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Bio-reduction of Antimonate by Anaerobic Methane Oxidation in a Membrane Biofilm Batch Reactor Chun-Yu Lai, Qiu-Yi Dong, Bruce E. Rittmann, and He-Ping Zhao Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02035 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

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Environmental Science & Technology

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Bio-reduction of Antimonate by Anaerobic Methane

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Oxidation in a Membrane Biofilm Batch Reactor

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Chun-Yu Lai1, 4, Qiu-Yi Dong1, Bruce E. Rittmann5, He-Ping Zhao1, 2, 3, *

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1. College of Environmental and Resource Science, Zhejiang University, Hangzhou,

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China. 2. Zhejiang Prov Key Lab Water Pollut Control & Envi, Zhejiang University, Hangzhou, Zhejiang, China.

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3. MOE Key Lab of Environmental Remediation and Ecosystem Health, College of

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Environmental and Resource Science, Zhejiang University, Hangzhou, China,

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310058. 4. Advanced Water Management Centre, The University of Queensland, St Lucia, Queensland 4072, Australia 5. Biodesign Swette Center for Environmental Biotechnology, Arizona State University, P.O. Box 875701, Tempe, Arizona 85287-5701, USA.

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* Correspondance to Dr. He-Ping Zhao. Tel (Fax): 0086-571-88982739, E-mail:

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[email protected]; [email protected]

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ACS Paragon Plus Environment


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Abstract

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Employing a special anaerobic membrane biofilm batch reactor (MBBR), we

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demonstrated antimonate (Sb(V)) reduction using methane (CH4) as the sole electron

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donor.

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(EDS), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and

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Raman and photoluminescence (PL) spectra identified that Sb2O3 microcrystals were

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the main reduced products.

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111-day experiment, which supports the enrichment of the microorganisms

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responsible for Sb(V) reduction to Sb(III).

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archaeal and bacterial 16 S rRNA genes increased in parallel. Clone library and

29

Illumina sequencing of 16S rRNA gene demonstrated that Methanosarcina became

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the dominant archaea in the biofilm, suggesting that Methanosarcina might play an

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important role in Sb(V) reduction in the CH4-based MBBR.

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Key words: Antimonate, bioreduction, methane, membrane biofilm batch reactor.

Scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy

The Sb(V) reduction rate increased continually over the

Copy numbers of the mcrA gene and

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2


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TOC

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37

Introduction

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Methane (CH4) is a potent greenhouse gas with a global warming potential 25 times

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higher than that of carbon dioxide over a 100-year horizon,1 and it also is an important

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by-product of anaerobic digestion in wastewater treatment, where it can be used for

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energy generation.2 Recent studies suggest that CH4 also can serve as an electron

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donor for bio-reduction of oxidized contaminants, including the oxyanions nitrate

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(NO3-),3 perchlorate (ClO4-),4 chromate (CrO42-),5-7 selenate (SeO42-),8, 9 and bromate

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(BrO3-).10

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organic electron donors (e. g., methanol, lactate, and acetate).3

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In past research with CH4, methanotrophs played a major role by activating CH4

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through aerobic methane oxidation (AMO).

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methanol,3 acetate,3, 5, 11 and citrate,12 were the direct electron donors for

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oxyanion-reducing bacteria.

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CH4 activation, increasing energy consumption.13 In contrast, anaerobic methane

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oxidation (AnMO) does not require O2, thus improving energy conservation and

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lowering the cost.14

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The number of electron acceptors documented to be reduced by AnMO is limited:

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sulfate (SO42-), NO3-, NO2-, Fe3+, and Mn4+.15-19 Kruger et al. reported that anaerobic

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methanotrophic archaea (ANME) were able to activate CH4 by reverse

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methanogenesis, which generated electrons that could be shuttled to sulfate-reducing

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bacteria in the absence of O2.18

This provides a low-cost electron donor to substitute for traditional

Activated intermediates, such as

Thus, a certain amount of oxygen (O2) was needed for

Haroon et al. confirmed AnMO coupled to NO34


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reduction by an ANME Methanoperedens nitroreducens, also through a

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reverse-methanogenesis pathway.17

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affiliated with the NC10 phylum carried out nitrite (NO2-) reduction and AnMO by an

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“intra-aerobic” pathway in which O2 was produced and consumed internally.16 More

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recently, Ettwig et al. observed that an archaeon related to Candidatus

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Methanoperedens nitroreducens was able to catalyze iron-dependent AnMO.19

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Antimony (Sb) is emerging as a heavy-metal contaminant in wastewaters.20

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occurrence of Sb contamination has been recorded in the waters near antimony mines

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in Corsican river and Western Carpathians, Slovakia.21,22

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industries that manufacture semiconductors and flame retardants,

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to humans and other organisms through its damage to the cardiac and gastrointestinal

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systems.23,24

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water is 6 µg/liter.25 Antimonate (Sb(V)) and antimonite (Sb(III)) are the most

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common states of Sb in water bodies.26 Sb(V) is toxic and highly soluble, while

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Sb(III) usually precipitates at neutral pH and can be easily removed by centrifugation

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or filtration.27 Therefore, bio-reduction of Sb(V) to Sb(III) is a promising

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Sb-removal process.20 Lactate and acetate have been applied as organic electron

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donors for Sb(V) reduction.28-30

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Until now, bio-reduction of Sb(V) using CH4 as the electron donor has not been

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reported.

Ettwig et al. demonstrated that a bacterial group

The

Derived from mining and Sb is highly toxic

The USEPA’s maximum contaminant level (MCL) for Sb in drinking

Considering that the Sb(OH)6-/Sb(OH)3 couple has a high redox potential

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(760 mV),31 we hypothesize that Sb(V) reduction can be coupled to AnMO based on

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the following stoichiometry and energetics:32

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CH4 + 4Sb(OH)6- + 4H+ = 4Sb(OH)3 + 10H2O + CO2

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(1)

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It is reported that the Sb(OH)3 is transformed to Sb2O3 microcrystals, which have

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applications in glass decolorizing, optoelectronic devices, flame-proof retardants,

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paint pigment, and polyvinyl chloride (PVC) stabilizers.23,33

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might open new options for nanotechnology and economic benefits.28

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The first objective of this study was to test the feasibility of Sb(V) bio-reduction using

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CH4 as the sole electron donor when the exogenous O2 supply was negligible.

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second objective was to identify the physical and chemical features of Sb2O3

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microcrystals using multiple characterization technologies.

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identify key microorganisms performing AnMO and Sb(V) reduction by using a

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combination of quantitative real-time PCR (qPCR), cloning, and high-throughput

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sequencing.

∆G0’=-481.52 kJ/molCH4

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Thus, Sb(V) reduction

The

In addition, we aimed to

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Materials and Methods

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Reactor Configuration.

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The membrane biofilm batch reactor (MBBR) (Figure S1) was composed of a 1-L

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glass vessel，32 main hollow fibers (280 µm OD, 180 µm ID, 15-cm length, model

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MHF-200TL, Mitsubishi, Ltd., Japan), 10 coupon fibers of the same type, Norprene

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tubing, and plastic caps.

The total membrane surface area was 4×10-3 m2, and all

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fibers were glued together by epoxy adhesive and sealed within Norprene tubing.

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The fibers have a CH4 permeability of 1.03 × 10−7 m3 CH4Ɵ - m membrane

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thickness/m2 hollow fiber surface area-d-bar (CH4Ɵ is defined as the volume of CH4 at

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standard temperature and pressure).4

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through the walls of the fibers at constant CH4 pressure (10 psig or 165 kpa).

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magnetic stirrer (HJ-1, Xinbao, Ltd., China) was applied to maintain homogeneity of

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the system and to enhance mass transfer.

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MBBR and was clamped by pinch cocks.

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The MBBR system was designed and constructed to preclude O2 penetration based on

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the following features: 1) AB gel was used to seal all openings (e. g., gaps between

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the plug, bottle, tubes, and caps); 2) Operation in a batch sequencing mode reduced

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the amount of O2 residual from the feed solution; and 3) The large ratio of liquid to

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gas (8:2) minimized the invasion of external O2. To assess if any O2 had

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accumulated in the reactor, we loosened the pinch cocks and sampled the gas in the

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bag every four days.

CH4 was supplied to the lumen and diffused A

A gas collection bag was connected to the

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Start up and Operation

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The MBBR was inoculated with 100 mL of a culture that had been enriched for 12

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months with CH4 and Sb(V) in a conventional bench-scale membrane biofilm reactor

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(MBfR) system.8

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contained (per L of demineralized water):

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MgSO4·7H2O 0.02 mmol, KH2PO4 3 mmol, Na2HPO4·12H2O 2.23 mmol, 1 mL acid

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trace element solution, and 1 mL alkaline trace element solution.4 We degassed the

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mixture with argon (Ar) for 30 min to create an anaerobic condition as much as

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possible.

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headspace.

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deionized water, kept anaerobic as described above, and stored in a sealed serum

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bottle.

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The MBBR was operated in batch mode for five successive Sb(V) amendments

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during which the initial concentration of Sb(V) in the reactor was 410, 410, 820, 820,

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and 1600 µmol Sb/L (stages 1A, 1B, 2A, 2B and 3, respectively).

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MBBR as a positive control by not providing CH4.

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measured by GC (Agilent Technologies GC system, model 7890A, Agilent

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Technologies Inc., U.S.A) throughout the whole experiment, and the concentration

133

was always below the detection limit (200 ppm for gas phase, equivalent to 0.25

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µmol/L dissolved O2 in liquids according to Henry’s Law) for both MBBRs.

We added to the MBBR ~700 mL of mineral salt medium that NH4Cl 0.37 mmol, CaCl2 0.009 mmol,

The working volume of the MBBR was 800 mL of liquid with 200 mL of A sterile stock solution of KSb(OH)6 (33 mmol Sb/L) was dissolved in

The surrounding temperature for the MBBR was maintained at 35 ± 1oC.

We also set up a

O2 in the reactor’s gas phase was

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Chemical Analysis

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We took liquid samples using a 200-mL gas-tight syringe and immediately filtered

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them through a 0.22-µm membrane filter (LC+PVDF membrane, Shanghai Xinya,

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China).

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any precipitate, diluted the samples with deionized water, and then determined Sb(V)

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and Sb(III) using HPLC-ICP-MS (Nexlon 300X, PekinElmer, USA).20,34

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was equipped with an anion exchange column (PRP-X100, 250 mm, 10 mm,

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Hamilton) to separate Sb(V) from Sb(III).

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hydrogen-phthalate at pH 4.5 was applied as the mobile phase, which was delivered at

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a constant flow rate of 1 ml/min.

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spectrometry (ICP-MS) was run in standard mode for Sb determination.

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Biofilm Sampling, DNA Extraction, and Imagining

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We took biofilm samples from the MBBR at the end of each stage by cutting off a

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~5-cm section from a coupon fiber for DNA extraction, and we sealed the remaining

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fiber by tying the open end into a knot.

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conducted in an anaerobic glove box (AW200SG, Electrotek, England). DNA was

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extracted from the biofilm using the DNeasy Blood and Tissue Kit (Qiagen,

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Germantown, MD).

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coupon fiber was cut off for SEM observation, as previous described by Lai et al,20

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and the suspended white precipitates in the bulk liquids were also collected for

156

characterization.

We centrifuged the filtered liquid samples at 15,000 g for 10 min to remove

The HPLC

20 mM EDTA and 2 mM potassium

The inductively coupled plasma-mass

The procedure for biofilm sampling was

At the end of Stage 3, an extra ~5 cm long section from the

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Phylogenetic Analysis Targeting Archaeal and Bacterial 16S rRNA Genes

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We applied primer pair 1106F (5’-TTWAGTCAGGCAACGAGC-3’)/1378R

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(5’-TGTGCAAGGAGCAGGGAC-3’),35 and 515F

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(5’-GTGCCAGCMGCCGCGG-3’)/ 907R (5’-CCGTCAATTCMTTTRAGTTT-3’)8

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to amplify partial 16S rRNA gene of methanogenic Archaea and Bacteria, respectively.

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The PCR products were purified by using AxyPrep DNA Gel Extraction Kit (Axygen

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Biosciences, USA) and sent to Novogene Technology (Beijing, China) for Illumina

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MiSeq sequencing. We processed the raw data using QIIME (version 1.9.1) as

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described by Lai et al.36 and predicted the metagenomic information using PICRUSt

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piplines as described by Langille et al.37 and Xie et al.38

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The primer set 344F (5’-ACGGGGYGCAGCAGGCGCGA-3’)/915R

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(5’-GTGCTCCCCCGCCAATTCCT-3’) targeting archaeal 16S rRNA gene was used

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for constructing clone libraries.

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Simple cloning kit (TransGen, China), and the positive clones were sequenced

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following screening with M13 universal primers.39

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and searched related comparing sequences in the NCBI website using BLAST.

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phylogenetic trees of these genes were constructed using Mega software. We

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aligned the sequences using the Clustal W program,40 and constructed the

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phylogenetic tree by the neighbor-joining method.41

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length of 554 bp was used for analysis.

The bootstrap analysis was used to provide

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statistical confidence for the branches.

We grouped the clones together which had

The amplicons were cloned by using a pEASY-T1

We selected 49 positive clones The

The final edited sequences with

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the sequence similarity > 99%.

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Quantitative Analysis of mcrA and Bacterial and Archaeal 16S rRNA Gene

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We used quantitative PCR (qPCR) to assess the abundance of the mcrA gene (encodes

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the α unit of methyl-coenzyme reductase, a key enzyme for methanogenesis and

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AnMO), and 16S rRNA genes from archaea and bacteria in our samples.

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primers and qPCR conditions are shown in Table S1.

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RNaseH Plus, Takara Bio Inc., Japan) was used to conduct qPCR as described by

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Zhao et al,42 and sterile water served as negative template.

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cloned, and then linearized plasmids containing the target sequences were used to

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construct standard curves.

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Sb2O3 recovery and Characterization

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The Sb2O3 microcrystals were recovered by the method from Abin et al.28 We added

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the precipitates to deionized water, 100% acetone, and deionized water in sequence,

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centrifuged samples at 10000 g for 2 min for each addition, and discarded the

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supernatant.

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in a freeze dryer (Alpha 1-2 LD, Martin Christ, Germany).

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spectroscopy (EDS, Hitachi Se3400N VP-SEM-EDS) was performed to confirm the

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elementary composition of the precipitates, while X-ray photoelectron spectroscopy

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(XPS) was applied to analyze the valence state of Sb through a Thermo ESCALAB

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250 electron spectrometer (Thermo Electron, USA).

The

SYBR Premix Ex Taq Kit (Tli

PCR products were

All reactions were performed in triplicate.

This process was repeated three times, and the precipitates were dried Energy dispersive X-ray

X-Ray diffraction (XRD,

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X’Pert3 Powder, PANalytical, Netherlands) analysis was performed to characterize

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the crystal structure of the precipitates with CoKα radiation (λ = 1.541 Å) operating at

200

40 kV and 40 mA, and photoluminescence (PL) spectrum was recorded with FLS920

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spectrofluorometer (Edinburgh Instruments, England) at an excitation wavelength of

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325 nm.

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Results and Discussion

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Sb(V) reduction in the CH4 -MBBR

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Figure 1 shows the concentration of Sb(V) in the MBBR for five cycles in the three

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stages.

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higher than the maximum theoretical CH4 demand for all stages (Table S2).

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added 410 µmol/L of Sb(V) was fully removed within 20 and 12 days in Stages 1A

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and 1B, respectively.

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within 20 and 16 days in Stages 2A and 2B, respectively.

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entirely removed within 34 days, even though more than 1600 µmol /L of Sb(V) was

213

introduced.

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mainly followed zero-order kinetics, and the increasing zero-order k values show that

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the Sb(V)-reduction rate continued to increase throughout the experiments, implying

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enrichment of microbial groups doing CH4 oxidation and Sb(V) respiration in the

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MBBR.

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The increase of Sb(III) in parallel with the loss of Sb(V) further supports that

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bio-reduction, rather than bio-adsorption, was the major process for Sb(V) removal.

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The decrease in total soluble Sb (~1200 µmol/L at the end of the experiment,

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compared to addition of 4100 µmol /L as Sb(V)), along with the occurrence of white

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precipitates (Sb2O3), were signs of Sb(III) precipitation.

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the control without CH4 supply (Figure S2), meaning that the Sb(V) reduction was

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driven by CH4 oxidation.

The CH4 supply was not limiting, since the CH4 delivery capacity was much The

Similarly, 820 µmol /L of Sb(V) was completely removed In Stage 3, Sb(V) was

A linear-regression model (Table S3) supports that Sb(V) reduction

Sb(V) loss was minimal in

By subtracting total soluble Sb (soluble Sb(V) plus 13



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soluble Sb(III)) from total added Sb(V), we calculated that the concentration of Sb(III)

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precipitated was 2900 µmol /L as Sb at the end of Stage 3.

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In our previous studies, we demonstrated Cr(VI) and Se(VI) reductions in CH4-based

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MBfRs having continuous influent feeding.5, 8 In those MBfRs, O2 remained at

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concentrations between 0.1-0.2 mg/L in liquid phase, and O2 seemed to have played

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an important role in activating CH4 to products that were utilized to drive Cr(VI) and

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Se(VI) bio-reduction.5, 8

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into the reactor, and the O2 concentration was below the detection limit at all times.

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The absence of oxygen implies that CH4 oxidation was carried out via AnMO.

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Sb2O3 characterization

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Multiple characterization technologies were applied to study the physical and

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chemical features of produced Sb(III) precipitates, and the results are shown in

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Figures 2 and 3.

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and complex fan-shaped (orthorhombic) microcrystals (Figures 2A＆B), and the sizes

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of these microcrystals ranged from 1 to 10 µm. XPS shows that trivalent Sb (Sb(III))

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was the predominant valence state for the microcrystals (Figure 3A).

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the microcrystals shows that Sb and O were the main chemical elements, and the ratio

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of Sb/O was near to 2/3, consistent with the ≥99% pure Sb2O3 standard sample

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(Figure 3B).

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The intense and sharp peaks shown in XRD (Figure S3) indicate that the Sb2O3 was

In this study, we employed the MBBR to preclude O2 entry

SEM shows that the precipitates were mainly composed of cubic

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EDS targeting

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well crystallized and a mixture of cubic (senarmontite, cell constants a=11.140 Å,

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b=11.140 Å, c=11.140 Å; JCPDS Card No. 75-1565) and orthorhombic (valentinite,

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cell constants a=4.911 Å, b=12.464 Å, c=5.412 Å; JCPDS Card No. 71-0383) Sb2O3

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microcrystals, in agreement with the SEM observations in Figure 2C. These results

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were confirmed by Raman spectrum (Figure 3C), in which the signals of the Sb2O3

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microcrystals fit well with the data of cubic and orthorhombic phase Sb2O3 described

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by Cody et al.43 and Mestl et al.44

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the maximum peak intensity of the Sb2O3 microcrystals was at 382 nm, consistent

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with synthesized Sb2O3 widely applied in industrial manufacturing.45-47

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All the characterization results confirm that Sb2O3 was the dominant reduced product,

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and this underscores the potential for producing commercially valuable microcrystals

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using AnMO of Sb.

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Enrichment of archaeal and bacterial genes

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Figure 4 shows the copy numbers of archaeal and bacterial 16S rRNA genes, along

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with the mcrA gene, in Stages 1-3 based on qPCR.

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continuously through the stages, in parallel to the elevated Sb(V)-reducing capability

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of the biofilm. The enrichment of archaeal 16S rRNA genes and the mcrA gene

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supports that archaea played a role in AnMO in the MBBR system.

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comprise one of the main groups contributing to AnMO,17, 18 while mcr has been

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demonstrated to be the key enzyme in the reverse-methanogenesis pathway used by

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ANME to activate CH4 under anaerobic conditions.48

Finally, the PL spectrum (Figure 3D) shows that

All gene copies increased

Archaea ANME

The larger copy numbers for

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the bacterial 16S rRNA gene over that of archaeal 16S rRNA gene (about 2 logs

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versus 1 log) is likely due to the inherently slower specific growth rate of

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methanogens compared to bacteria.49

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Microbial Community Structure

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Figure 5 shows the dominant methanogenic archaea identified by Illumina sequencing

271

targeting the methanogenic 16S rRNA gene.

272

were Methanosarcina and Methanolobus.

273

decreased from 42% to 19% from Stage 1 to Stage 3, while the relative abundance of

274

Methanosarcina increased from 33% to 75%.

275

that resemble Methanosarcina (Figure 2C).

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produce methane, it is also able to perform methane oxidation and release acetate or

277

methanol, which can be used as electron donors to support respiration for co-existing

278

bacteria.10,50

279

couple AnMO to Fe3+ reduction.51 Furthermore, Luo et al. reported that

280

Methanosarcina appeared to be the methane-oxidizing microorganism to generate

281

acetate in a CH4-based MBfR supplied with BrO3- as the electron acceptor.10

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We compared the clone library targeting the archaeal 16S rRNA gene for the MBBR

283

biofilm to known ANME gene sequences derived from NCBI; Figure 6 gives the

284

phylogenetic tree.

285

affiliated to Methanosarcina, in agreement with Illumina sequencing analysis.

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clone sequences were phylogeneticly related to ANME-3, which is affiliated with

The two main methanogenic archaea

The relative abundance of Methanolobus

The SEM images also showed cocci Although Methanosarcina are known to

Moran et al. reported that Methanosarcina acetivorans was able to

Most of the gene sequences from the MBBR biofilm were

16


These

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Methanosarcinales and implicated in manganese-dependent AnMO.14 The

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enrichment of Methanosarcina in the CH4-fed MBBR implies that Methanosarcina

289

might play a role in AnMO.

290

Figure S4 shows the bacterial community structure of the MBBR biofilm based on

291

Illumina sequencing.

292

methylotroph Methylophilus were detected throughout all stages, but decreased

293

significantly, from 37% to 10% and from 22% to 7%, respectively, from Stage 1 to

294

Stage 3.

295

in the MBBR.

296

biofilm after 111 days, which is consistent with the occurrence of rod-shaped cells

297

observed by SEM (Figure 2D).

298

The persistence of aerobic methanotrophs in the anaerobic MBBR probably occurred

299

because aerobic methanotrophs coming from the micro-aerobic inoculum could

300

tolerate the MBBR’s anaerobic environment.

301

type strain FJG1 has been reported to oxidize methane coupled to NO3- reduction

302

during hypoxia,52 while Danilova et al. found that the type Ib methanotroph

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Methylococcaceae tended to live in zones where CH4 was enriched but O2 was

304

scarce.53 Furthermore, Naqvi et al. found the co-existence of nitrite-dependent

305

anaerobic methanotrophs (NC10 phylum) and aerobic mathanotrophs with high

306

abundance (up to 13.9%) in anoxic freshwater reservoirs.54

307

However, as aerobic methanotrophs have versatile functions, we cannot completely

The aerobic methanotroph Methylomonas and the

Their declining trend supports that these aerobes were being out-competed However, these two genera still occupied a large proportion in the

Methylomonas denitrificans sp. nov.

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rule out a contribution of these microorganisms to AnMO.

Martinez-Cruz et al.

309

incubated sub-Arctic lake sediments in anaerobic serum bottles and found that the

310

type I methanotroph Methylobacter was the predominant genera involving AnMO.55

311

Similarly, Bar-Or et al. supplied an anoxic slurry with 13C-labeled CH4 and iron

312

oxides and, conducting anaerobic incubations, found significant 13C enrichment into

313

aerobic methanotrophs and increased gene copies of pmoA.56

314

the door to aerobic methanotrophs having played some role in AnMO, although the

315

function is unknown.

316

In this research, we achieved Sb(V) reduction in an anaerobic MBBR fed with CH4 as

317

the sole electron donor and carbon source.

318

crystalline Sb2O3.

319

played a role in AnMO through reverse methanogenesis, since its genus’s abundance

320

and the copy numbers of mcr gene increased in parallel to an increasing rate of Sb(V)

321

reduction.

322

reductase has not been purified, further study is needed to verify the role of

323

Methananosarcina or other microorganisms.

These evidences open

The major reduction product was

Methanosarcina, a well-known methanogen, seems to have

As the Sb(V) reduction mechanism is unknown and a microbial Sb(V)

324

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Acknowledgments

326

Authors greatly thank the “National Key Technology R&D Program

327

(2017ZX07206-002)”, the “National Natural Science Foundation of China (Grant No.

328

21577123)”, the “Natural Science Funds for Distinguished Young Scholar of Zhejiang

329

Province (LR17B070001)”, and the “Fundamental Research Funds for the Central

330

Universities (2016QNA6007, 2017XZZX010-03)” for their financial support.

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Figure 1.

Concentrations of Sb(V) and Sb(III) in the CH4-based MBBR.

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At the

end of each cycle, the total soluble Sb was consistently less than the total Sb(V) added, 4100 µmol/L by the end of the experiment.

1


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Figure 2.

SEM of Sb2O3 microcrystals and microbial cells. Sb2O3 microcrystals

formed after Sb(V) reduction with cubic (A) and orthorhombic (B) crystal structures; Aggregation of Methanosarcina–like cells is observed at 20000-X magnification (C); Bacteria with rod-shaped cells dominated in the biofilm observed at 10000-X magnification (D).

2



Figure 3.

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Characterizations of Sb2O3 microcrystals produced in the biofilm.

XPS

of purified Sb2O3 in the MBBR biofilms and Sb2O3 standard sample (A).

EDX

pattern of the Sb2O3 standard sample and precipitates in the biofilm (B).

Raman

spectra of the purified Sb2O3 in the biofilms (C).

PL spectra obtained from the

purified Sb2O3 in the biofilms; the λex is at 325 nm (D).

3


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Figure 4.

Gene copies of mcrA and 16S rRNA genes of archaea and bacteria in the

CH4-based MBBR biofilms using qPCR. Results are presented as the mean values plus standard deviations from three replicate qPCR reactions.

4



Figure 5.

Relative abundances of dominant methanogenic archaea in the biofilm at

each of the three stages, based on Illumina sequencing.

5


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Figure 6.

Phylogenetic tree of archaeal 16S rRNA gene sequences retrieved from

the biofilm compared to ANME sequences derived from NCBI. This is a neighbor-joining tree calculated using program Clustal W. Support for branches was determined from 1000 bootstrap iterations. The scale bar indicated 2% divergence. Solid circles indicate the nodes with a bootstrap support greater than 75%.

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