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Energy and the Environment

High-rate Production of Short-chain Fatty Acids from Methane in a Mixed-culture Membrane Biofilm Reactor Hui Chen, Lei Zhao, Shihu Hu, Zhiguo Yuan, and Jianhua Guo Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.8b00460 • Publication Date (Web): 19 Oct 2018 Downloaded from http://pubs.acs.org on October 21, 2018

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Environmental Science & Technology Letters

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High-rate Production of Short-chain Fatty Acids from Methane in a Mixed-culture

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Membrane Biofilm Reactor

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Hui Chen, Lei Zhao, Shihu Hu, Zhiguo Yuan*, Jianhua Guo*

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Advanced Water Management Centre, The University of Queensland, St Lucia, Queensland 4072,

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Australia.  

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*Corresponding author: 1. Jianhua Guo, Phone: + 61 7 3346 3222; FAX: + 61 7 3365 4726; E-

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mail: [email protected]. 2. Zhiguo Yuan, Phone: +61 7 3365 4374; FAX: +61 7 3365 4726;

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E-mail: [email protected].

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Keywords: methane bioconversion; short-chain fatty acid; membrane biofilm reactor.

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ABSTRACT

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Methane bioconversion to liquid chemicals has attracted much attention. However, the production

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rate reported to date has been far lower than what required for economical viability. This is partly

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due to the low solubility of methane, low mass transfer rate and low microbial activities. This study

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demonstrates a production rate of close to 10 g/L.d of short-chain fatty acids (SCFAs) by mixed-

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culture biofilm growing in a membrane biofilm reactor (MBfR). Hollow fiber membranes were

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used both to deliver a high flux of methane and to provide a surface to slow-growing

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microorganisms to form biofilms with intensified activities. The rate achieved is nearly two-orders

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of magnitude higher than the SCFAs production rate reported to date, and is close to the rates

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required for practical applications (~12-120 g/L.d). The consortium in biofilm was dominated by

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methanogens

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Sporolactobacillus and Propionispora, suggesting likely roles of these organisms in methane

Methanosarcina

and

Methanobacterium,

1 ACS Paragon Plus Environment

and

acid-producing

bacteria


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bioconversion into SCFAs. This work shows methane-based MBfR represents a promising

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technology for achieving high-rate chemical production from methane.

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INTRODUCTION

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The chemical industry is a key sector in the global economy, responsible for the conversion of raw

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materials into tens of thousands of products critical to modern society. The world’s chemical

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market reached $3 trillion in 2013 and is growing at a rate of 8% per annum,1 with crude oil being

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by far the dominant feedstock for the production of chemicals. However, the remaining reserves

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of non-renewable crude oil resources are diminishing and these will become increasingly difficult

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and costly to extract over the coming decades.2 In contrast, huge natural gas reserves have become

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economically recoverable over the last decade (globally estimated at 7.2 × 103 trillion ft3) due to

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new horizontal drilling and efficient extraction technologies.3 In light of this, there is an emerging

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trend in using methane as the source of hydrocarbon for chemical production to take advantage of

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the huge deposits of methane.4

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Although methane can be converted to the market-required chemicals or biofuels through chemical

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processes, the existing technologies (e.g. the Fischer-Tropsch gas-to-liquid process) are capital-

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and energy-intensive, requiring high temperature, high pressure and expensive catalyst.5 These

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technologies are not economically viable for small-scale methane sources, including biogas, which

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is a distributed source of methane with a relatively low supply. Compared with chemical processes,

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microbial conversion processes are more attractive due to its high product specificities, mild

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operational conditions and low energy consumption.6 The biocatalysts are microorganisms able to

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oxidize methane, including both aerobic and anaerobic methanotrophs, which carry natural

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enzymes that can efficiently activate the strong C-H bond in methane.7 Aerobic methanotrophs


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activate the strong C-H bond with enzyme pMMO/sMMO (soluble/particulate methane

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monooxygenases). These enzymes require electrons to activate the C-H bond, which are supplied

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through the oxidation of part of the downstream products to CO2. Therefore, aerobic processes

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have relatively low energy and carbon efficiencies due to the loss of electrons to O2 and loss of

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carbon to CO2. For example, the conversion of methane to n-butanol has a theoretical energy and

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carbon efficiency of 51% and 67%, respectively.5 In contrast, anaerobic methanotrophs oxidize

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methane through the reverse methanogenesis pathway, where methane is activated by MCR

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(methyl-coenzyme M reductase) without the need for electrons or oxygen, thus overcoming the

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carbon and energy efficiency barriers associated with oxygen-dependent processes.

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Recently, there have been a number of attempts directed towards microbially converting methane

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to chemicals, including methanol8, 9 and organic acids10-12. For example, organic acids (e.g., acetate

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and propionate) are important chemical building blocks that can be used as precursors for biofuel

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production.13-15 Additionally, these organic acids, which are readily usable carbon source, can be

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used as electron donors to remove anion-contaminates (e.g., nitrate, nitrite, and sulfate) from

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wastewater or groundwater.16, 17 The previous studies mainly employed pure cultures to achieve

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methane bioconversion. However, pure culture gas fermentation suffers from problems such as

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high operational cost, strain degeneration and contamination.18 In this work, we explore the use of

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mixed-culture for the conversion of methane to short-chain fatty acids (SCFAs).

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MATERIALS AND METHODS

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MBfR Reactor Design

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A lab-scale MBfR with 50 mL working volume was set up to achieve methane bioconversion

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(Figure S1). The MBfR had gas-permeable hollow-fibre membranes (300 μm o.d. and 200 μm i.d.,

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STERAPORE™ 2000 series, composite polymer materials, manufactured by Mitsubishi). One 3 ACS Paragon Plus Environment


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end of the fibers is open, but glued and packed into a polyester tube, while another end is sealed

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with epoxy resin and thus becomes a dead end. The total membrane surface area was 2.41×10-3

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m2, giving an A/V ratio of 48 m2/m3 in the MBfR. Mixed gas (95% CH4 + 5% CO2, Coregas,

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Australia) was delivered to the lumen of the fibers from the open end at 1.25 atm controlled by the

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regulator connected to the gas cylinder. Influent, with composition to be further described, was fed

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to the MBfR at a daily flow rate of 50 mL/d using a peristaltic pump. The reactor was mixed

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continuously by recirculating liquid with a peristaltic pump (Masterflux, USA) at 12.5 mL/min. A

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100 mL overflow bottle with 75 mL headspace was setup for liquid sampling, online pH

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monitoring and final effluent discharge. A water seal bottle was connected to the overflow port of

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the overflow bottle to release effluent and gases (nitrogen, CO2, and residual methane), and also

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to prevent air from getting into the system.

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Operational Conditions and Medium

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The MBfR was operated for about 420 days at room temperature (22 ± 2 ℃). The reactor was

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operated as a sequencing batch reactor (SBR) with a cycle time of 12 h including a 3 min feeding,

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during which 25 mL growth medium was supplied, and a 717 min of biological reaction phase.

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The recirculation pump was stopped during the feeding period, and 25 mL effluent was discharged

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from the overflow bottle in the feeding period. Such an operation was chosen to provide the growth

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medium at a rate of 50 mL/d (0.035 mL/min), which as it is practically difficult to deliver such a

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low flow rate accurately for through continuous operation by of a peristaltic pump. The hydraulic

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retention time (HRT) is calculated to be 1 day, without considering the liquid volume in the

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overflow bottle due to negligible presence of biomass and hence negligible biological activity in

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the bottle. pH of the reactor was monitored by a pH meter (Oakton, Australia) placed in the

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overflow bottle, and was maintained at 7-7.5 by manual dosing of 1 M HCl or 1 M NaOH solution. 4 ACS Paragon Plus Environment

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The medium fed to the MBfR contained (per liter): KH2PO4, 0.075 g; CaCl2·2H2O, 0.3 g;

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MgCl2·7H2O, 0.23 g; NaNO3, 0.4 g; NH4Cl, 0.09 g; acidic trace element solution, 0.5 mL; alkaline

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trace element solution, 0.2 mL.19 Nitrate and ammonium were added to the medium to obtain

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concentration of 300 mg N/L and 90 mg N/L, respectively. The influent was degassed with N2 for

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30 min prior to use.

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Analytical Methods

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Liquid samples of 2 mL was taken from the MBfR twice per week via the overflow bottle with a

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syringe, and were filtered immediately through a 0.22 μm disposable sterile millipore filter (Merck)

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for chemical analysis. The concentrations of NO3--N, NO2—N, and NH4+-N were assayed with a

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Lachat QuickChem8000 Flow Injection Analyser (Lachat Instrument, Milwaukee, WI). The short-

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chain fatty acids (SCFAs) (acetate, propionate, iso-butyrate, butyrate, valerate, iso-valerate, and

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caproate) concentrations were measured with a Shimadzu HPLC system with a Bio-Rad HPLC

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Column (300×7.8mm) and a RID detector. Dissolved oxygen (DO) concentration in the MBfR

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was measured periodically with a DO probe (HACH HQ40d) in the overflow bottle (Figure S1).

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Dissolved oxygen was always below the detection limit. The methane concentration in the liquid

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phase was measured with the method described in Shi et al. (2013)20.

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For microbial community analysis, biofilm samples were collected for DNA extraction on Day

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129 and Day 345 to identify biofilm communities. DNA was extracted using the FastDNA SPIN

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Kit for Soil (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer’s instructions.

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The extracted DNA was amplified, sequenced and analyzed based on the procedures described in

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the Supporting Information.

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RESULTS AND DISCUSSION

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Short-chain Fatty Acids Production in Methane-based MBfR Reactor 5 ACS Paragon Plus Environment


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A membrane biofilm reactor (MBfR) (Figure 1, Figure S1) was used in this study. A bundle of

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hollow fibre membranes was packed in a sealed bioreactor. Membranes were used to deliver

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methane to the reactor, and also to provide support for the methane utilising organisms, which

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typically grow slowly,21 to grow in biofilms. We inoculated the reactor with an enriched culture20

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performing anaerobic methane oxidation coupled to nitrate reduction, which was dominated by

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two known anaerobic methane oxidation organisms namely Candidatus ‘Methanoperedens

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nitroreducens’ and Candidatus ‘Methylomirabilis’. A layer of biofilm gradually grew on, and

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covered the surface of the hollow fiber membranes after 124-day operation. Liquid samples were

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since collected regularly (2-3 times per week) to monitor the SCFAs concentration in the effluent.

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Acetate, propionate, iso-butyrate, butyrate, valerate, iso-valerate, and caproate were detected, with

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a total SCFAs production rate of 1.72 g/L.d on Day 138 (Figure 1B). Acetate and propionate,

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produced at a rate of 1.27 g/L.d and 0.42 g/L.d, respectively, were the major products, accounting

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for 97.7% of the total SCFAs. The acetate and propionate production rates increased to 4.80 g/L.d

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and 1.84 g/L.d, respectively, on Day 348, raising the total SCFAs production rate to 6.88 g/L.d.

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After Day 348, the acetate production rate remained stable at 4.99 ±0.19 g/L.d, while the

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propionate production rate continued to increase, reaching 3.61 g/L.d on Day 418. The total SCFAs

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production rate steadily increased to 9.63 g/L.d on Day 404. C4, C5, and C6 acids were also

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produced, with the peak production rate for butyrate, iso-butyrate, valerate, iso-valerate, and

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caproate being 0.16 g/L.d (Day 348), 0.05 g/L.d (Day 418), 0.07 g/L.d (Day 418), 0.05 g/L.d (Day

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418), and 0.02 g/L.d (Day 376), respectively. Similar SCFAs production was also observed in a

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parallel reactor operated similarly (Figure S3), confirming the reproducibility of the results.


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Figure 1. The conceptual MBfR system (A) (see Figure S1 for the detailed experimental setup);

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Production rates of total SCFAs, acetate, and propionate (B) and Production rates of iso-butyrate,

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butyrate, valerate, iso-valerate, and caproate (C).

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Replacing the widely used bubbling columns, we employed an MBfR, where gas-permeable

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membranes were used to achieve efficient methane supply and retention/accumulation of methane-

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oxidising organisms. Using this innovative approach, we achieved an SCFAs production rate of

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9.63 g/L.d with methane as the sole organic carbon supplied. This rate is far higher than all

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methane-supported SCFAs production rates reported to date, which ranged from 0.02 to 0.19g/L.d

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(Table S1). In addition, formation of butyrate, iso-butyrate, valerate, iso-valerate, and caproate has

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not been observed in previous studies (Table S1).10,

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various processes for liquid chemicals production from methane (e.g. methanol, acetate, and

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formate), along with the operational conditions applied. To our knowledge, the production rate

22

Table S1 summarizes performance of



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achieved in this work is the highest rate reported to date. It should be noted that this is our first

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attempt to produce liquid chemicals from methane using an MBfR. While our rate is still out of

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the economically viable range of 12 – 120 g-products/L.d,5 we expect that the rate could be

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substantially enhanced through process optimisation. For example, the methane transfer

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coefficient can be substantially enhanced by increasing the density of membranes in the reactor,

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increasing the methane pressure in the lumen of the membrane, or using more efficient membranes.

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Microbial Community Structure and Potential Players in SCFAs production

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We characterized the microbial community of biofilm samples collected on Day 0 (i.e., the

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inoculum), Day 129, and Day 345, using high throughput 16S rRNA gene sequencing. We

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generated an average of ~16600 high-quality 16S rRNA gene sequences per sample (n=3) from

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which we picked 581 operational taxonomic units (OTUs, 97% similar identity) and assigned

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taxonomy. Microbial community composition and their relative abundances in the phylum level

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and genus level are shown in Figure 2A and B, respectively. Phylotypes related to Planctomycetes,

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the NC10 phylum, Euryarchaeota, Chloroflexi, Chlorobi, and Proteobacteria dominated the

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inoculum, accounting for 37.8%, 19.5%, 19.2%, 5.8%, 5.6%, and 4.2%, respectively, of the total

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community (Figure 2A). However, on Day 129, microbial communities shifted and were

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dominated by Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. These four phyla also

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dominated on Day 345. Euryarchaeota also reoccurred in the Day 345 sample, at an abundance of

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14.6%.


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Figure 2. Taxonomic profiles of the microbial communities according to dominant microorganism

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in the inoculum and the MBfR biofilm. A) phyla-level abundance; B) genera-level abundance.

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Minor phyla groups accounting for less than 1% of the total sequence are presented as “others”.

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In the inoculum, both Candidatus ‘Methanoperedens nitroreducens’ and Candidatus

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‘Methylomirabilis’ were detected, with an abundance of 21.0% and 11.6%, respectively (Figure

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2B). Candidatus ‘M. nitroreducens’ and Candidatus ‘Methylomirabilis’ are known methane-

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utilizing organisms using nitrate and nitrite as electron acceptor, respectively.23, 24 However, these

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anaerobic methanotrophs gradually disappeared (Figure 2B). An aerobic methanotroph

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Methylomonas was detected in the Day 129 sample, with an abundance of 1.9%, but disappeared

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in the Day 345 sample. Intriguingly, the phylum Euryarchaeota, which was not detected in the Day

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129 sample but reappeared on Day 345 with a high percentage of 14.6%, comprised

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Methanosarcina at 13.5% and Methanobacterium at 1.1% (Figure 2B). These two are the only

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known methane-relevant microorganisms in the community on Day 345. However, both are known

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as methanogens instead of methanotrophs.25, 26 9 ACS Paragon Plus Environment


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The community data show that neither known anaerobic methanotrophs nor known aerobic

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methanotrophs were present in the bioreactor in periods when high SCFAs production rates were

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observed, eliminating the possibility that these populations contributed to methane activation. In

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contrast, a relatively high abundance of known methanogens was detected, indicating methanogens

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may have been involved in methane activation. The ability of methanogens, e.g. Methanosarcina

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acetivorans, to anaerobically oxidise methane has been observed in some previous studies.27, 28

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Additionally, acetate was found to occur as oxidation products in methanogenic cultures.29-31

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Schilov et al. (1999)29 reported the production of acetate by mixed cultures dominated by the

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methanogens Methanosarcina and Methanosaeta cultivated on methane and bicarbonate. Luo et

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al. (2018)32 implied a possible role of Methanosarcina in the production of acetate. In addition,

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methanogens have also been reported to be able to both produce and consume hydrogen.33 These

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observations suggest that methanogenic microorganisms might be versatile and flexible in their

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metabolic capacity and have the potential to activate methane, which warrants further studies.

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Although acid-producing bacteria were at a negligible level in the inoculum, several typical acid-

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producing microorganisms were found on Day 129, and even higher abundance was observed on

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Day 345. At phylum level, microorganisms affiliated to Bacteroidetes, Firmicutes, and

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Actinobacteria accounted for 14.0%, 3.5%, and 0.9%, respectively, of the community in the Day

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129 sample (Figure 2A). The abundance of Firmicutes and Actinobacteria increased to 20.4% and

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1.6%, respectively, while the abundance of Bacteroidetes slightly dropped to 6.9% on Day 345. A

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few members within Firmicutes, Actinobacteria, and Bacteroidetes are known as anaerobic

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fermenters, which have been reported to be capable of fermenting organic carbons (e.g.

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monosaccharide) to produce SCFAs.34-36 Our analysis showed the enrichment of Propionicimonas,

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Proteiniphilum, and Dysgonomonas over time, and these microorganisms are known for their 10 ACS Paragon Plus Environment

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putative capability of producing acids, e.g., acetic and propionic acids, as fermentation products.37-

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39

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abundance of 12.1% and 6.1%, respectively, on Day 345. Sporolactobacillus and Propionispora

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has been reported to be capable of fermenting organics (e.g. fructose and glucose) to produce

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acids.40, 41 Similar to Sporolactobacillus and Propionispora, Pelosinus that was enriched with an

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abundance of 1.8% on Day 129, can produce acids from glucose or other sugars.42

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The relatively high abundance of methanogens (14.7%) and acid-producing bacteria (25.1%) in

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the community suggests a possibility that anaerobic methanogens and acid-producing bacteria

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jointly converted methane into SCFAs. These two kinds of microorganisms could have formed a

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carbon sharing partnership, where methane was converted by the methanogens into some organic

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intermediates, which were further degraded into SCFAs by the acid-producing bacteria. Similar

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microbial communities including both methanogens and acid-producing bacteria were also

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observed in a parallel reactor operated (Figure S5), indicating the reproducibility of the 16S rRNA

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gene sequencing results.

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Possible Reactions Involved in Methane Conversion to SCFAs

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In the liquid feed to the reactor, we provided nitrate at a concentration of 300 mg N/L. This

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provision was based on our previous observation that Candidatus ‘M. nitroreducens’ can oxidise

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methane to acetate as well, using nitrate as an electron acceptor.

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10CH4 + 8NO3- + 8H+ → 5CH3COOH + 4N2 + 14H2O; △G0' = -393 kJ mol-1 CH4

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The MBfR was operated with a hydraulic retention time (HRT) of 1 day, giving a nitrate loading

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rate of 300 mg N/L.d. Nitrate in the effluent was negligible (Figure S4), which means nitrate was

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consumed at a rate of approximately 300 mg N/L.d. Even assuming that all nitrate was used in

Sporolactobacillus and Propionispora, affiliated to Firmicutes, were enriched with a high


(1)


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Reaction (1), i.e., nitrate was not used to oxidise methane or SCFAs to CO2,24 acetate would be

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produced at a rate 804 mg/L.d, according to the reaction stoichiometry, which is far lower than

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what measured (Figure 1).

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To provide good mixing in the MBfR, we included external recirculation (Supporting Information,

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Figure S1). We later identified that the tubing (Tygon E-Lab tubing, internal diameter 3.1 mm,

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Masterflex, Cole-Parmer) used for the medium recirculation was permeable to air. We

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experimentally determined an oxygen transfer rate of 10.0 mg/L.d (Supporting Information). The

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provision of oxygen could have triggered aerobic methane oxidation to organic acids, although

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dissolved oxygen was not detected in the MBfR:

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2CH4 + 2O2 → CH3COOH + 2H2O; △G0' = -387 kJ mol-1 CH4

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However, even assuming that oxygen thus provided was not used for methane or SCFAs oxidation

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to CO2, the acetate production rate is estimated to be 9.4 mg/L.d, which is negligible compared to

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the measured SCFAs production rate. The above mass balance analysis suggests that CO2, the only

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other potential electron acceptor in the MBfR, could have been involved in the reaction:

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CH4 + CO2 → CH3COOH; △G0' = 36 kJ mol-1 CH4

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CO2 was available in the reactor because the gas feed contained CO2 at 5%. Although this reaction

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is thermodynamically unfavourable, the limited amount of energy required for this process could

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have been made available by coupling with other electron accepting pathways (e.g. nitrate and

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oxygen reduction) (Reaction 1 and Reaction 2). A thermodynamically feasible pathway has been

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proposed in a genome-scale metabolic model of M. acetivorans for the co-utilization of methane

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and bicarbonate in the presence of Fe3+, NO3-, SO42-, and MnO2 as external electron acceptors,

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which makes an overall negative △G to ensure the thermodynamic feasibility of the 12 ACS Paragon Plus Environment

(2)

(3)

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methanogenesis reversal.43 Furthermore, Soo et al. (2016)44 have confirmed the co-metabolism of

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methane and bicarbonate into acetate using Fe3+ as the electron acceptor through

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labelling of bicarbonate and methane, catalysed by an engineered methanogen Methanosarcina

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acetivorans. It is worth noting that the use of CO2 as the primary electron sink, if proven, would

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dramatically enhance both the energy and carbon efficiencies in methane bioconversion to SCFAs.

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Practical Implications and Future Investigations

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Natural gas, consisting primarily of methane, is being increasing used as a feedstock for the

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production of liquid chemicals to augment the petroleum-dominated chemical market. However,

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the chemical-engineering-based technologies (e.g. Fischer-Tropsch process) used currently to

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achieve this conversion are capital- and energy-intensive, requiring high temperature, high

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pressure and expensive catalysts. These technologies are not suitable for the small and distributed

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methane sources that are being increasingly discovered. Consequently, small gas sources are

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currently not utilized (vented or flared), causing not only significant resource wastage but also

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greenhouse gas emissions. Alternatively, microbial production of liquid chemicals using methane

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as feedstock incurs much lower capital and operational costs. For the first time, a methane-based

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MBfR was successfully applied to achieve methane bioconversion to SCFAs by mixed cultures.

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We obtained an SCFAs production rate of 9.63 g/L.d, which is two orders of magnitude higher

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than the SCFAs production rate reported in previous studies and is close to the rates required for

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industrial applications. The methane-based MBfR represents a highly promising technology for

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both efficient methane delivery and effective retention of slow-growing microorganisms,

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potentially overcoming the kinetic limitation for methane bioconversion to liquid chemicals.

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However, it should be noted that more studies are needed to elucidate the detailed reactions

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involved and reveal the functions of the key members of a surprising microbial community in our 13 ACS Paragon Plus Environment

13C

isotopic


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reactor. Firstly, further experiments (e.g. isotopic labelling test, and metagenomic and

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metatranscriptomic analyses) are needed to confirm the co-metabolism of methane and CO2 in the

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reactor. In addition, it is still unclear whether bioconversion of CH4 to SCFAs is a direct process,

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or an indirect process where CH4 was firstly converted into intracellular (e.g. glycogen and

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polyhydroxyalkanoates) or extracellular compounds (e.g. extracellular polymeric substances),

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then these intermediate compounds were further converted to SCFAs. Furthermore, the enigmatic

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role of key members of the microbial community (e.g. methanogens) should be identified by

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multiple molecular methods. For example, it is worthwhile to further verify the quantified results

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obtained from 16S rRNA sequencing by using quantitative polymerase chain reaction (qPCR). In-

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depth metagenomic and metatranscriptomic studies are required to verify this hypothesis and to

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reveal the metabolic pathways involved in methane conversion to SCFAs. Answers to these

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research questions will provide insights to support further enhancement of methane bioconversion

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to liquid chemicals.

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CONFLICTS OF INTEREST

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There are no conflicts to declare.

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ASSOCIATED CONTENT

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Additional method details and supporting data in figures and tables.

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ACKNOWLEDGEMENTS

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

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project Australian Laureate Fellowship (FL170100086). Jianhua Guo would like to acknowledge


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the Australian Research Council Future Fellowship (FT170100196). Hui Chen would like to

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acknowledge The University of Queensland for her IPRS scholarship support.

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