DSpace at VNU: Methanogenesis Facilitated by Geobiochemical Iron Cycle in a Novel Syntrophic Methanogenic Microbial Community

Article pubs.acs.org/est Methanogenesis Facilitated by Geobiochemical Iron Cycle in a Novel Syntrophic Methanogenic Microbial Community Shenghua Jiang,†,‡ Sunhwa Park,† Younggun Yoon,† Ji-Hoon Lee,§ Wei-Min Wu,‡ Nguyen Phuoc Dan,∥ Michael J Sadowsky,⊥ and Hor-Gil Hur*,† † School of Environmental Science and Engineering, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea § Korea Institute of Geoscience and Mineral Resources, Daejeon 305-350, Republic of Korea ‡ Department of Civil and Environmental Engineering, Stanford University, Stanford, California 94305, United States ∥ Faculty of Environment, Ho Chi Minh City University of Technology, Ho Chi Minh City, Vietnam ⊥ BioTechnology Institute and Department of Soil, Water, and Climate, University of Minnesota, St Paul, Minnesota 55108, United States S Supporting Information * ABSTRACT: Production and emission of methane have been increasing concerns due to its significant effect on global climate change and the carbon cycle Here we report facilitated methane production from acetate by a novel community of methanogens and acetate oxidizing bacteria in the presence of poorly crystalline akaganeite slurry Comparative analyses showed that methanogenesis was significantly enhanced by added akaganeite and acetate was mostly stoichiometrically converted to methane Electrons produced from anaerobic acetate oxidation are transferred to akaganeite nanorods that likely prompt the transformation into goethite nanofibers through a series of biogeochemical processes of soluble Fe(II) readsorption and Fe(III) reprecipitation The methanogenic archaea likely harness the biotransformation of akaganeite to goethite by the Fe(III)−Fe(II) cycle to facilitate production of methane These results provide new insights into biogeochemistry of iron minerals and methanogenesis in the environment, as well as the development of sustainable methods for microbial methane production ■ INTRODUCTION Biological methanogenesis widely occurs in natural environments including soils, water, deep sea and digestive systems of some animals and plays an important role in global carbon cycling.1−5 Methane producing microorganisms, mainly methanogenic archaea, which obtain energy for growth by converting a limited number of substrates to methane, are common in anaerobic environments where organic matter undergoes decomposition.6,7 The main precursor for methane production by methanogens is often acetate, which is one of the most abundant products from anaerobic digestion of organic matter via bacterial catabolism.6,8,9 Two mechanisms for methane generation from acetate by methanogens have been known, such as an aceticlastic reaction10 and a two-step process in which acetate is first oxidized to H2 and CO2, followed by their subsequent conversion to methane by methanogens.9,11 In the latter process, although some methanogens, such as Methanosarcinaceae, are capable of independently oxidizing acetate by themselves,11 various syntrophic bacteria are also involved in multistep processes such as acetogenic fermentation, syntrophic acetate oxidation, and hydrogenotrophic methane production.12 © 2013 American Chemical Society In the syntrophic methanogenic system, acetate-oxidation is thermodynamically inferior to the aceticlastic pathway and can only proceed under low H2 partial pressures exerted by hydrogenotrophic methanogenesis.8,13 This suggests that interspecies H2 transfer between acetate-oxidizer and hydrogenotrophic methanogens is an essential process.14 Recently, Kato et al.15 found that methanogens in cultures of soil microbes were significantly facilitated by supplemented nanosized iron oxide minerals that appeared to promote interspecies interaction between dissimilatory metal reducing bacteria (DMRB) and hydrogenotrophic methanogens.15 They proposed that an electric syntrophism mediated by semiconductive iron oxide nanominerals acting as electron conduits could be involved in methanogenesis.15−17 Given global concerns of methane emissions from the environments, we examined the influence of the geobiochemical cycling of iron minerals on syntrophic methane production and hydrogenotrophic methReceived: Revised: Accepted: Published: 10078 May 30, 2013 July 26, 2013 August 6, 2013 August 6, 2013 dx.doi.org/10.1021/es402412c | Environ Sci Technol 2013, 47, 10078−10084 Environmental Science & Technology Article Figure Methane production, Fe(III) reduction and acetate consumption in methanogenic community in the presence of diverse forms of iron in 150 mL septum vials (a) and (b) Methane accumulation in the 100 mL headspace and concentration of acetate in the 50 mL aqueous phase cultures supplemented with akaganeite slurry; (c) and (d) accumulation of methane in 100 mL headspace and concentration of Fe(II) in the 50 mL aqueous phase of cultures supplemented with diverse forms of iron HEPES-buffered anaerobic basal salts medium amended with 20 mM sodium acetate and 50 mM akaganeite for enrichment incubation Cultures were incubated at 30 °C and transferred into fresh medium every month After two years of enrichment incubation, mL of inoculum from the stable enrichment culture was added into 50 mL new medium containing 20 mM acetate and 50 mM of the akaganeite slurry Phylogenetic Analyses The microbial populations in the syntrophic methanogenic system were analyzed by using domain-specific 27F/1492R18 and 109F/915R,21 and PCR amplification of 16S rRNA gene fragments for genomic DNA from bacteria and archaea, respectively DNA fragments were individually cloned into the pGEM-Teasy vector and then 188 and 48 colonies containing bacterial and archaeal DNA, respectively, were isolated from clone libraries Analyses of Fe(II) and Acetate in the Aqueous Phase Reduced iron, Fe(II) produced by the bacterial reduction of akaganeite, was quantified using the ferrozine assay method.22 The concentration of acetate was determined by an HPLC (Shimadzu, Tokyo, Japan), equipped with a SPD-10A UV detector (Shimadzu, Tokyo, Japan) and a Shodex RSpak KC811 (8.0 mm ID × 300 mm) column (Shodex, Tokyo, Japan) The mobile phase was mM sulfuric acid at a flow rate of 0.5 mL/min, and the UV detection was performed at 210 nm Characterization of the Nanominerals The minerals produced by the microbes were collected from the cultures using syringes after incubation and triple-washed with deionized water (DI H2O) The washed minerals were subsequently dried under anaerobic conditions in a glovebox XRD analysis was performed using a Rigaku D/MAX Ultima III high-resolution X-ray diffractometer (Rigaku, Tokyo, Japan) with Cu−Kα irradiation The generator was operated at 40 kV and 40 mA The samples were scanned between 2θ = 10 and 70°, at a scan speed of 2° min−1 anogenesis via anaerobic acetate-oxidation in a newly isolated microbial community ■ EXPERIMENTAL SECTION Media and Culture Conditions HEPES-buffered defined medium and standard techniques for culturing anaerobic bacteria were used in this study.18 The culture medium was boiled and flushed for 30 with N2 gas (100%) to remove dissolved O2 An aliquot (50 mL) of the medium was dispensed into 150 mL serum bottles under N2 (100%), which were capped with thick butyl rubber stoppers and aluminum seals (Bellco Glass, Vineland, NJ) Sodium acetate was added as the substrate at a final concentration of 20 mM by inserting an aseptic syringe into the bottled culture medium Poorly crystalline akaganeite was synthesized via the hydrolysis of FeCl3 solution.19 A dark-brown solution was obtained from partial neutralization of 100 mL of M FeCl3 by 75 mL of M NaOH solution This solution was left to stand at room temperature for 50 h, followed by the addition of 20 mL of 10 M NaOH solution The mixture was then heated at 70 °C for days X-ray diffraction (XRD) analyses identified the nanorod-like product as akaganeite.20 The suspension was anaerobically prepared as a ∼0.7 M stock solution The synthetic poorly crystalline akaganeite was added as an electron acceptor to growth medium at a final concentration of 50 mM using an aseptic syringe As precursors for comparative experiments, goethite nanorods and ferrihydrite nanoparticles were also prepared according to the methods suggested by Schwertmann and Cornell with slight modifications.19 The sediment sludge sample was collected from Tham Luong Canal, a tributary of Sai Gon river, Tan Binh District, Ho Chi Minh City, Vietnam, 2010, which was heavily contaminated with wastewater effluent from the local dye companies The sludge sample (5 g of wet weight) was inoculated into 150 mL sealed serum bottles containing 50 mL 10079 dx.doi.org/10.1021/es402412c | Environ Sci Technol 2013, 47, 10078−10084 Environmental Science & Technology Article nm), can be considered to be an electron acceptor for anaerobic acetate oxidation (Figure S1b, Supporting Information), it could also serve as an inhibitor of methanogenesis due to the high redox potential of Fe(ferrihydrite)/Fe(II), which could competitively consume electrons produced from the anaerobic acetate oxidation.15,24,25 Interestingly, when mM FeCl2 was added to the syntrophic culture, there was 0.6 mM Fe(II) still remained in the liquid culture after 30 days incubation This suggested that Fe(II) was not consumed as the electron shuttle for production of methane from CO2 because of the much higher redox potential of Fe(III)/Fe(II) (+0.77 V) than HCO3−/CH4 (−0.24 V) in liquid phase.24 Despite a significant decrease of methane production, as compared with that in culture containing akaganeite, there was still considerable methane accumulation in cultures supplemented with Fe(II) (FeCl2) This result suggested that the presence of Fe(II) iron probably enhanced methane production by decreasing the redox potential of the cultures.26 Microbes in the Syntrophic Methanogenic System Analysis of microbial populations in the syntrophic methanogenic system based on sequencing of 16S rRNA genes of bacteria and archaea showed that over two years incubation period, the population level of Clostridium spp (Table S1, Supporting Information) in the community increased from 30 to 75% (Figure 2), and the methanogens were dominated by Methanosarcina barkeri (99% identical to Genbank accession NC_007355.1) Transmission electron microscopy (TEM) analyses were performed on the collected samples in order to observe the mineralogical morphologies Precipitate in the bacterial culture medium was collected and triple-washed with DI H2O prior to electron microscopy of specimens The specimens were dried on Cu-grids under an ambient condition for TEM (JEOL, Tokyo, Japan) analyses Measurement of Methane and Hydrogen Gases For analyzing methane accumulated in the head space, 0.1 mL of gas was taken from the vial head space with a gastight syringe and directly injected into a gas chromatograph (GC, ACME 6100, Young Lin Instrument Co., Anyang, Korea) equipped with a thermal conductivity detector (TCD) and a Carboxen1000 (Supelco, Bellefonte, PA) column The GC operation condition was oven temperature initially maintained at 60 °C for and increased to 215 °C at a rate of 30 °C/min, and the injector and detector temperatures at 150 and 200 °C, respectively Helium gas was used as the carrier gas with a flow rate of 30 mL min−1 For analyzing hydrogen gas accumulated in the head space, the same volume of gas was detected by a gas chromatograph (GC-2010, Shimadzu, Kyoto, Japan) equipped with a thermal conductivity detector (TCD) and a CPPoraPLOT Q fused silica capillary column (Agilent Technologies, CA) The gas chromatography was run with nitrogen as the carrier gas with a flow rate of 5.0 mL min−1 The column temperature was maintained at 110 °C for The injector and detector temperatures were 33 and 110 °C, respectively ■ RESULTS AND DISCUSSION Methane Production by Methanogenic Community After two years of enrichment incubation of the sediment sludge in acetate/akaganeite amended medium, a mL aliquot from the stable enrichment culture was added into 50 mL of medium containing 20 mM acetate (1 mmol acetate in total) and 50 mM akaganeite slurry (2.5 mmol Fe in total) Compared to the minimum production of methane in the cultures without added akaganeite, methane production was significantly facilitated by addition of 50 mM akaganeite slurries (Figure 1a) The concentration of acetate in the cultures containing akaganeite significantly decreased whereas almost no acetate consumption was observed in the cultures without supplementary akaganeite (Figure 1b) Methane began to accumulate in the 100 mL headspace after days incubation of the cultures containing akaganeite (Figure 1a) Akaganeite was reduced and Fe(II) was released into the liquid phase coupling with consumption of acetate (Figure 1d) Under these cultural conditions, methanogenesis most likely occurred through the two-step reactions in which Fe(III) reduction by acetate oxidation bacteria followed by subsequently conversion of HCO3− and H2 to CH4 like previous reports.9 In order to investigate the influence of diverse iron minerals on methanogenesis, a series of comparative incubations were performed with 50 mM goethite, 50 mM ferrihydrite, and mM FeCl2, respectively After 30 days incubation, almost no methane was produced in cultures containing either 50 mM goethite or 50 mM ferrihydrite (Figure 1c) The concentration of soluble Fe(II) released into aqueous medium was also low (Figure 1d) The chemically synthesized goethite nanorods with lower redox potential of Fe(goethite)/Fe(II)23 and well crystalline large structure (>200 nm) (Figure S1a, Supporting Information) appear to contribute as a poor electron acceptor for the bacterial anaerobic oxidation of acetate.24 Although particulate ferrihydrite, amorphous with much smaller size (