biofilm

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

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

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

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

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[email protected]

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# These authors contributed equally to this work.

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ABSTRACT

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

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

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

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

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

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

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

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sequencing indicates that Candidatus Methanoperedens and Candidatus Methylomirabilis remained

ACS Paragon Plus Environment


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

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

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

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INTRODUCTION

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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materials of the earth's crust.


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

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

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

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

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

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

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

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

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

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elemental selenium (Se0).21,

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

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

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

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

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

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

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

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AOM process could occur in such environments.

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

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

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

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

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

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

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

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carried out to characterize the microbes involved.

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


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and a


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MATERIALS AND METHODS

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Experimental Setup

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

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

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

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28, 29

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

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

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

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

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

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

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

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according to the previous method

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

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

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

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

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maintained at 22±2 oC.

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MBfR Operation

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

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

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

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

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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

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, and the maximum methane delivery capacity at 150 kPa was


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

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

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

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

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

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

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

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0.242, SeO2 0.067) 0.2 mL/L.

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

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

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

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

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and Day 442, respectively (see methods below).

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Batch Tests

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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selenate as described below.

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Chemical Analyses

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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(1486.6 eV) at 150 W.

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DNA Extraction and 16S rRNA Gene Sequencing

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

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

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

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

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

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

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

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

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

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

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

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

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

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representative OTU sequences were taxonomically BLASTed against Greengenes 16S rRNA

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

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sequences was output as the main analysis results.

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FISH

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

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

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

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



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

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

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RESULTS AND DISCUSSION

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Enriching DAMO Microorganisms in an MBfR Reactor

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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where methane was delivered from the


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

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archaeal DAMO.38

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Achieving Microbial Selenate Reduction Coupled to AOM

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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40

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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



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

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

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

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

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

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

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

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

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

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extracellular polymeric substances on the cytomembrane.

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

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

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

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

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

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

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

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

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

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

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

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Microbial Community Structure and Potential Players in Selenate Bio-reduction

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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Methanoperedens and Candidatus Methylomirabilis were the only known methane-oxidising

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

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

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S1) were not present in the reactor.

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

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

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

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

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

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

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

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unlikely selenate reducers. Their roles remain to be clarified.

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Possible Roles of Candidatus Methanoperedens and Candidatus Methylomirabilis



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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iron/manganese10,

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

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

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

12

reduction. It is also suspected that DAMO microorganisms might generate


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

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with other unidentified selenate reducers.

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Environmental Implications

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

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

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

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

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

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

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

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

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

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

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

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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).



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.


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Nitrate removal rate (mmol/L/d)

Nitrate/nitrite concentration (mmol/L)


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.



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.



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).


Page 21 of 22


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.



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.


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Microbial Selenate Reduction Driven by a Denitrifying Anaerobic

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