Methane-producing Microorganisms Research Articles

Fumarate reductase drives methane emissions in the genus Oryza through differential regulation of the rhizospheric ecosystem

The emergence of waterlogged Oryza species ∼15Mya (million years ago) supplied an anoxic warm bed for methane-producing microorganisms, and methane emissions have hence accompanied the entire evolutionary history of the genus Oryza. However, to date no study has addressed how methane emission has been altered during Oryza evolution. In this paper we used a diverse collection of wild and cultivated Oryza species to study the relation between Oryza evolution and methane emissions. Phylogenetic analyses and methane detection identified a co-evolutionary pattern between Oryza and methane emissions, mediated by the diversity of the rhizospheric ecosystems arising from different oxygen levels. Fumarate was identified as an oxygen substitute used to retain the electron transport/energy production in the anoxic rice root, and the contribution of fumarate reductase to Oryza evolution and methane emissions has also been assessed. We confirmed the between-species patterns using genetic dissection of the traits in a cross between a low and high methane-emitting species. Our findings provide novel insights on the evolutionary processes of rice paddy methane emissions: the evolution of wild rice produces different Oryza species with divergent rhizospheric ecosystem attributing to the different oxygen levels and fumarate reductase activities, methane emissions are comprehensively assessed by the rhizospheric environment of diversity Oryza species and result in a co-evolution pattern.

Abstract 1403 Metabolic response to light-induced stress in methane-producing microorganisms

Abstract 1403 Metabolic response to light-induced stress in methane-producing microorganisms

A low-methane rice with high-yield potential realized via optimized carbon partitioning

Global rice cultivation significantly contributes to anthropogenic methane emissions. The methane emissions are caused by methane-producing microorganisms (methanogenic archaea) that are favoured by the anoxic conditions of paddy soils and small carbon molecules released from rice roots. However, different rice cultivars are associated with differences in methane emission rates suggesting that there is a considerable natural variation in this trait. Starting from the hypothesis that sugar allocation within a plant is an important factor influencing both yields and methane emissions, the aim of this study was to produce high-yielding rice lines associated with low methane emissions. In this study, the offspring (here termed progeny lines) of crosses between a newly characterized low-methane rice variety, Heijing 5, and three high-yielding elite varieties, Xiushui, Huayu and Jiahua, were selected for combined low-methane and high-yield properties. Analyses of total organic carbon and carbohydrates showed that the progeny lines stored more carbon in above-ground tissues than the maternal elite varieties. Also, metabolomic analysis of rhizospheric soil surrounding the progeny lines showed reduced levels of glucose and other carbohydrates. The carbon allocation, from roots to shoots, was further supported by a transcriptome analysis using massively parallel sequencing of mRNAs that demonstrated elevated expression of the sugar transporters SUT-C and SWEET in the progeny lines as compared to the parental varieties. Furthermore, measurement of methane emissions from plants, grown in greenhouse as well as outdoor rice paddies, showed a reduction in methane emissions by approximately 70 % in the progeny lines compared to the maternal elite varieties. Taken together, we report here on three independent low-methane-emission rice lines with high yield potential. We also provide a first molecular characterisation of the progeny lines that can serve as a foundation for further studies of candidate genes involved in sugar allocation and reduced methane emissions from rice cultivation.

Performance and interaction mechanism of integrating alkali pretreatment and immobilized electrodes on enhanced sludge digestion for methane production

Introduction of voltage or conductive materials have been proven to be effective strategies for enhancing the stability and efficiency of sludge digestion for methane recovery. This experiment simulated a two-phase anaerobic system, consisting of an acid-producing phase with alkali pretreatment and a methane-producing phase with immobilized electrodes and voltage enhancement. Various effective and comparative experiments were conducted to investigate the crucial roles of immobilized electrodes like carbon brush (CB) and nickel foam (NF), electroactive biofilm and voltage during the sludge methane-producing phase. The experimental results indicate that directly application of immobilized CB and NF electrode to promote sludge anaerobic digestion achieves better performance than additional voltage supply. The electrodes can increase methane production by 38% to 94%, whereas under additional voltage supply, methane production only increases by 22%–53% compared to the control. Through the analysis of key transformations in organic substrates and changes in microbial community structure, the inherent advantages of electrode materials in promoting the syntrophic methanogenesis processes are confirmed. Compared to suspended sludge, the types and relative abundance of typical syntrophic methane-producing microorganisms, Methanosarcina, and acetogenic bacteria, Syntrophomonas on the electrode surface significantly increase by 8.5% and 1.7%. This process is notably superior to the methane generation process dominated by hydrogenotrophic methanogensis after applying voltage. The results indicate that CB and NF are suitable conductive materials to promote anaerobic methane recovery, and their effectiveness surpasses that of applying voltage. These research outcomes provide new insights for the development of novel anaerobic digestion enhancement systems.

Microbial methane formation in deep aquifers associated with the sediment burial history at a coastal site

Abstract. Elucidating the mechanisms underlying microbial methane formation in subsurface environments is essential to understanding the global carbon cycle. This study examined how microbial methane formation (i.e., methanogenesis) occurs in natural-gas-bearing sedimentary aquifers throughout the sediment burial history. Water samples collected from six aquifers of different depths exhibited ascending vertical gradients in salinity from brine to fresh water and in temperature from mesophilic to psychrophilic conditions. Analyses of gas and water isotopic ratios and microbial communities indicated the predominance of methanogenesis via CO2 reduction. However, the hydrogen isotopic ratio of water changed along the depth and salinity gradient, whereas the ratio of methane changed little, suggesting that in situ methanogenesis in shallow sediments does not significantly contribute to methane in the aquifers. The population of methane-producing microorganisms (methanogens) was highest in the deepest saline aquifers, where the water temperature, salinity, and total organic carbon content of the adjacent mud sediments were the highest. Cultivation of the dominant hydrogenotrophic methanogens in the aquifers showed that the methanogenesis rate was maximized at the temperature corresponding to that of the deepest aquifer. These results suggest that high-temperature conditions in deeply buried sediments are associated with enhanced in situ methanogenesis and that methane that forms in the deepest aquifer migrates upward into the shallower aquifers by diffusion.

Methanotrophs dominate methanogens and act as a methane sink in a subterranean karst cave

Karst caves are potential sinks of atmospheric methane due to microbial consumption. However, knowledge gaps on methanogens (methane producing microorganisms) and their interaction with methane-oxidizing bacteria (MOB) hinder our further understanding about methane dynamics in karst caves. Here we reported methanogenic community composition and their interaction with MOBs in the Heshang Cave to comprehensively understand methane cycling in subsurface biosphere. MOBs in karst cave were dominated by high-affinity MOB, upland soil cluster (USC), with USCγ pmoA gene abundance within the range of 1.34 × 104 to 1.8 × 107 copies·g−1 DW. In contrast, methanogens were dominated by Methanoregula and cluster ZC-I. The mcrA numbers were 7.21 × 103 to 8.31 × 104 copies·g−1 DW, 1–3 orders of magnitude lower than those of MOB. The inter-domain network analysis indicated that MOBs and methanogens cooperated more in the interior of the cave. Despite of the higher number of methanogenic nodes in the network, MOB dominated the keystone taxa, suggesting a leading functional role of MOB. MOB in caves showed a comparable with or higher potential methane oxidizing rate (PMOR, 0.63 ng CH4·g−1 DW·h−1 in sediment versus 11.02 ng CH4·g−1 DW·h−1 in weathered rock) than those in soils, whereas methane produced by methanogens was undetected. Collectively, high absolute abundances of MOB, high PMORs, the dominance of methanotrophic keystone taxa in the inter-domain network confirmed the superiority of MOBs over methanogens in the oligotrophic karst cave, mounting new evidence on caves as an important methane sink in terms of the interaction between methanogens and MOBs.

Cytochrome-mediated direct electron uptake from metallic iron by Methanosarcina acetivorans.

Methane-producing microorganisms accelerate the corrosion of iron-containing metals. Previous studies have inferred that some methanogens might directly accept electrons from Fe(0), but when this possibility was more intensively investigated, H2 was shown to be an intermediary electron carrier between Fe(0) and methanogens. Here, we report that Methanosarcina acetivorans catalyzes direct metal-to-microbe electron transfer to support methane production. Deletion of the gene for the multiheme, outer-surface c-type cytochrome MmcA eliminated methane production from Fe(0), consistent with the key role of MmcA in other forms of extracellular electron exchange. These findings, coupled with the previous demonstration that outer-surface c-type cytochromes are also electrical contacts for electron uptake from Fe(0) by Geobacter and Shewanella species, suggest that the presence of multiheme c-type cytochromes on corrosion surfaces might be diagnostic for direct metal-to-microbe electron transfer and that interfering with cytochrome function might be a strategy to mitigate corrosion.

Fusion of Vermicompost and Sewage Sludge as Dark Fermentative Biocatalyst for Biohydrogen Production: A Kinetic Study

The present study explores the synergy between vermicompost and the anaerobic sewage sludge as inoculum for biohydrogen production using food waste as a substrate. Experiments were designed and performed in two phases of operation. In the first phase, the vermicompost (VC) was used as inoculum and food waste as substrate at three different organic loading rates of 10 gVS/L (VC1), 20 gVS/L (VC2), and 30 gVS/L (VC3). In the second phase of operation, the inoculums were combined with a proportion of 50% (VC+AS). The study showed an effective biohydrogen production of 20 gVS/L when the mixing ratio of vermicompost and anaerobic sludge was 50:50. The results inferred that effective synergy was observed between the combined consortia of the inoculum, which induces a more effective metabolic pathway for enhanced hydrogen production. H2 production was 33 mL/gVS (VC1), 48 mL/gVS (VC2), 35 mL/gVS (VC3), 46 mL/gVS (AS), and 50 mL/gVS (VC+AS). Heat pretreatment (100–120 °C) of the inoculum suppresses the methane-producing microorganisms and increases the hydrogen-producing microbes. In addition to hydrogen production, different metabolites are formed in the liquid phase, such as acetic acid, butyric acid, and propionic acid of 2.957 g/L, 4.286 g/L, and 2.123 g/L, respectively, with an energy content of 257 J/day with VC+AS. In addition, a kinetic model was studied for the cumulative hydrogen production curves using the modified Gompertz model, and the fit infers that the experimental data fitted well, with high coefficients of determination for VC+AS (R2 (G) > 0.99).

Microbial mechanism underlying high methane production of coupled alkali-microwave–H2O2–oxidation pretreated sewage sludge by in-situ bioelectrochemical regulation

The stabilization and disposal of the large amounts of sewage sludge pose a worldwide challenging problem. To solve this problem, the technical feasibility of coupled alkali-microwave–H2O2–oxidation pretreatment with in-situ bioelectrochemical regulation was used for enhancing sludge biodegradability and methane productivity. The optimum condition of the combined pretreatment was pH 10.0 ± 0.1, microwave 700 W and H2O2 0.4 g/g TS. In this case, soluble chemical oxygen demand (SCOD) of pretreated sludge was increased from initial 330.9 ± 10.0 to 3328.8 ± 49.6 mg/L. The highest accumulative methane yield of 234.3 mL CH4/g VS was obtained for pretreated sludge at the cathodic potential of −0.8 V vs. Ag/AgCl, increasing by 4.3-, and 1.9-fold compared with the raw and pretreated sludge, respectively. The microbial community analysis further provided a compelling evidence that bioelectrochemical regulation stimulated the growth of the functional microorganisms, especially in protein-degrading (Firmicutes), polysaccharides-utilizing (Chloroflexi), electroactive (Geobacter, and Desulfomicrobium) and methane-producing (Methanobacterium) microorganisms. In addition, pH adjustment of pretreated sludge by addition of H2SO4 could further enrich the abundance of microbial community and build a strong syntrophic interaction, accordingly provoking the hydrolysis and subsequent methane production. The result of this study will contribute to the establishment of an efficient sludge stabilization and bioenergy recovery strategy.

Subtle early-warning indicators of landfill subsurface thermal events

It is generally accepted that landfills undergo at least four phases of decomposition, 1) an initial aerobic phase, 2) an anaerobic acid phase, 3) an initial methanogenic phase, and 4) a stable methanogenic phase. While landfill fires are common, recently anoxic subsurface heating events (ASHEs) have been recognized, potentially catalyzed by aluminum waste. This paper reviews two such events to forensically examine if there are predictable early warning signs in the gas record prior to the onset of an ASHE that could facilitate early action. At the Countywide Recycling and Disposal Facility in East Sparta, Ohio, decreasing methane (CH4) in landfill gas was identified in a post-mortem analysis as an early indicator parameter (IP) of an abnormal landfill condition because the ASHE inhibited methane-producing microorganisms. Here the CH4:CO2 ratios also decreased to < 0.8 at least one year prior to onset of the full-fledged ASHE in some gas wells. Landfill A was a municipal solid waste landfill that operated through December 2004. The landfill gas and temperature profiles appeared normal after closure until 2009, when landfill gas characteristics in several wells started to evolve toward an upset condition, with decreasing methane (CH4 <43%), increasing carbon monoxide (CO > 500 ppm), increasing balance gas (>20%) and a CH4/CO2 ratio decreasing below 0.8. The evolution in sequencing of ASHE IPs was, 1) declining CH4, followed by, 2) increasing CO2 and % balance gas and, then 3) CO before onset of the ASHE. Subsequently, 4) leachate volumes increased as did, 5) the BOD/COD ratios and benzene in leachate, and finally 6) release of landfill contaminants to groundwater. These multiple lines of evidence suggest that operators should carefully evaluate data trends as indicators of potential upset conditions. The gas profiles in many Landfill A gas wells were similar to those at Countywide and appear to be predictable by a Landfill Fire Index (LFI) based on temperature, CO2 and CH4 patterns. While there were sporadic exceedances of the NSPS temperature (131 °F), and above the Higher Operating Value (140 °F) in some wells, the change in landfill gas IP signature and the LFI appears to have pre-dated the major thermal event by between ½ to 3 years, depending on the well.

Spectral and Chemical Characterization of Copiapite and Rozenite from Padinjarathara in Wayanad, Southern India: Implications for Mars Exploration

Copiapite (Fe2+Fe3+4(SO4)6(OH)2 ∙ 20 H2O) and rozenite (Fe2+(SO4) ∙ 4H2O) are secondary hydrous sulfate minerals, mostly formed because of oxygenation and weathering events of primary sulfides on Earth. Acidophiles, mainly archaea and bacteria, and specifically anaerobic methanogens (methane-producing microorganisms) have been detected in regions where alteration of primary sulfide phases to secondary sulfates are predominant. These secondary sulfate minerals are significant in defining past geological environments on Earth and other rocky planets such as Mars. We discuss here the results of chemical and spectral analyses of copiapite and rozenite found in massive sulfide zones associated with banded iron formations (BIFs) in the Wayanad region in Kerala, southern India. The results from different spectroscopic techniques — VNIR reflectance, TIR (emitted radiation), FTIR, Laser Raman, and X-ray diffraction — were recorded and matched with existing literatures and databases. Different matching algorithms like Spectral Feature Fitting (SFF), Spectral Similarity Value (SSV), Modified Spectral Angle Similarity (MSAS), and Pearsonian Correlation Coefficient (PCC) were utilized in order to quantitatively match the reflectance spectra with the reference spectra (USGS and RELAB). The genesis of copiapite and rozenite in the area is through seasonal and temporary development of an acidic environment, with the high elevations of the Wayanad plateau providing the necessary low-humidity condition during the dry season. Copiapite and rozenite were detected as alteration products in different regions on Mars, which can provide insights into environmental conditions that prevailed during the biogeochemical evolution of Mars. The study of copiapite and rozenite formation in a terrestrial environment will therefore enhance our understanding of similar mineral precipitation, genesis, and fluvial processes on Mars. Future missions to Mars could also use the data obtained from the present study to calibrate similar instruments to detect these secondary sulfate minerals.

Lower-than-expected CH4 emissions from rice paddies with rising CO2 concentrations.

Elevated atmospheric CO2 (eCO2 ) generally increases carbon input in rice paddy soils and stimulates the growth of methane-producing microorganisms. Therefore, eCO2 is widely expected to increase methane (CH4 ) emissions from rice agriculture, a major source of anthropogenic CH4 . Agricultural practices strongly affect CH4 emissions from rice paddies as well, but whether these practices modulate effects of eCO2 is unclear. Here we show, by combining a series of experiments and meta-analyses, that whereas eCO2 strongly increased CH4 emissions from paddies without straw incorporation, it tended to reduce CH4 emissions from paddy soils with straw incorporation. Our experiments also identified the microbial processes underlying these results: eCO2 increased methane-consuming microorganisms more strongly in soils with straw incorporation than in soils without straw, with the opposite pattern for methane-producing microorganisms. Accounting for the interaction between CO2 and straw management, we estimate that eCO2 increases global CH4 emissions from rice paddies by 3.7%, an order of magnitude lower than previous estimates. Our results suggest that the effect of eCO2 on CH4 emissions from rice paddies is smaller than previously thought and underline the need for judicious agricultural management to curb future CH4 emissions.

Redox-flow battery design for a methane-producing bioelectrochemical system

Methane production at biocathodes is an innovative approach of storing renewable electrical energy in chemical energy via the biological conversion of carbon dioxide. Methane-producing microorganisms use electricity to catalyze the conversion of carbon dioxide into methane; a form of carbon-neutral natural gas. However, the rates of methane production remain too low for practical application. To improve performance, high area-to-volume ratio with good mass transfer is required. In this study, we used the design of redox flow-batteries with a high area-to-volume ratio of 2.0 cm2/cm3 and an external capillary manifold for flow distribution. Current densities up to 35 A/m2 were applied, resulting in volumetric methane production rates of up to 12.5 L CH4/L/d, three times higher than rates reported so far. The highest energy efficiency of 30% was obtained at 25 A/m2. Even with a low relative abundance of methanogens in the microbial community (20%), dense biofilm growth was observed on the outer surface of the biocathode. Flow-battery cell design shows promising performance for application of methane-producing biocathodes.

Limited potential of harvest index improvement to reduce methane emissions from rice paddies.

Rice is a staple food for nearly half of the world's population, but rice paddies constitute a major source of anthropogenic CH4 emissions. Root exudates from growing rice plants are an important substrate for methane-producing microorganisms. Therefore, breeding efforts optimizing rice plant photosynthate allocation to grains, i.e., increasing harvest index (HI), are widely expected to reduce CH4 emissions with higher yield. Here we show, by combining a series of experiments, meta-analyses and an expert survey, that the potential of CH4 mitigation from rice paddies through HI improvement is in fact small. Whereas HI improvement reduced CH4 emissions under continuously flooded (CF) irrigation, it did not affect CH4 emissions in systems with intermittent irrigation (II). We estimate that future plant breeding efforts aimed at HI improvement to the theoretical maximum value will reduce CH4 emissions in CF systems by 4.4%. However, CF systems currently make up only a small fraction of the total rice growing area (i.e., 27% of the Chinese rice paddy area). Thus, to achieve substantial CH4 mitigation from rice agriculture, alternative plant breeding strategies may be needed, along with alternative management.

DETAILED DESIGN OF A FIBER GLASS BASED MEDIUM SCALE PLUG FLOW REACTOR FOR BIOGASIFICATION OF BIODEGRADABLE FOOD WASTE FOR RURAL AND URBAN INDIA

Plug flow reactor vessel is a long narrow (typically a 2:1 ratio; 2 times as long as the width) insulated inside and heated tank made of reinforced concrete, steel or fiberglass with a gas tight seal cover to collect the biogas. These digesters operate at a mesophilic or thermophilic temperature range. Retention time is usually ranges between 15 to 20 days. Fiber Glass is the material used for construction of the digester tank. The methanogens (methane producing micro-organisms) can digest sugar, starch, fats, cellulose and proteins etc. Their digestive mechanism is similar to that of many other organisms, up to the point of converting the food into acetic acid. Methanogens convert acetic acid into methane and carbon dioxide. (CH3COOH = CH4+CO2). They work optimally at the temperature of 38°C and a pH value of 7. At this temperature and pH, they can convert food waste into biogas within a cycle of about 24 hours.1 kg (dry weight) of food waste yields about 1 kg (about 800 litres) of biogas. To get the same quantity of biogas from dung, one needs about 40 kg dung, and a fermentation period of about 20 days. The calorific value of biogas will be about 4000 kCal per kg. The waste material to be fed into a biogas plant should contain only biodegradable organic material. Other organic products like plastic, rubber, petroleum, hooves or wood cannot be digested by the methanogens. Introduction of any toxic or bactericidal material into the biogas plant would kill the methanogens and the system would stop producing methane. Increase in the population density of non-methanogenic organisms causes reduction in that of the methanogens, inhibiting the formation of methane.

Reduction of butyrate- and methane-producing microorganisms in patients with Irritable Bowel Syndrome.

The pathophysiology of irritable bowel syndrome (IBS) remains unclear. Here we investigated the microbiome of a large cohort of patients to identify specific signatures for IBS subtypes. We examined the microbiome of 113 patients with IBS and 66 healthy controls. A subset of these participants provided two samples one month apart. We analyzed a total of 273 fecal samples, generating more than 20 million 16S rRNA sequences. In patients with IBS, a significantly lower microbial diversity was associated with a lower relative abundance of butyrate-producing bacteria (P = 0.002; q < 0.06), in particular in patients with IBS-D and IBS-M. IBS patients who did not receive any treatment harboured a lower abundance of Methanobacteria compared to healthy controls (P = 0.005; q = 0.05). Furthermore, significant correlations were observed between several bacterial taxa and sensation of flatulence and abdominal pain (P < 0.05). Altogether, our findings showed that IBS-M and IBS-D patients are characterized by a reduction of butyrate producing bacteria, known to improve intestinal barrier function, and a reduction of methane producing microorganisms a major mechanism of hydrogen disposal in the human colon, which could explain excess of abdominal gas in IBS.

English

This study was aimed at determining the effect of temperature on the stabilization of organic solid waste conjugated with sewage sludge in anaerobic batch reactors (ABR), and to estimate the efficiency of the process in producing biogas and methane. The substrate contained 36.2 g L-1 of total solids comprising residues from fruits and vegetables enriched with anaerobic sludge from sanitary sewage (4:1, w/w). The reactors were of 1.15 L capacity and were operated at 25.5, 40 and 50°C (ABR1, ABR2 and ABR3, respectively) for 160 days. The efficiencies of transformation of total volatile solids in ABR1, ABR2 and ABR3 were, respectively, 43.2, 34.2 and 32%, and the transformation of chemical oxygen demand showed a similar tendency with efficiencies of 39.5, 33.6 and 16.6%, respectively. The mean volumes of biogas accumulated by ABR1, ABR2 and ABR3 were 28.85, 21.24 and 20.54 L, respectively, while the respective mean volumes of methane were 9.04, 7.11 and 1.11 L. The results demonstrate that the activity of methane-producing microorganisms was inhibited at higher process temperatures. It is concluded that anaerobic digestion at ambient temperature represents an economical and environmentally viable strategy for the disposal of municipal solid wastes. Key words: Plant residues, anaerobic digestion, biofuel, methane

Biowaste to Biofuel: Using Methane-Producing Microorganisms Found in Soil Samples from Local Wetlands

In this study, we investigated whether methane- producing microorganisms are present in the soil samples obtained from local bogs and wetlands in the Puget Sound region of Washington state. Furthermore, we determined the temperature at which these methane- producing microorganisms can be used to produce methane, a biofuel, from kitchen and yard waste. Producing methane for making biofuel as renewable energy is better than sending kitchen and yard waste to landfills, which do not utilize or collect these wastes for energy production. We show that methane-producing microorganisms are indeed present in the soil samples we collected and that they remain active at both indoor (68 °F) and outdoor (36 to 70 °F) temperatures. A significant amount of methane was detected after 45–75 days of soil incubation with fed biowaste. Importantly, our work establishes the potential of using these methane-producing microorganisms for the production of biogas in colder climates of the Pacific NW and paves the way for the future research and design of a kitchen- scale biodigester for use in colder climates.

Rapid Biogas Production by Compact Multi-Layer Membrane Bioreactor: Efficiency of Synthetic Polymeric Membranes

Entrapment of methane-producing microorganisms between semi-permeable synthetic membranes in a multi-layer membrane bioreactor (MMBR) was studied and compared to the digestion capacity of a free-cell digester, using a hydraulic retention time of one day and organic loading rates (OLR) of 3.08, 6.16, and 8.16 g COD/L·day. The reactor was designed to retain bacterial cells with uprising plug flow through a narrow tunnel between membrane layers, in order to acquire maximal mass transfer in a compact bioreactor. Membranes of hydrophobic polyamide 46 (PA) and hydroxyethylated polyamide 46 (HPA) as well as a commercial membrane of polyvinylidene fluoride (PVDF) were examined. While the bacteria in the free-cell digester were washed out, the membrane bioreactor succeeded in retaining them. Cross-flow of the liquid through the membrane surface and diffusion of the substrate through the membranes, using no extra driving force, allowed the bacteria to receive nutrients and to produce biogas. However, the choice of membrane type was crucial. Synthesized hydrophobic PA membrane was not effective for this purpose, producing 50–121 mL biogas/day, while developed HPA membrane and the reference PVDF were able to transfer the nutrients and metabolites while retaining the cells, producing 1102–1633 and 1016–1960 mL biogas/day, respectively.

Discovery and Characterization of the First Archaeal Dihydromethanopterin Reductase, an Iron-Sulfur Flavoprotein from Methanosarcina mazei

The microbial production of methane by methanogenic archaea is dependent on the synthesis of the pterin-containing cofactor tetrahydromethanopterin (H4MPT). The enzyme catalyzing the last step of H4MPT biosynthesis (dihydromethanopterin reductase) has not previously been identified in methane-producing microorganisms. Previous complementation studies with the methylotrophic bacterium Methylobacterium extorquens have indicated that an uncharacterized archaeal-flavoprotein-like flavoprotein (AfpA) from Methylobacillus flagellatus or Burkholderia xenovorans can replace the activity of a phylogenetically unrelated bacterial dihydromethanopterin reductase (DmrA). We propose that MM1854, a homolog of AfpA from Methanosarcina mazei, catalyzes the last step of H4MPT biosynthesis in methane-producing microorganisms. To test this hypothesis, a six-histidine (His6)-tagged version of MM1854 was produced. Bioinformatic analysis revealed the presence of one flavin mononucleotide (FMN)-binding site and two iron-sulfur cluster sites, consistent with an oxidoreductase enzyme. Purified His6-MM1854 occurred as a homodimer of 29-kDa subunits, and the UV-visible spectrum of the purified protein showed absorbance peaks at 380 and 460 nm, characteristic of oxidized FMN. NAD(P)H was incapable of directly reducing the flavin cofactor, but dithionite eliminated the FMN peaks, indicating successful electron transfer to MM1854. An electron transfer system of NADPH, spinach NADPH-ferredoxin oxidoreductase, and ferredoxin could also reduce the FMN peaks. A newly developed assay indicated that dithiothreitol-reduced MM1854 could transfer electrons to dihydromethanopterin. This assay was also effective with a heat-stable DmrX analog from Methanocaldococcus jannaschii (MJ0208). These results provide the first biochemical evidence that MM1854 and MJ0208 function as archaeal dihydromethanopterin reductases (DmrX) and that ferredoxin may serve as an electron donor.