Article Cite This: Environ. Sci. Technol. 2019, 53, 7371−7379
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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*,† †
Advanced Water Management Centre, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia School of Resource and Safety Engineering, Central South University, Changsha 410083, China § Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia Downloaded via BUFFALO STATE on July 19, 2019 at 06:46:11 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
‡
S Supporting Information *
ABSTRACT: There is great interest in microbial conversion of methane, an abundant resource, into valuable liquid chemicals. While aerobic bioconversion of methane to liquid chemicals has been reported, studies of anaerobic methane bioconversion to liquid chemicals are rare. Here we show that a microbial culture dominated by Candidatus ‘Methanoperedens nitroreducens’, an anaerobic methanotrophic archaeon, anaerobically oxidizes methane to produce acetate, indirectly via reaction intermediate(s), when nitrate or nitrite is supplied as an electron acceptor under a rate-limiting condition. Isotopic labeling tests showed that acetate was produced from certain intracellular storage compounds that originated from methane. Fluorescence in situ hybridization and Nile red staining demonstrated that polyhydroxyalkanoate in M. nitroreducens was likely one of the intracellular storage compounds for acetate production, along with glycogen. Acetate is a common substrate for the production of more valuable chemicals. The microbial conversion discovered in this study potentially enables a new approach to the use of methane as a feedstock for the chemical market.
1. INTRODUCTION The global interest in the use of methane as a resource is rapidly growing. This is due to a number of factors, including increasing environmental concerns regarding the extraction and utilization of conventional energy sources (e.g., oil and coal), the enormous global reserve of methane as natural gas, shale gas, and gas hydrate, and the technological advances in exploiting these deposits.1,2 The application of methane in transportation is considered one viable option for methane utilization.3 However, the gaseous form of methane at ambient temperature hinders its adoption in the transportation sector due to its low volumetric energy density and the lack of compatible infrastructure for fueling and end use.4 Conversion of methane to fuel in liquid form could potentially overcome the aforementioned disadvantages.5 In addition, there is also potential for conversion of methane to a wide range of compounds for broad-spectrum applications, which would substantially add value to methane as an energy source.6,7 These applications, nevertheless, could be achieved only through the development of applicable methane conversion technologies. The Fischer−Tropsch process has been employed for the conversion of methane to liquid chemicals at an industrial scale. This state-of-the-art technology involves a complex, multistep process consisting of the conversion of methane to syngas (consisting primarily of carbon monoxide and hydro© 2019 American Chemical Society
gen), catalytic formation and subsequent cracking of longchain hydrocarbons, and, finally, separation of end products.3 Chemical plants require large-scale facilities to manage the process due to technological complexities such as the numerous changes in temperature and pressure necessary for the catalytic reactions to proceed, thus requiring a large capital investment.3 This complex, chemical technology is also disadvantaged by low energy and carbon efficiencies.8 Biological methane conversion has the potential to circumvent the disadvantages of chemical processing as bioconversion can proceed under mild operating conditions and at small scale.5 Bacterially mediated aerobic methanotrophy represents one of the routes for methane bioconversion.3 Here, methane activation is catalyzed by methane monooxygenases (MMOs), resulting in the formation of methanol.9 Methanol is further oxidized to formaldehyde, which can be assimilated via the ribulose monophosphate (RuMP) pathway or the serine pathway for the synthesis of metabolic building blocks.10,11 However, like chemical conversion of methane, bioconversion of methane via native aerobic methanotrophic pathways displays low energy and carbon efficiencies, attributed to Received: Revised: Accepted: Published: 7371
January 4, 2019 April 23, 2019 June 4, 2019 June 4, 2019 DOI: 10.1021/acs.est.9b00077 Environ. Sci. Technol. 2019, 53, 7371−7379
Article
Environmental Science & Technology inefficient methane activation and formaldehyde conversion.3 To date, aerobic methanotrophic bacteria, either native or engineered, have been investigated for the conversion of methane to a variety of products such as methanol,12 biopolymers,13 and lipids.14 Compared with aerobic oxidation of methane, anaerobic oxidation of methane (AOM) represents another promising bioprocess for methane conversion.15 Recently, a metabolically engineered methanogen “Methanosarcina acetivorans” was shown to convert methane and CO2 to acetate using Fe(III) as an electron acceptor.16 Genes (mcrBGA) of a homologue of methyl-coenzyme M reductase (MCR) derived from anaerobic methanotrophic (ANME) archaeal population 1 were introduced into M. acetivorans, which enabled this anaerobic archaeon to initiate the reverse methanogenesis pathway.16 Isotopic labeling with 13C revealed that acetate was produced from methane and CO2, possibly via reversal of the aceticlastic pathway.16 M. acetivorans was further engineered by transforming a plasmid (containing mcrBGA and other genes for butanol formation) into the cells.17 Unexpectedly, lactate, instead of butanol, was produced and secreted by M. acetivorans.17 Meanwhile, the level of methane consumption remained similar while the level of acetate production decreased dramatically in comparison to that of M. acetivorans without genes for butanol formation.17 It was proposed that methane was converted to lactate via acetate by this M. acetivorans.17 In addition, 3-hydroxybutyryl-CoA dehydrogenase (Hbd) was determined to be responsible for lactate production.17 Another work reported that a synthetic consortium consisting of M. acetivorans and “Geobacter sulfurreducens” could produce electricity from methane in a microbial fuel cell.18 It was proposed that the electrical current was produced through a synergic relationship, in which M. acetivorans converted methane to acetate and G. sulfurreducens captured electrons from acetate and delivered them to the anode.18 Native ANME archaea also have the potential to convert methane into liquid chemicals.19−21 Metagenomic analyses showed that genes encoding all proteins required for carbon assimilation from methane and CO2 via the reductive acetylCoA pathway are present in ANME-1.20,21 The presence of genes encoding a homologue of the acetyl-CoA synthetase (Acd) indicated that the production of acetate from methane oxidation is genetically possible.20,21 Expression of mRNA of the α-subunit of Acd (AcdA) further indicated that the AcdA homologue might be functionally involved in acetate production.20 Furthermore, the presence and activity of formate dehydrogenase implied that production of formate by ANME-1 is possible.20 ANME archaea (ANME-1, ANME2a/c, and ANME-3) are commonly found to coexist with sulfate-reducing bacteria (SRB).22 Although physiological studies have not definitively shown that acetate and/or formate functions as an intermediate for electron transfer between ANME archaea and SRB,23,24 the possibility of ANME archaea producing these liquid chemicals could not be completely excluded. It has been shown that Candidatus “Methanoperedens nitroreducens”, an archaeon affiliated with ANME-2d, can couple AOM to nitrate reduction.19 M. nitroreducens oxidizes methane to CO2 via the reverse methanogenesis pathway, supplying electrons for the reduction of nitrate to nitrite.19 More recently, a M. nitroreducens-like archaeon has also been shown to catalyze the dissimilatory nitrate reduction to
ammonium (DNRA) via nitrite.25 Intriguingly, in addition to harboring genes for complete reverse methanogenesis and DNRA, genes encoding the reductive acetyl-CoA pathway and Acd were also found in M. nitroreducens.19 This observation suggested that M. nitroreducens has the potential to convert methane to acetate, consistent with the previous prediction for ANME archaea.20,21 In this study, we used a microbial culture dominated by M. nitroreducens to determine if acetate can be produced anaerobically from methane by this archaeon and to determine the conditions that promote acetate production. Conversion of carbon compounds, including methane, acetate, and other volatile fatty acids (VFAs), and potential intracellular storage compounds, namely, polyhydroxyalkanoate (PHA) and glycogen, was monitored. 13C-labeled methane (13CH4) was used to trace carbon conversion.
2. MATERIALS AND METHODS 2.1. Biomass Source. The biomass used in this study was taken from a 5.6 L bioreactor (working volume of 4.6 L), dubbed the “parent bioreactor”, which was previously reported to be dominated by M. nitroreducens with the anammox bacterium of Candidatus “Kunenenia stuttgartiensis” and many other bacteria forming a flanking community.19 The detailed operating conditions of the bioreactor were described previously19 and are described in the Supporting Information. The performance of the parent bioreactor was stable during this study (Figure S1). Biomass samples were taken from the parent bioreactor at the time when the batch tests reported below were completed [day 940 (Figure S1)] for microbial community analyses using 16S rRNA gene sequencing and fluorescence in situ hybridization (FISH) (Supporting Information). These results were consistent with those of the previous study,19 in which M. nitroreducens was identified as the only methane oxidizer in the culture (Figures S2 and S3). The 16S rRNA gene sequences obtained in this study were deposited in the SRA database in NCBI with accession number SRR8893850. 2.2. Batch Tests. 2.2.1. Experiment 1: Acetate Production. It was hypothesized in a previous study that M. nitroreducens could produce acetate.19 To stimulate the production of acetate from the culture, a substrate-limiting condition was created by supplying the electron acceptor (i.e., nitrate or nitrite) at a limited rate compared to the normal rate of consumption of nitrate in the parent bioreactor. Nitrate has been demonstrated to be the primary electron acceptor for M. nitroreducens.19 More recently, M. nitroreducens has also been shown to catalyze DNRA via nitrite,25 indicating that nitrite may be an alternative electron acceptor. Therefore, in this experiment, both nitrate and nitrite were employed as potential electron acceptors to increase the likelihood of acetate production by M. nitroreducens, with the following hypothesized reactions under electron acceptor-limiting conditions: 1 1 1 1 NO3− + CH4(aq) + H+ → C2H4O2 + NH4 + + H 2O 2 2 2 2 (ΔG °′ = −223 kJ/mol of CH4) (1) 2 4 1 2 1 NO2− + CH4(aq) + H+ → C2H4O2 + NH4 + + H 2O 3 3 2 3 3 (ΔG°′ = −215 kJ/mol of CH4) 7372
(2) DOI: 10.1021/acs.est.9b00077 Environ. Sci. Technol. 2019, 53, 7371−7379
Article
Environmental Science & Technology
Figure 1. Experimental design for acetate production batch tests (tests A−D) and isotopic labeling batch tests (tests E−H). Potential carbon sources (CH4 and/or CO2) for acetate production were present in all tests. In tests E−G, 13C-labeled CH4 was added, which represented 10% of the total CH4 added. In test H, 13C-labeled CO2 (as bicarbonate) was added, which represented 10% of the total CO2 present. Nitrate or nitrite was supplied continuously in each test. The loading rate of nitrate or nitrite in tests A, B, and D was ∼36 μmol L−1 h−1. The loading rate of nitrite in tests C and E−H was ∼29 μmol L−1 h−1. In tests E−H, the “preincubation” phase was for the formation of the intracellular storage compound and the “incubation” phase was for the conversion of the intracellular storage compound to acetate. Green dots in tests E−H indicate injections of 13 CH4 or 13CO2 (13C-labeled bicarbonate).
in all of the tests (tests A−C) using both methane feeding strategies (batch and continuous). Nitrate (test A) or nitrite (tests B−D) was loaded continuously into each batch reactor through a rubber stopper using a precise programmable syringe pump (New era, NE1600). The loading rate of nitrate or nitrite for tests A, B, and D was ∼36 μmol L−1 h−1, and the loading rate of nitrite for test C was slightly lower (∼29 μmol L−1 h−1). In all of the tests, the total liquid volume added was
