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Biochar-mediated Anaerobic Oxidation of Methane Xueqin Zhang, Jun Xia, Jiaoyang Pu, Chen Cai, Gene W. Tyson, Zhiguo Yuan, and Shihu Hu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b01345 • Publication Date (Web): 17 May 2019 Downloaded from http://pubs.acs.org on May 19, 2019

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Environmental Science & Technology

Biochar-mediated Anaerobic Oxidation of Methane

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Xueqin Zhang1, Jun Xia1, Jiaoyang Pu1, Chen Cai1, Gene W Tyson2, Zhiguo Yuan1 & Shihu

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Hu1,*

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1Advanced

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

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2Australian

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University of Queensland, Brisbane, QLD, Australia

Water Management Centre, Faculty of Engineering, Architecture and Information

Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, The

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Corresponding Author

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* Tel: +61 7 3346 3230. Fax: +61 7 336 54726. Email: [email protected]

1

ACS Paragon Plus Environment


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Table of Contents (TOC) Art

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ABSTRACT: Biochar was recently identified as an effective soil amendment for CH4 capture.

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Corresponding mechanisms are currently recognized to be from physical properties of biochar

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providing a favourable growth environment for aerobic methanotrophs which perform aerobic

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methane (CH4) oxidation. However, our study shows that the chemical reactivity of biochar

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can also stimulate anaerobic oxidation of CH4 (AOM) by anaerobic methanotrophic archaea

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(ANME) of ANME-2d, which proposes another plausible mechanism for CH4 mitigation by

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biochar amendment in anaerobic environments. It was found that by adding biochar as the sole

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electron acceptor in an anaerobic environment, CH4 was biologically oxidised, with CO2

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production of 106.3 ± 5.1 μmol g-1 biochar. In contrast, limited CO2 production was observed

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with chemically reduced biochar amendment. This biological nature of the process was

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confirmed by mcr gene transcript abundance as well as sustained dominance of ANME-2d in

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the microbial community during microbial incubations with active biochar amendment.

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Combined FTIR and XPS analyses demonstrated that the redox activity of biochar relates to

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its oxygen-based functional groups. Based on microbial community evolution as well as

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intermediates production during incubation, different pathways in terms of direct or indirect

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interactions between ANME-2d and biochar were proposed for biochar-mediated AOM.

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INTRODUCTION

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Environmental and social concerns over global methane (CH4) emissions relate to its high

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global warming potential (GWP) which is 25 times that of CO2 over a period of 100 years.1

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Most CH4 released to the atmosphere (69%) is microbially derived, from natural sources

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including oceans, wetlands, sediments, soils, and from anthropogenic sources such as rice

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paddies, sewage treatment plants, landfill.2 In an attempt to mitigate the adverse effects on

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climate caused by increased CH4 emission, efforts have targeted low-level CH4 emissions,

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mainly from anthropogenic sites. A popular mitigation strategy is to enhance microbial CH4

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oxidation in situ by applying amendments including inorganic materials such as sand3 and slag4,

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and organic materials such as sludge5, compost6, and wood pellets7 to fields which would

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otherwise emit significant CH4.

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Biochar was recently proposed as an alternative amendment for CH4 mitigation, and it has

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been shown to decrease CH4 emission from landfill7, 8 and paddy soils.9, 10 Biochar is produced

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via pyrolysis or gasification of organic substances under oxygen-limited conditions.11 It

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features merits of microbial-decomposition recalcitrance to organic amendments9 and

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microbial affinity to inorganic amendments,1 which catalyse renewed interests in mechanisms

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of biochar amendment decreasing CH4 emissions. Biochar’s high surface area and porosity

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enable a high water-retention capacity, creating favourable conditions for the colonization of

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CH4-affiliated organisms.7,

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microbial CH4 oxidation kinetics by increasing gas diffusion.9, 10

9

In addition, biochar’s porosity and adsorbability facilitate

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Biochar is generally considered to be chemically inert toward adsorbates,12 and all

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aforementioned mechanisms are based on our common understanding of biochar’s physical

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properties in terms of favourable surface properties, porosity and adsorbability. However,

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recent studies have demonstrated biochar is also chemically reactive. Characteristics of biochar

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such as a graphitic structure and/or the presence of redox-active functionalities such as (hydro)

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quinones, facilitate abiotic and biological redox reactions.12-14 Therefore, biochar-amended soil

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can potentially act as a carbon sink by stimulating microbial CH4 respiration, or microbial

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metabolism of CH4 via other pathways, as yet undetermined.

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Moreover, the current understanding of mechanisms by which biochar impacts CH4 fluxes

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is limited to its influences on aerobic CH4 oxidation by methanotrophs.7,

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anaerobic oxidation of methane (AOM) by anaerobic methanotrophs also plays a significant

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role in global CH4 emission mitigation.15, 16 Due to the unfavourable gas accessibility of soil,

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biochar amended into deep fields may involve itself into active mediation of anoxic CH4

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oxidation, the feasibility and mechanism of which have yet to be explored.

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However,

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Therefore, the aim of the present study was to investigate the chemically reactive role of

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biochar amendment on anoxic CH4 oxidation for the mitigation of CH4 emission. A mixed

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microbial culture dominated by anaerobic methanotrophs was coupled to biochar amendment,

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and CH4 turnover and AOM gene transcription were monitored. In addition, biochar as a direct

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electron sink for mitigating global CH4 emissions was evaluated based on globally available

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biomass-feedstocks which could be converted into biochar, and as an electron shuttle indirectly

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facilitating AOM coupled to other ubiquitous electron acceptors was assessed based on the

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standard reduction potential of biochar.

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MATERIALS AND METHODS

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Biochar. The commercial Triple R Biochar (Triple R Co, AUS) used here was produced by

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pyrolysis at 600-650 oC from sustainably sourced plant material. The detailed properties of the

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biochar are summarized in Table S1 of the Supporting Information. The biochar was ground,

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and a fraction was then sieved (40 µm). The powder was then rinsed with Milli-Q water three

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times to remove organics before being oven-dried at 80 oC. Total organic carbon (TOC)

79

analysis suggested the washed biochar contained little dissolved organic carbon (Figure S1).

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Inoculum. The mixed culture which was used as inoculum was taken from a 5 L parent

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reactor performing nitrate reduction coupled to AOM.17 The mixed culture is most dominated

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by an anaerobic methanotrophic archaeon, known as Candidatus ‘Methanoperedens

83

nitroreducens’ or ANME-2d.18

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For microbial inoculation preparation, biomass was collected from the parent reactor and

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centrifuged 6000 rpm for 15 min. The biomass pellet was then washed three times

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anaerobically with fresh medium to eliminate residual nitrate, followed by resuspension in

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fresh medium with double volume for downstream inoculations. The medium consisted of

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ingredients modified from that used in the parent reactor17 (0.15 g L-1 NH4Cl, 0.015 g L-1

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CaCl2·2H2O, 0.1 g L-1 MgCl2·6H2O, trace elements,17 and 25 mM PBS to buffer pH variation

90

caused by biochar amendment), and was anaerobically prepared by sparging with nitrogen for

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30 min.

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Test Incubations for Biochar-mediated CH4 Oxidation. Incubations were performed in

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4.5-mL Exetainer vials (Labco, UK) with sacrificial sampling. Biochar with exact quantities

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(0.04 or 0.08 g) was added to vials as the potential electron acceptor. Before inoculation, vials

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containing biochar were placed in a vacuum chamber overnight to eliminate absorbed oxygen.

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Biomass suspension was then distributed to aliquots of 2 mL in Exetainers, resulting in

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biochar concentrations of 40 g L-1 and 20 g L-1, respectively. The abiotic control test was

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prepared by adding 2 mL fresh medium into 0.08 g biochar-amended vials.

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To test our hypothesis that biochar can play a role as an electron sink to medicate microbial

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CH4 oxidation, another control incubation was prepared with chemically reduced biochar.

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Vials containing 0.08 g biochar were filled with 4 mL 50 mM sodium bisulfite solution, and

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were shaken overnight in an anaerobic chamber (Coy Laboratory Products Inc., USA). The

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chemically reduced biochar was then washed anaerobically with fresh medium three times

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before inoculating with 2 mL of biomass.

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To confirm the role of CH4 as electron donor, a control test was performed without CH4

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feeding. These vials contained 0.08 g biochar and biomass inoculation, and were pressurized

107

with 2.0 mL argon gas.

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All other Exetainer vials were sealed and pressurized by injecting 2.0 mL CH4 (also provided

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as electron donor). All inoculation procedures were carried out anaerobically in an anaerobic

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chamber. Inoculated vials were then transferred to a shaker-incubator (30 oC and 120 rpm,

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Multitron, INFPRS HT, Switzerland).

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The CH4 oxidation activity was quantified by determining inorganic carbon (CO2)

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production as well as CH4 consumption. To quantify CO2 accumulation, three vails were

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harvested every 3 days and acidified with 0.2 mL anaerobically prepared HCl stock solution (1

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M) to extract dissolved CO2. Vials were then kept for at least 1 h for headspace equilibration

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before CO2 was analysed by gas chromatography. CH4 consumption was determined by CH4

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concentration change in the both headspace and liquid of incubation vails. CH4 concentration

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in the headspace was determined by direct gas chromatography measurement and that in the

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liquid was calculated form headspace concentration based on Henry’s Low. Vials were also

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harvested on a regular basis from duplicated incubations (with 0.08 g biochar amendment) to

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test for acetate production test. To test for biological CH4 oxidation, parallel vails containing

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0.08 g biochar or 0.08 g chemically reduced biochar were harvested at time intervals for mcr

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gene transcriptomic analysis by reverse transcription PCR (RT-PCR), and microbial

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community analysis by 16S rRNA amplicon pyrosequencing.

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Electrochemical Characterizations of Biochar. Cyclic voltammetry (CV) was used to

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characterize the redox properties of biochar. Biochar-based electrodes were prepared by

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coating conductive carbon cloth (AvCarb 1071 HCB, FuelCellStore, USA) with biochar

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powder (prepared as previously described). Carbon cloth was cut into a square piece (2cm 

129

2cm), and then cleaned by rinsing in 1 M HCl followed by rinsing in pure acetone, each for 24

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h. Afterwards, cloth pieces were washed thoroughly with Milli-Q water and dried at room

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temperature before use. For the coating process, biochar powder (5 mg) was mixed with 1000

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uL absolute ethanol and 100 uL Nafion (5%, Sigma, USA) under sonication to obtain a coating

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ink. The ink was then spread evenly on both sides of the carbon cloth, and dried completely.

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Finally, the biochar-coated cloth was connected to titanium wires for use as electrodes for CV

135

tests. A PBS solution (50 mM) with 1 M KCl was used as the electrolyte for CV tests, with

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scans recorded from -0.8 V to + 0.8 V, and varied sweep rates from 1 mV s-1 to 50 mV s-1.

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The biochar’s oxidative state was further evaluated and quantified by direct electrochemical

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reduction, performed in a sealed electrolysis cell (50 mL). Biochar-based electrodes with mass

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loadings of 2.5 mg, 5.0 mg, 7.5 mg, 10 mg, 12.5 mg, respectively, were used as working

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electrodes, and a titanium wire was applied as the counter electrode. The electrolysis cell was

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filled with 40 mL electrolyte solution (as above) that had been sparged for 30 min to eliminate

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dissolved oxygen before sealing the vessel. Electrochemical reduction was achieved with a

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potentiostat (BioLogic VSP, Claix, France) by applying a reductive potential of -0.4 V versus

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an Ag/AgCl reference electrode (RE-5B, BASi, USA). Reductive current responses were

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recorded. Each electrochemical reduction was equilibrated once reductive current was

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decreased to baseline (current response of electrochemical reduction of carbon cloth without

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biochar loading). The resulting current peaks were integrated to determine electron transfer (Q)

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for the reduction of biochar with different masses: 𝑡

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Q=

∫𝑡0𝐼𝑑𝑡 𝐹

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Where I is the baseline-calibrated reductive currents recorded, t is time, and F (=96485 C mol-1

151

e-) is the Faraday constant. The electron storage capacity (ESC) of biochar was defined by

152

integrated electrons transfer per unit biomass mass, which was derived by fitting the slope of

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the regression line of reductive electron transfer versus biochar mass.

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Chemical Analyses. Quantities of CH4 and CO2 in the headspace were determined by gas

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chromatography. 100 µL of headspace gas sample was collected by gas-tight syringe (1710SL,

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Hamilton, USA) and the concentration was quantified with an Agilent Gas Chromatograph

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(GC, 7890A, Agilent, USA) equipped with a HayeSepQ column (2440  2.0mm) and a thermal

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conductivity detector. Argon was used as carrier gas at a flow rate of 28 mL min-1. The injector,

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column and detector temperatures were maintained at 110, 45 and 170 ℃ respectively during

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tests. Gases were identified according to their retention times, and their concentrations were

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calculated against standards.

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Liquid samples were taken from vails using a syringe, and then filtered immediately through

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a 0.22 μM disposable sterile millipore filter (Merck). Acetate concentration was measured

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using a Shimadzu HPLC system with a Bio-Rad HPLC Column (300×7.8 mm) and a refractive

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index detector. Peaks corresponding to those of organic acids were confirmed by retention time,

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co-elution with standards, and by comparing absorbance spectra with those of the standards.

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Total organic carbon (TOC) was measured by using a TOC Analyzer (Shimadzu 5000 A).

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Fourier Transform Infrared Spectroscopy (FTIR). FTIR analysis was performed using

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an infrared spectrometer (Nicolet 6700, Thermo Scientific, USA) equipped with a Diamond

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ATR crystal (Attenuated Total Reflection). The spectrum was collected in the frequency

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range 2,000–600 cm−1.

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X-ray Photoelectron Spectroscopy (XPS). XPS spectra were collected by a Kratos Ultra

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Axis XPS with Al Kα X-ray. Spectra calibrated on the 538.1 (O1s) and 296.1 (C1s) eV peaks

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were analyzed using XPSPEAK.

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RT-PCR. Biomass was collected from each incubation vial (2 mL), and total cell RNA was

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extracted using RNeasy Mini kit (Qiagen, Germany) according to manufacturer’s protocol. An

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additional DNase treatment was performed (provided within the Qiagen RNase-Free DNase

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Set). RNA quality was checked by agarose-gel electrophoresis, and the RNA concentration 9



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was measured with a spectrophotometer Nanodrop (ND-1000, Thermo Fisher, USA). RNA

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was reverse transcribed to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen) via

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mcrA gene primer 345R. Quantitative real-time PCR (qPCR) amplification was performed by

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using a QuantStudio real-time PCR system (Thermo Fisher Scientific, USA) with the following

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composition: 0.1 μL mix of 20 μM of the mcrA gene primers 159F and 345R, 5 μL of SYBR

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Green PCR Master Mix (Thermo Fisher Scientific), 2 μL DNA (1ng/ μL) and 3 μL of Milli-Q

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water. The PCR cycle conditions were as follows: 95 °C for 10 min, followed by 40 cycles of

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95 °C for 15 s, 60 °C for 1 min, then a final melt curve stage at 95 °C for 15 s, 60 °C for 1 min

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and 95 °C for 15s. The standard curve was constructed from a series of 10-fold dilution series

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of plasmid DNA of a known copy number, with R2 values of at least 0.99 for all assays.

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16S rRNA Gene Sequencing. At the end of the incubation course, biomass (1 mL) was

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collected from incubation vials, and total cell DNA was extracted using FastDNA SPIN for

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

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DNA concentration was quantified with NanoDrop 2000 (Thermo Fisher Scientific, USA). The

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16S

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AAACTYAAAKGAATTGACGG-3 ′ ) and 1392R (5 ′ -ACG- GGCGGTGTGTRC-3 ′ ). A

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

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were employed to purify and quantify the PCR products, respectively. Amplicons were pooled

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in equimolar concentration and sequenced with an Illumina sequencer (Illumina, USA). Raw

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sequencing data were quality-filtered and demultiplexed using Trimmomatic, with poor-

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

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similarity were clustered into operational taxonomic units (OTUs) using QIIME with default

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parameters, and representative OTU sequences were taxonomically aligned against

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Greengenes 16S rRNA database. An OTU table consisting of the taxonomic classification and

rRNA

gene

was

amplified

using

10

universal


primer

set

926F

(5 ′ -

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OTU representative sequences was generated as the main analysis results, based on which a

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microbial community heatmap was based (Rstudio).

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RESULTS AND DISCUSSION

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CH4 Oxidation Activity Assessed by CO2 Accumulation and CH4 Consumption. As shown

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in Figure 1a, , there was no CO2 production detected in abiotic incubation tests (without

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biomass inoculation). This suggested that chemical catalysis of CH4 by biochar was not

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occurring. Limited CO2 was produced in the presence of biomass without CH4 feeding, with

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an accumulation of 1.26 ± 0.05 µmol CO2 over 21 days. Other potential sources of CO2, for

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example, from biochar intrinsic organics or biomass lysis, were excluded as biochar rinse

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solutions contained no detectable TOC after 3 washes (Figure S1). In addition, the stable

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ammonium concentration observed over time (data not shown) indicated negligible microbial

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lysis was occurring. Interestingly, acetate accumulation was traced during the initial incubation

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period (within 4 days) regardless of the presence of CH4, presumably as a result of

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decomposition of microbial intracellular storage compounds that were generated whilst in the

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parent reactor (Figure S2).18 A gradual decrease in acetate concentration after day 5 indicated

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that acetate may be released as an intermediate supporting biochar-mediated microbial-

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respiration and CO2 production (Figure S2).

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In contrast, with CH4 feeding, vials containing 0.08 g biochar rapidly accumulated CO2

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reaching a maximum concentration at 15 days (Figure 1a, biochar + biomass). The amount of

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CO2 in the reactor became stable after 15 days, implying that all biochar added have been

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reduced and the reaction stopped. The supply of CH4 resulted in faster CO2 production rate as

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well as much higher accumulation (8.50 ± 0.41 µmol) to that without CH4 feeding, indicating

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that the CO2 was produced from CH4. As all other potential electron acceptors (including

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oxygen, nitrate and sulphate) had been eliminated from incubations, biochar was likely the sole

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candidate mediating CH4 oxidation. This hypothesis is supported by the limited CO2

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accumulation (1.34 ± 0.28 µmol) in the incubation containing chemically reduced biochar

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(Figure 1a, 0.08 g reduced biochar + biomass). The small amount of CO2 production in this

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case probably originated from incomplete chemical reduction of biochar, and the CO2

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accumulation gap mediated by reduced biochar demonstrated that reactive biochar played a

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role as electron sink for AOM. Furthermore, the incubation with 0.04 g biochar demonstrated

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a similar CO2 production trend to the 0.08 g biochar incubation, but with approximately half

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the amount of CO2 accumulation (4.09 ± 0.41 µmol), which was a quantitative support of

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biochar’s active role mediating AOM processes. The CH4 oxidation capacities of biochar

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calculated based on the CO2 production rates are 106.3 ± 5.1 μmol g-1 and 102.3 ± 10.3 μmol

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g-1, separately, during these two tests.

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Figure 1. CO2 production (a) and CH4 consumption (b) in incubations with biochar only, 0.08

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g biochar with biomass, 0.04 g biochar with biomass, 0.08 g chemically-reduced biochar with

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biomass, and 0.08 g biochar with biomass without CH4. Data represent the mean values of

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triplicate incubations ± standard deviation of the mean.

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CH4 consumption was monitored as a more direct way to describe CH4 oxidation activity

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mediated by biochar. It should be noted that due to the technical difficulty of accurately

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controlling manual feeding of 2 mL CH4 to incubation vials, CH4 concentrations were

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measured with a relatively wide confidence interval (Figure 1b). Even so, an increasing trend

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in CO2 production was observed with a concomitant decreasing trend in CH4 consumption in

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biochar amended biotic incubations. A total of 6.35 ± 0.52 µmol and 4.12 ± 0.96 µmol CH4

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was consumed in 0.08 g and 0.04 g biochar amendments, respectively. CH4 consumption was

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related to CO2 production with a corresponding balance error of 25.3% and 0.7%, respectively

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(calculation details in Supporting Information). In contrast, incubations with biochar only

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(without microbial inoculum) or with chemically reduced biochar with microbial inoculum

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(Figure 1b, reduced biochar + biomass) demonstrated limited CH4 consumption over time.

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Together, these results support the hypothesis that biochar can mediate anaerobic CH4

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oxidation.

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Tracking Microbial CH4 Oxidation through mcr Gene Transcription and Community

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Analysis. The inoculum used here contains ANME-2d which anaerobically activates CH4

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through reverse methanogenesis mediated by the mcr gene.18 Thus we used mcr transcript

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abundance to indicate the bio-activity of AOM. We measured mcr expression in microbial

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incubations amended with biochar and chemically reduced biochar, respectively, to determine

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how the redox capacity of biochar affects AOM activity.

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As shown in Figure 2a, actively growing biomass demonstrated high transcript levels for the

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mcr gene (ca. 5.1  108 copies, day 0). A significant drop in transcript abundance was observed

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on day 3 in 0.08 g biochar-amended incubations. As the biomass used here was originally

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cultured with nitrate as the electron acceptor, the transcript drop was likely due to the less

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favourable electron transfer to solid biochar rather than to soluble nitrate. Nevertheless, it still

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remained at a considerable level (4.6  108 copies) in comparison with the reduced biochar

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amendment (6.0  107 copies), demonstrating the contribution of biochar to preservation of

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AOM. Consistent with the gradual decrease in CO2 accumulation (Figure 1a), the mcr gene

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transcript abundance decreased steadily over time (Figure 2a), which was likely due to 13



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saturation of the biochar electron-accepting capacity. In contrast, microbial incubation with

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reduced biochar showed relatively limited mcr transcription from 3 days of incubation onwards,

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indirectly demonstrating the dependence of AOM on the biochar redox capacity.

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Figure 2. (a) Transcript abundances of the mcr gene for microbial incubations with 0.08 g

276

biochar and 0.08 g chemically reduced biochar, respectively. Error bars represent one standard

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deviation from three triplicate incubations; (b) microbial community diversity in incubations

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sampled on day 21: A, inoculum; B, 0.08 g biochar amendment; C, 0.08 g chemically reduced

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biochar amendment.

280

To further interpret the relationship between AOM and biochar chemical reactivity,

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microbial community evolution was compared in non-reduced and chemically reduced biochar

282

amended incubations, respectively (Figure 2b). In the non-reduced biochar amendment,

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methanotrophic activator ANME-2d dominated the community (Figure 2b, B, 31.0%),

284

comparable to that of the inoculum (32.2%). In contrast, ANME-2d represented only 6.6% of

285

the microbial community in the reduced biochar amendment, a decrease of almost 80% from

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the inoculum (Figure 2b, C). A legitimate explanation for this is that because reduced biochar

287

has limited electron-accepting capacity, ANME-2d could not gain enough energy to survive

288

due to interruption of the electron transport chain. Thus microbial community evolution results

289

further confirmed that chemically reactive biochar contributed to anaerobic CH4 oxidation.

290

It is worth noting that Geobacteraceae were enriched in incubations with both non-reduced 14


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(7.9%) and reduced biochar (4.3%) (Figure 2b, B and C, respectively), likely stimulated by the

292

production of substrate, acetate (Figure S2). The extracellular electron transfer capability of

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Geobacteraceae has been widely recognized and reported,19 and redox-active biochar can

294

mediate microbial respiration via extracellular electron transfer.20 Combined, these

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observations indicate that biochar can also stimulate Geobacteraceae to metabolize acetate.

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The presence of Geobacteraceae implied that biochar mediated AOM perhaps occurs through

297

syntrophic interaction between ANME-2d and Geobacteraceae.

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Redox Properties of Biochar Characterized by Electrochemical Analysis. Combined,

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the results suggest that biochar is able to mediate AOM depending on its oxidation state, with

300

biochar losing the capacity when reduced. Other studies have also attributed the ability of

301

biochar to mediate biological reactions to its redox properties.12, 13 In our study, the redox

302

properties of biochar were assessed by cyclic voltammetry (Figure 3a). The bare carbon cloth

303

showed a smooth curve with a weak current response, while the biochar-coated carbon cloth

304

displayed a remarkable current increase with obvious redox peaks. Cyclic voltammetry

305

performed at high scan rates above 5 mV s-1 displayed a dramatic reductive current increase

306

once the potential negatively reached -0.25 V (Figure 3a, inset), caused by interactions on the

307

interface of biochar and electrolyte. Given the existence of overpotential for practical H2

308

evolution by electrical reduction, reduction current turnovers shown on CV curves were not

309

likely from H2 evolution as the reduction turnover potential observed here (around -0.45 V vs.

310

SHE) was highly close to theoretical H2 evolution potential (-0.421 V vs. SHE; pH = 7).

311

Accordingly, oxidative peaks were observed at all scan rates (Figure 3a, inset). Together, these

312

results imply that redox-active sites are present on the biochar contributing to its chemical

313

reactivity. More specifically, the CV conducted at the lowest rate of 1 mV s-1 revealed an

314

obvious redox couple centred at a formal potential of -0.045 V vs. Ag/AgCl. This formal

315

potential is more positive than the midpoint potentials of general cytochromes known to be

15



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involved in microbial extracellular electron transfer (EET).21 Combined, these CV results

317

indicate that biochar is chemically active as an electron sink, and its standard reduction

318

potential range enables biochar to mediate microbial EET.

319

To further evaluate and quantify the capacity of biochar to act as an electron sink for

320

mediating AOM, direct electrochemical reductions of biochar were carried out (Figure 3b). All

321

biochar samples initially generated a strong reductive current response, and then approached

322

equilibrium, as the biochar became saturated with electrons. Increasing biochar mass loading

323

resulted in correspondingly stronger current peaks and a linear increase in respective reduction

324

charge, all of which demonstrate the positive oxidation state of biochar and its capacity to

325

accept electrons. The linear relationship between biochar reduction charge and the biochar

326

mass revealed the electron storage capacity of biochar to be 0.74 mmol e- g-1 (Figure 3b, inset),

327

and this was comparable to the value (0.85 mmol e- g-1) calculated from biochar reduction

328

coupled to microbial CH4 oxidation (calculation detailed in Supporting Information).

329 330

Figure 3. Electrochemical characterizations of biochar (a) Cyclic voltammetry (CV) of carbon

331

cloth alone (CC support), and biochar-coated (5 mg biochar loading) carbon cloth (Biochar),

332

at scan rates ranging from 1 mV s-1 to 50 mV s-1 (as indicated). (b) Current responses to

333

electrical reduction (at -0.4 V vs. Ag/AgCl) with different biochar masses (as indicated), and

334

the relationship between respective biochar mass and reduction charges (insert). The slope of

335

the linear regression line corresponds to the reduction charge of per biochar mass, defined as 16


Page 16 of 28

Page 17 of 28

336 337


electron storage capacity (ESC) of biochar. Characterization of Biochar by XPS and FTIR. Previous studies have showed that

338

biochar’s chemical reactivity is normally related its redox-active moieties.12-14,

339

biochar’s functional groups were characterized through XPS and FTIR to understand the active

340

source that potentially drives AOM.

341

22

Thus,

The qualitative survey detected carbon and oxygen (Figure S3), the latter being commonly

342

attributed in literature to biochar oxygenic moieties.13,

343

resolution core-line scan for C elements (C1s) revealed that the most prominent peak occurred

344

at 285.1 eV for the C-C bond.24, 25 The C1s XPS spectra also demonstrated peaks at 286.5 eV

345

and 288.9 eV for C-O and C=O bonds, respectively.25,

346

containing bonds was also verified by high-resolution O1s XPS spectra (Figure S4).24, 25

23

As shown in Figure 4a, the high

26

The existence of these oxygen-

347 348

Figure 4. (a) High-resolution C1s XPS spectra of biochar; and (b) FTIR profile of biochar

349

Further FTIR characterization revealed two C-C absorbance peaks stretching from 800-

350

1,200 cm-1 (Figure 4b).27, 28 Meanwhile, notable vibrations of C-O at 1,440 cm-1 and C=O at

351

1,570 cm-1 were detected, which was indicative of oxygenic moieties in terms of carboxylic

352

acid groups and quinone structures.13,

353

including biochar is widely recognized to be derived from electron-unsaturated oxygenic

27, 28

The chemical reactivity of carbon materials

17



354

moieties such as (hydro)quinones,

355

especially C=O moiety, revealed by XPS and FTIR characterizations suggests that these

356

functional groups could actively mediate AOM. CH4 oxidation mediated by biochar could be

357

expressed by the following stoichiometric equation:

358

CH4 + 8 C=O + 2H2O  8 C-OH + CO2

12-14, 22

thus the existence of oxygen-based functionalities,

359

Mechanisms for Biochar-mediated AOM. The current study shows that biochar feasibly

360

mediates AOM due to inherent redox-active functionalities. Both abiotic- and biological

361

reduction of biochar have long been reported,12, 29 while our study, to the best our knowledge,

362

is the first report of microbial reduction of biochar by anaerobic methanotrophs. Importantly,

363

our study also provides evidence that anaerobic methanotrophic archaea (ANME) utilize an

364

EET mechanism, a concept which has been proposed but not yet fully confirmed.30, 31 The

365

ANME-2d used in our incubations encodes numerous (38 species) multiheme c-type

366

cytochromes providing a genetic basis for biochar respiration via EET,18 as c-type cytochromes

367

can function as electron shuttles from within the cell to solid electron acceptors external to the

368

cell.32, 33 It was noteworthy that Geobacteraceae also evolved as an important member of the

369

microbial consortium with biochar potentially playing a role as sole electron acceptor (Figure

370

2b). Geobacteraceae is well-recognized for its EET ability,32 and it has been reported to be

371

able to establish syntrophic associations with other microbes via direct interspecies electron

372

transfer (DIET).34 Therefore, we propose that direct EET occurs from ANME-2d to biochar,

373

but direct interspecies electron transfer as a syntrophic coupling mechanism between ANME-

374

2d and Geobacteraceae may also be occurring during biologically mediated biochar reduction.

375

Moreover, as previously mentioned the production of acetate during incubation provides

376

another potential electron transfer mechanism based on interspecies transfer via metabolic

377

intermediates. Based on results from the incubation control without CH4 (Figure 1), the

18


Page 18 of 28

Page 19 of 28


378

presence of acetate was likely due to its release from intercellular storages of the inoculum.18

379

It is also possible that real-time acetate production occurred from direct oxidation of CH4 via

380

the reductive acetyl CoA pathway in ANME-2d.18 In our study, ANME-2d was also potentially

381

linked to Geobacteraceae through a diffusible metabolite with acetate acting as an intermediate,

382

rather than through direct electrical connections. For this mechanism, electrons originating

383

from CH4 are not necessarily transferred from the cytoplasm to the outer membrane in ANME-

384

2d. However, in this cases, the biosynthesis of acetate from CH4 oxidation thermodynamically

385

requires extra electron acceptors.35 As no other electron acceptors were added to our

386

incubations, the only pathway that could produce acetate would be via EET directly or

387

indirectly to biochar (Figure 5, dotted box), and an intermediate based electron transfer

388

mechanism could be applicable. Therefore, we propose that ANME-2d achieves AOM via an

389

EET dependent pathway, potentially through three mechanisms (Figure 5).

390 391

Figure 5. Proposed mechanisms for biochar-mediated AOM 1) Proposed direct electron

392

transfer from ANME-2d to biochar; 2) Indirect extracellular electron transfer from ANME-2d

393

to biochar based on direct interspecies electron transfer between ANME-2d and

394

Geobacteraceae; 3) indirect extracellular electron transfer from ANME-2d to biochar based on

395

interspecies electron transfer between Geobacteraceae and ANME-2d via metabolism of

396

intermediate, acetate (direct electron transfer from ANME-2d to biochar is the

397

thermodynamical premise for biosynthesis of acetate from CH4 oxidation in this case) 19



398

Practical Implications. Biochar amendment has been reported to be an effective approach to

399

capturing CH4 emissions by stimulating aerobic methanotrophic activity.9, 10 Our study shows

400

that biochar amendment can mediate CH4 oxidation in anoxic environments, and offers

401

plausible mechanisms for stimulation of microbial CH4 oxidation by biochar based on

402

interaction with ANME. In our study, biochar acted as an electron sink for CH4 oxidation

403

providing a reservoir for anaerobic CH4 fixation with potential applications for global CH4

404

emission mitigation.

405

Large-scale applications of biochar are not prevalent, and are limited by the challenges of

406

commercial production necessary to achieve high biochar yield through biomass pyrolysis.36

407

The majority of biochar is currently sourced from non-commercial, non-engineered production,

408

that is known as ‘black carbon’, and produced from open biomass burning.37 Electron

409

equivalent estimations (see Supporting Information for details) indicate that combined, all

410

current biochar production provides a negligible electron sink for CH4 oxidation as compared

411

with global CH4 emission (Table 1), even assuming all black carbon is coupled to AOM in

412

anoxic sites of natural environments. So it is difficult to say biochar nowadays is contributing

413

a lot to decrease CH4 emission by mediating AOM via direct supply of electron sink.

414

However, our report may provide scientific support for the promotion of large-scale biochar

415

application and production.36 Biochar provides a sustainable strategy to decrease CO2, CH4 and

416

N2O emissions, sequestering organics through biomass pyrolysis38. Widespread application of

417

biochar to agricultural soils could further decrease greenhouse gas emissions by providing a

418

substantial electron sink for AOM. On a global scale, if the maximum sustainable amount of

419

available biomass feedstock is 2.27 Pg carbon per year, and approximately 49% C yield is

420

achievable for biochar production from biomass pyrolysis,38 we can expect that a year of

421

biochar production provides a potential electron reservoir of at least 1.11  1012 mol e-

422

(electron-accepting capacity of 0.85 mmol e- g-1 biochar) and up to 9.10 1012 mol e- based on 20


Page 20 of 28

Page 21 of 28


423

the maximum biochar electron-accepting capacity of 7 mmol e- g-1 (Table 1; calculation details

424

in Supporting Information). In coupling to CH4 emission, an electron reservoir of this capacity

425

would effectively mitigate global CH4 emissions assuming that biochar is broadly applied to

426

CH4 sources such as landfill and rice paddies (Table S2).39 For example, based on electron-

427

accepting capacity of biochar in this study, we estimate that 341.8 t biochar amendment per ha

428

per year would be able to effectively control CH4 emission from rice paddy (calculation details

429

in Supporting Information). However, it is noteworthy that these are theoretical estimations,

430

with several premises: 1) maximum biochar yield from sustainable biomass resources,38 2) all

431

of the biochar produced can be used at landfill sites and paddy soils to reduce methane emission,

432

3) proper design and installation can ensure biochar-mediated AOM process in subsurface

433

CH4-producing environments. The effectiveness of biochar amendments on anaerobic CH4

434

mitigation at landfill sites should be further evaluated and relevant technical design during

435

practical applications warrant following investigations.

436

Through its chemical reactivity, biochar can shuttle electron transfer from microbes to

437

extracellular electron acceptors.40 The evidence here of extracellular electron transfer between

438

ANME-2d and biochar would suggest that AOM driven by other electron acceptors could be

439

promoted by biochar. For instance, Fe(III) minerals have been reported to be able to be directly

440

coupled to AOM.41 The redox potentials of iron minerals are strongly influenced by

441

environmental conditions, and the wide redox potential range over which different forms of

442

Fe(III) exist (Eo -314 mV to 770 mV vs. SHE) implies that biochar (Eo = 152 mV vs. SHE)

443

could act as an intermediary during AOM.42 A similar shuttling process may also be applicable

444

to manganese-driven AOM due to the highly positive standard reduction potential of MnO2 (Eo

445

= 1.23 mV vs SHE), although direct or indirect electron transfer from methanotrophs to MnO2

446

has not been confirmed.41

21



Page 22 of 28

447

Table 1. Evaluation of electron-donating capacity of global CH4 production and electron-

448

accepting capacity of biochar Annual biochar production ( g yr-1) current black carbon production (Mt)

large-scale commercial biochar production (Pg)

8.4012

1.31a Electron-accepting capacity of biochar (mol e- yr-1)

based on electron storage capacity in this study

based on biochar’s maximum electron storage capacity12

based on electron storage capacity in this study

based on biochar’s maximum electron storage capacity

6.46  1010

5.32  1011

1.11  1012

9.10  1012

Annual global CH4 emission (g yr-1) b emission from rice fields

emission from landfills

total global emission

9.50  1013

5.00  1013

5.20  1014

Electron-donating capacity (mol e- yr-1) 4.75  1013

2.50  1013

Calculation based on annual biomass-feedstock availability; available sites for large-scale biochar amendment

a

2.60  1014 b

assume rice paddies and landfills would be

449 450

Recent evidence indicates that sulfate-dependent AOM occurs through direct interspecies

451

electron transfer between ANME and sulfate-reducing bacteria (SRB).30, 31 Hence, the capacity

452

of biochar to promote interspecies electron transfer38, 43 suggests it could also promote sulfate-

453

driven AOM in sediment. Given the ubiquity of iron/manganese minerals and sulfate in anoxic

454

environments, an abundant electron reservoir exists for AOM. In this scenario, biochar is not

455

limited by its inherent electron-accepting capacity for direct mediation of CH4 oxidation; rather,

456

biochar is more likely to contribute to global CH4 emissions reduction as an electron shuttle to

457

promote AOM coupled to other electron acceptors. Logical next experiments are herein

458

desirable to affirm this propose.

459 460

SUPPORTING INFORMATION

461

Calculation details and additional tables and figures, as mentioned in the text, are available in 22


Page 23 of 28


462

the supporting information section available free of charge via the Internet at

463

http://pubs.acs.org.

464 465

AUTHOR INFORMATION

466

Corresponding Author

467

Tel: +61 7 3346 3230. Fax: +61 7 336 54726. Email: [email protected]

468

Notes

469

The authors declare no competing financial interest.

470 471

ACKNOWLEDGMENTS

472

The Authors thank Dr Eloise Larsen for careful editing of the manuscript. We are grateful to

473

The AWMC Analytical Services Laboratory (ASL) for all chemical analysis. This work is

474

supported by the Australian Research Council (ARC) through the projects of Australian

475

Laureate Fellowship (FL170100086) and Discovery Project (DP170104038), and the U.S.

476

Department of Energy’s Office of Biological Environmental Research (DE-SC0010574). X.Z.

477

is supported by The University of Queensland International Scholarship (UQI); J.X. is

478

supported by the UQ Research Training Scholarship J.P. is supported by The University of

479

Queensland International Scholarship and China Scholarship Council Scholarship; S.H. is

480

supported by the Advanced Queensland Research Fellowship.

23



481

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Biochar-Mediated Anaerobic Oxidation of Methane | Environmental

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