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Environmental Processes
Acetate production from anaerobic oxidation of methane via intracellular storage compounds Chen Cai, Ying Shi, Jianhua Guo, Gene W. Tyson, Shihu Hu, and Zhiguo Yuan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b00077 • Publication Date (Web): 04 Jun 2019 Downloaded from http://pubs.acs.org on June 7, 2019
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Acetate production from anaerobic oxidation of methane via intracellular
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storage compounds
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Chen Cai,†,§ Ying Shi,†,‡,§ Jianhua Guo,† Gene W. Tyson,⊥ Shihu Hu,*,† and Zhiguo Yuan*,†
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†Advanced
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Queensland 4072, Australia
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‡School
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China
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⊥Australian
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University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia
Water Management Centre, The University of Queensland, St Lucia, Brisbane,
of Resource and Safety Engineering, Central South University, Changsha 410083,
Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, The
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Abstract
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There is great interest in microbial conversion of methane, an abundant resource, into valuable
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liquid chemicals. While aerobic bioconversion of methane to liquid chemicals has been
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reported, studies on anaerobic methane bioconversion to liquid chemicals are rare. Here we
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show that a microbial culture dominated by Candidatus ‘Methanoperedens nitroreducens’, an
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anaerobic methanotrophic (ANME) archaeon, anaerobically oxidizes methane to produce
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acetate, indirectly via reaction intermediate(s), when nitrate or nitrite was supplied as an
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electron acceptor under a rate-limiting condition. Isotopic labelling tests showed that acetate
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was produced from certain intracellular storage compounds which originated from methane.
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Fluorescence in-situ hybridization (FISH) and Nile red staining demonstrated that
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polyhydroxyalkanoate (PHA) in M. nitroreducens was likely one of the intracellular storage
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compounds for acetate production, along with glycogen. Acetate is a common substrate for the
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production of more valuable chemicals. The microbial conversion discovered in this study
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potentially enables a new approach to the use of methane as a feedstock for the chemical market.
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1 Introduction
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The global interest in the use of methane as a resource is rapidly growing. This is due to a
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number of factors including increasing environmental concerns regarding the extraction and
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utilization of conventional energy sources (e.g. oil and coal), the enormous global reserve of
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methane as natural gas, shale gas and gas hydrate, as well as the technological advances in
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exploiting these deposits1, 2. The application of methane in transportation is considered one
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viable option for methane utilization3. However, the gaseous form of methane at ambient
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temperature hinders its adoption in transportation sector due to its low volumetric energy
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density and the lack of compatible infrastructure for fueling and end-use4. Conversion of
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methane to fuel in liquid form could potentially overcome the aforementioned disadvantages5.
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Further, there is also a potential for conversion of methane to a wide range of compounds for
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broad-spectrum applications, which would substantially add value to methane as an energy
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source6, 7. These applications, nevertheless, could only be achieved through the development
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of applicable methane conversion technologies.
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The Fischer-Tropsch process has been employed for methane conversion to liquid chemicals
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at an industrial scale. This state-of-the-art technology involves a complex, multistep process
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consisting of methane conversion to syngas (consisting primarily of carbon monoxide and
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hydrogen), catalytic formation and subsequent cracking of long-chain hydrocarbons, and
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finally, separation of end-products3. Chemical plants require large-scale facilities to manage
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the process due to technological complexities such as numerous changes of temperature and
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pressure necessary for the catalytic reactions to proceed, thus requiring a large capital
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investment3. This complex, chemical technology is also disadvantaged by low energy- and
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carbon efficiencies8.
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Biological methane conversion has the potential to circumvent the disadvantages of chemical
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processing as bioconversion can proceed under mild operating conditions, and at small scale5.
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Bacterially mediated aerobic methanotrophy represents one of the routes for methane
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bioconversion3. Here, methane activation is catalyzed by methane monoxygenases (MMOs)9,
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resulting in the formation of methanol. Methanol is further oxidized to formaldehyde, which
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can be assimilated via the ribulose monophosphate (RuMP) pathway or the serine pathway for
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the synthesis of metabolic building blocks10, 11. However, like chemical conversion of methane,
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bioconversion of methane via native aerobic methanotrophic pathways displays low energy-
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and carbon efficiencies, attributed to inefficient methane activation and formaldehyde
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conversion3. To date, aerobic methanotrophic bacteria, either native or engineered, have been
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investigated for conversion of methane to a variety of products such as methanol12,
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biopolymers13, and lipids14.
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Compared with aerobic oxidation of methane, anaerobic oxidation of methane (AOM)
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represents another promising bioprocess for methane conversion15. Recently, a metabolically
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engineered methanogen ‘Methanosarcina acetivorans’ was shown to convert methane and CO2
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to acetate using Fe(III) as an electron acceptor16. Genes (mcrBGA) of a homolog of methyl-
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coenzyme M reductase (MCR) derived from anaerobic methanotrophic (ANME) archaeal
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population 1 were introduced into M. acetivorans, which enabled this anaerobic archaeon to
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initiate the reverse methanogenesis pathway16. Isotopic labelling with 13C revealed that acetate
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was produced from methane and CO2, possibly via reversal of the aceticlastic pathway16. M.
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acetivorans was further engineered through transforming a plasmid (containing mcrBGA and
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other genes for butanol formation) into the cells17. Unexpectedly, lactate, instead of butanol,
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was produced and secreted by M. acetivorans17. Meanwhile, methane consumption remained
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at a similar level while acetate production decreased dramatically in comparison to the M.
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acetivorans without genes for butanol formation17. It was proposed that methane was converted
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to lactate via acetate by this M. acetivorans17. In addition, 3-hydroxybutyryl-CoA
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dehydrogenase (Hbd) was determined to be responsible for lactate production17. Another work
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reported that a synthetic consortium consisting of M. acetivorans and ‘Geobacter
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sulfurreducens’ could produce electricity from methane in a microbial fuel cell18. It was
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proposed that the electrical current was produced through a synergic relationship, in which M.
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acetivorans converted methane to acetate and G. sulfurreducens captured electrons from
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acetate and delivered them to anode18.
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Native ANME archaea also have the potential to convert methane into liquid chemicals19-21.
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Metagenomic analyses showed that genes encoding all proteins required for carbon
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assimilation from methane and CO2 via the reductive acetyl-CoA pathway are present in
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ANME-120, 21. The presence of genes encoding a homolog of the acetyl-CoA synthetase (Acd)
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indicated that acetate production from methane oxidation is genetically possible20,
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Expression of mRNA of the α-subunit of Acd (AcdA) further indicated that the AcdA homolog
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might be functionally involved in acetate production20. Furthermore, the presence and activity
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of formate dehydrogenase implied that production of formate by ANME-1 is possible20. ANME
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archaea (ANME-1, ANME-2a/c, ANME-3) are commonly found to coexist with sulfate-
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reducing bacteria (SRB)22. Although physiological studies have not definitively shown that
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acetate and/or formate function as intermediates for electron transfer between ANME archaea
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and SRB23, 24, the possibility of ANME archaea to produce these liquid chemicals could not be
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completely excluded.
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It has been shown that Candidatus ‘Methanoperedens nitroreducens’, an archaeon affiliated
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with ANME-2d, can couple AOM to nitrate reduction19. M. nitroreducens oxidizes methane to
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CO2 via the reverse methanogenesis pathway, supplying electrons for nitrate reduction to
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nitrite19. More recently, M. nitroreducens-like archaeon has also been shown to catalyze
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dissimilatory nitrate reduction to ammonium (DNRA) via nitrite25. Intriguingly, in addition to
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harboring genes for complete reverse methanogenesis and DNRA, genes encoding the
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reductive acetyl-CoA pathway and Acd were also found in M. nitroreducens19. This
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observation suggested that M. nitroreducens has the potential to convert methane to acetate,
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consistent with the previous prediction for ANME archaea20, 21.
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In this study, we used a microbial culture dominated by M. nitroreducens to determine if acetate
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can be produced anaerobically from methane by this archaeon, and to determine the conditions
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that promote acetate production. Conversion of carbon compounds including methane, acetate
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and other volatile fatty acids (VFAs), and potential intracellular storage compounds, namely
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polyhydroxyalkanoate (PHA) and glycogen, were monitored.
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was used to trace carbon conversion.
13C-labelled
methane (13CH4)
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2 Materials and Methods
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2.1 Biomass source
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The biomass used in this study was taken from a 5.6 L bioreactor (working volume of 4.6 L),
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dubbed the ‘parent bioreactor’, which was previously reported to be dominated by M.
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nitroreducens with the anammox bacterium of Candidatus ‘Kunenenia stuttgartiensis’ and
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many other bacteria forming a flanking community19. The detailed operating conditions of the
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bioreactor were described previously19 and in the Supporting Information (SI). The
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performance of the parent bioreactor was stable during this study (Figure S1). Biomass samples
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were taken from the parent bioreactor at the time when the batch tests reported below were
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ended (Day 940, Figure S1) for microbial community analyses using 16S rRNA gene
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sequencing and fluorescence in-situ hybridization (FISH) (SI). These results were consistent
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with the previous study19, in which M. nitroreducens was identified as the only methane
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oxidizer in the culture (Figure S2, S3). The 16S rRNA gene sequences obtained in this study
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were deposited to SRA database in NCBI with accession number SRR8893850.
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2.2 Batch tests
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Experiment 1: Acetate production
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It was hypothesized in a previous study that M. nitroreducens could be able to produce acetate19.
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To stimulate acetate production from the culture, a substrate-limiting condition was created by
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supplying the electron acceptor (i.e. nitrate or nitrite) at a limited rate compared to the normal
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nitrate consumption rate in the parent bioreactor. Nitrate has been demonstrated to be the
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primary electron acceptor for M. nitroreducens19. More recently, M. nitroreducens has also
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been shown to catalyze dissimilatory nitrate reduction to ammonium (DNRA) via nitrite25,
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indicating that nitrite may be an alternative electron acceptor. Therefore, in this experiment
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both nitrate and nitrite were employed as potential electron acceptors to increase the likelihood
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of acetate production by M. nitroreducens, with the following hypothesized reactions under
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electron acceptor-limiting conditions:
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1/2 NO3- + CH4(aq) + H+ → 1/2 C2H4O2 + 1/2 NH4+ + 1/2 H2O (∆Go’ = -215 kJ mol-1 CH4) (1)
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2/3 NO2- + CH4(aq) + 4/3 H+ → 1/2 C2H4O2 + 2/3 NH4+ + 1/3 H2O (∆Go’ = -207 kJ mol-1 CH4)
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(2)
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As outlined in Figure 1, nitrate was continuously fed to the batch reactor in Test A at a rate of
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~36 µmol L-1 h-1, which was much lower than the nitrate consumption rate (56 µmol L-1 h-1) of
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the parent bioreactor. In comparison, methane was supplied in excess by keeping the methane
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partial pressure in the reactor headspace at > 90% atm. As will be shown in Results, ammonium
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accumulation was observed, indicating the triggering of DNRA under such conditions.
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In Tests B and C, nitrite rather than nitrate was supplied as the rate-limiting substrate (29-36
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µmol L-1 h-1) (Figure 1). To further clarify the role of methane in acetate production, Test D
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was conducted by supplying nitrite (~36 µmol L-1 h-1) without methane (Figure 1). The
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concentrations of both nitrate and nitrite were near zero during all the batch tests.
A B C D
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Incubation (80 h) CH4 + CO2; NO3-: 36 µmol L -1 h-1 CH4 + CO2; NO2-: 36 µmol L -1 h-1 CH4 + CO2; NO2-: 29 µmol L -1 h-1 CO2; NO2-: 36 µmol L -1 h-1
Incubation (80 h, NO 2-: 29 µmol L -1 h-1)
Pre-incubation (240 h)
E
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F
12
G H
CH4 (100%) + 12CO2 (100%)
CH4 (10%) + 12CH4 (90%) + 12CO2 (100%)
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CH4 (100%) + 12CO2 (100%)
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CH4 (10%) + 12CH4 (90%) + 12CO2 (100%)
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CH4 (10%) + 12CH4 (90%) + 12CO2 (100%)
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CO2 (100%)
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CH4 (100%) + 13CO2 (10%) + 12CO2 (90%)
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CH4 (100%) + 12CO2 (100%)
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Figure 1. Experimental design for acetate production batch tests (Tests A-D) and isotopic
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labelling batch tests (Tests E-H). Potential carbon sources (CH4 and/or CO2) for acetate
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production provided were present in all tests. In Tests E-G, 13C-labelled CH4 was added, which
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represented 10% of the total CH4 added. In Test H,
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added, which represented 10% of the total CO2 present. Nitrate or nitrite was supplied
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continuously in each test. The nitrate or nitrite loading rate in Tests A, B and D was ~36 µmol
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L-1 h-1. The nitrite loading rate in Tests C and E-H was ~29 µmol L-1 h-1. In Tests E-H, the ‘Pre-
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incubation’ phase was for intracellular storage compound formation and the ‘Incubation’ phase
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was for intracellular storage compound conversion to acetate. Green dots in Tests E-H indicate
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injections of 13CH4 or 13CO2 (13C-labelled bicarbonate).
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In each batch test, 200 mL of biomass was drawn from the parent bioreactor and distributed
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evenly into 4 falcon tubes (50 mL biomass to each tube). The samples were centrifuged at
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3,267 ×g for 10 min. The supernatant was discarded, and the biomass pellets from the four 9
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tubes were collectively placed in one 330 mL batch reactor. The falcon tubes and the batch
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reactor were flushed with nitrogen gas before and during the biomass transfer to minimize
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oxygen contamination. Each batch reactor was filled to 200 mL using mineral medium with
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same composition for the parent bioreactor, leaving a headspace of 130 mL. Each batch reactor
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was placed on a magnetic stirrer (Labtek, Australia) and mixed at a speed of 200 rpm.
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For Tests A and B, the batch reactors were flushed with mixed gas (CH4:N2:CO2 = 90:5:5%)
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for 10 min before the tests. While for Test C, mixed gas (CH4:N2:CO2 = 90:5:5%) was
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continuously fed into the batch reactor via an air stone at a rate of 8 mL min-1 controlled by a
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gas flow meter (Bronkhorst, Netherlands). The gas outlet was connected to a water-sealed
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bottle to keep the batch reactor oxygen-free. Methane concentration in the headspace was
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maintained above 90% atm in all the tests (Tests A-C) using both methane feeding strategies
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(batch/continuous).
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Nitrate (Test A) or nitrite (Tests B-D) was loaded continuously to each batch reactor through a
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rubber stopper using a precise programmable syringe pump (New era, NE-1600, USA). The
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nitrate/nitrite loading rate for Tests A, B and D was ~36 µmol L-1 h-1 and the nitrite loading
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rate for Test C was slightly lower at ~29 µmol L-1 h-1. In all tests, the total liquid volume added
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was less than 3 mL (i.e.
