Methanogenesis and Salt Tolerance Genes of a Novel Halophilic Methanosarcinaceae Metagenome-Assembled Genome from a Former Solar Saltern

Anaerobic archaeal methanogens are key players in the global carbon cycle due to their role in the final stages of organic matter decomposition in anaerobic environments such as wetland sediments. Here we present the first draft metagenome-assembled genome (MAG) sequence of an unclassified Methanosarcinaceae methanogen phylogenetically placed adjacent to the Methanolobus and Methanomethylovorans genera that appears to be a distinct genus and species. The genome is derived from sediments of a hypersaline (97–148 ppt chloride) unrestored industrial saltern that has been observed to be a significant methane source. The source sediment is more saline than previous sources of Methanolobus and Methanomethylovorans. We propose a new genus name, Methanosalis, to house this genome, which we designate with the strain name SBSPR1A. The MAG was binned with CONCOCT and then improved via scaffold extension and reassembly. The genome contains pathways for methylotrophic methanogenesis from trimethylamine and dimethylamine, as well as genes for the synthesis and transport of compatible solutes. Some genes involved in acetoclastic and hydrogenotrophic methanogenesis are present, but those pathways appear incomplete in the genome. The MAG was more abundant in two former industrial salterns than in a nearby reference wetland and a restored wetland, both of which have much lower salinity levels, as well as significantly lower methane emissions than the salterns.

Metagenomic Analysis Reveals Microbial Interactions at the Biocathode of a Bioelectrochemical System Capable of Simultaneous Trichloroethylene and Cr(VI) Reduction

Bioelectrochemical systems (BES) are attractive and versatile options for the bioremediation of organic or inorganic pollutants, including trichloroethylene (TCE) and Cr(VI), often found as co-contaminants in the environment. The elucidation of the microbial players’ role in the bioelectroremediation processes for treating multicontaminated groundwater is still a research need that attracts scientific interest. In this study, 16S rRNA gene amplicon sequencing and whole shotgun metagenomics revealed the leading microbial players and the primary metabolic interactions occurring in the biofilm growing at the biocathode where TCE reductive dechlorination (RD), hydrogenotrophic methanogenesis, and Cr(VI) reduction occurred. The presence of Cr(VI) did not negatively affect the TCE degradation, as evidenced by the RD rates estimated during the reactor operation with TCE (111±2 μeq/Ld) and TCE/Cr(VI) (146±2 μeq/Ld). Accordingly, Dehalococcoides mccartyi, the primary biomarker of the RD process, was found on the biocathode treating both TCE (7.82E+04±2.9E+04 16S rRNA gene copies g−1 graphite) and TCE/Cr(VI) (3.2E+07±2.37E+0716S rRNA gene copies g−1 graphite) contamination. The metagenomic analysis revealed a selected microbial consortium on the TCE/Cr(VI) biocathode. D. mccartyi was the sole dechlorinating microbe with H2 uptake as the only electron supply mechanism, suggesting that electroactivity is not a property of this microorganism. Methanobrevibacter arboriphilus and Methanobacterium formicicum also colonized the biocathode as H2 consumers for the CH4 production and cofactor suppliers for D. mccartyi cobalamin biosynthesis. Interestingly, M. formicicum also harbors gene complexes involved in the Cr(VI) reduction through extracellular and intracellular mechanisms.

Effects of combined tannic acid/fluoride on sulfur transformations and methanogenic pathways in swine manure

Livestock manure emits reduced sulfur compounds and methane, which affect nature and the climate. These gases are efficiently mitigated by addition of a tannic acid-sodium fluoride combination inhibitor (TA-NaF), and to some extent by acidification. In this paper, TA-NaF treatment was performed on swine manure to study the treatment influence on methanogenic pathways and sulfur transformation pathways in various laboratory experiments. Stable carbon isotope labeling revealed that both untreated and TA-NaF treated swine manures were dominated by hydrogenotrophic methanogenesis. However, in supplementary experiments in wastewater sludge, TA-NaF clearly inhibited acetoclastic methanogenesis, whereas acidification inhibited hydrogenotrophic methanogenesis. In swine manure, TA-NaF inhibited s-amino acid catabolism to a larger extent than sulfate reduction. Conversely, acidification reduced sulfate reduction activity more than s-amino acid degradation. TA-NaF treatment had no significant effect on methanogenic community structure, which was surprising considering clear effects on isotope ratios of methane and carbon dioxide. Halophile sulfate reducers adapted well to TA-NaF treatment, but the community change also depended on temperature. The combined experimental work resulted in a proposed inhibition scheme for sulfur transformations and methanogenic pathways as affected by TA-NaF and acidification in swine manure and in other inocula.

Microbial assemblages and methanogenesis pathways impact methane production and foaming in manure deep-pit storages

Foam accumulation in swine manure deep-pits has been linked to explosions and flash fires that pose devastating threats to humans and livestock. It is clear that methane accumulation within these pits is the fuel for the fire; it is not understood what microbial drivers cause the accumulation and stabilization of methane. Here, we conducted a 13-month field study to survey the physical, chemical, and biological changes of pit-manure across 46 farms in Iowa. Our results showed that an increased methane production rate was associated with less digestible feed ingredients, suggesting that diet influences the storage pit’s microbiome. Targeted sequencing of the bacterial 16S rRNA and archaeal mcrA genes was used to identify microbial communities’ role and influence. We found that microbial communities in foaming and non-foaming manure were significantly different, and that the bacterial communities of foaming manure were more stable than those of non-foaming manure. Foaming manure methanogen communities were enriched with uncharacterized methanogens whose presence strongly correlated with high methane production rates. We also observed strong correlations between feed ration, manure characteristics, and the relative abundance of specific taxa, suggesting that manure foaming is linked to microbial community assemblage driven by efficient free long-chain fatty acid degradation by hydrogenotrophic methanogenesis.

Early response of methanogenic archaea to H2 as evaluated by metagenomics and metatranscriptomics

Background The molecular machinery of the complex microbiological cell factory of biomethane production is not fully understood. One of the process control elements is the regulatory role of hydrogen (H2). Reduction of carbon dioxide (CO2) by H2 is rate limiting factor in methanogenesis, but the community intends to keep H2 concentration low in order to maintain the redox balance of the overall system. H2 metabolism in methanogens becomes increasingly important in the Power-to-Gas renewable energy conversion and storage technologies. Results The early response of the mixed mesophilic microbial community to H2 gas injection was investigated with the goal of uncovering the first responses of the microbial community in the CH4 formation and CO2 mitigation Power-to-Gas process. The overall microbial composition changes, following a 10 min excessive bubbling of H2 through the reactor, was investigated via metagenome and metatranscriptome sequencing. The overall composition and taxonomic abundance of the biogas producing anaerobic community did not change appreciably 2 hours after the H2 treatment, indicating that this time period was too short to display differences in the proliferation of the members of the microbial community. There was, however, a substantial increase in the expression of genes related to hydrogenotrophic methanogenesis of certain groups of Archaea. As an early response to H2 exposure the activity of the hydrogenotrophic methanogenesis in the genus Methanoculleus was upregulated but the hydrogenotrophic pathway in genus Methanosarcina was downregulated. The RT-qPCR data corroborated the metatranscriptomic Results H2 injection also altered the metabolism of a number of microbes belonging in the kingdom Bacteria. Many Bacteria possess the enzyme sets for the Wood-Ljungdahl pathway. These and the homoacetogens are partners for syntrophic community interactions between the distinct kingdoms of Archaea and Bacteria. Conclusions External H2 regulates the functional activity of certain Bacteria and Archaea. The syntrophic cross-kingdom interactions in H2 metabolism are important for the efficient operation of the Power-to-Gas process. Therefore, mixed communities are recommended for the large scale Power-to-Gas process rather than single hydrogenotrophic methanogen strains. Fast and reproducible response from the microbial community can be exploited in turn-off and turn-on of the Power-to-Gas microbial cell factories.

Procaryotic Diversity and Hydrogenotrophic Methanogenesis in an Alkaline Spring (La Crouen, New Caledonia)

(1) Background: The geothermal spring of La Crouen (New Caledonia) discharges warm (42 °C) alkaline water (pH~9) enriched in dissolved nitrogen with traces of methane, but its microbial diversity has not yet been studied. (2) Methods: Cultivation-dependent and -independent methods (e.g., Illumina sequencing and quantitative PCR based on 16S rRNA gene) were used to describe the prokaryotic diversity of this spring. (3) Results: Prokaryotes were mainly represented by Proteobacteria (57% on average), followed by Cyanobacteria, Chlorofexi, and Candidatus Gracilibacteria (GN02/BD1-5) (each > 5%). Both potential aerobes and anaerobes, as well as mesophilic and thermophilic microorganisms, were identified. Some of them had previously been detected in continental hyperalkaline springs found in serpentinizing environments (The Cedars, Samail, Voltri, and Zambales ophiolites). Gammaproteobacteria, Ca. Gracilibacteria and Thermotogae were significantly more abundant in spring water than in sediments. Potential chemolithotrophs mainly included beta- and gammaproteobacterial genera of sulfate-reducers (Ca. Desulfobacillus), methylotrophs (Methyloversatilis), sulfur-oxidizers (Thiofaba, Thiovirga), or hydrogen-oxidizers (Hydrogenophaga). Methanogens (Methanobacteriales and Methanosarcinales) were the dominant Archaea, as found in serpentinization-driven and deep subsurface ecosystems. A novel alkaliphilic hydrogenotrophic methanogen (strain CAN) belonging to the genus Methanobacterium was isolated, suggesting that hydrogenotrophic methanogenesis occurs at La Crouen.

Recirculation of H2, CO2, and ethylene improves carbon fixation and carboxylate yields in anaerobic fermentation

Anaerobic fermentation with mixed cultures has gained momentum as a bioprocess for its promise to produce platform carboxylates from low-value biomass feedstocks. Anaerobic fermenters are net carbon emitters and their carboxylate yields are limited by electron donor availability. In a new approach to tackle these two disadvantages, we operated two bioreactors fed with acetate and lactate as a model feedstock while recirculating H2/CO2 to stimulate concomitant autotrophic activity. After 42 days of operation, hydrogenotrophic methanogenesis was predominant and ethylene (≥1.3 kPa) was added to one of the reactors, inhibiting methanogenesis completely and recovering net carbon fixation (0.20 g CO2 L-1 d-1). When methanogenesis was inhibited, exogenous H2 accounted for 17% of the consumed electron donors. Lactate-to-butyrate selectivity was 101% (88% in the control without ethylene) and lactate-to-caproate selectivity was 17% (2.3% in the control). Community analysis revealed that ethylene caused Methanobacterium to be washed out, giving room to acetogenic bacteria. In contrast to 2-bromoethanosulfonate, ethylene is a scalable methanogenesis inhibition strategy that did not collaterally block i-butyrate formation. By favoring the bacterial share of the community to become mixotrophic, the concept offers a way to simultaneously increase selectivity to medium-chain carboxylates and to develop a carbon-fixing chain elongation process.

Process Performance and Microbial Community Variation in High-Rate Anaerobic Continuous Stirred Tank Reactor Treating Palm Oil Mill Effluent at Temperatures Between 55 °C and 70 °C

Palm oil mill effluent (POME) is wastewater with a relatively high temperature (80 – 90 °C) that is generated from the extraction of oil from palm fruit and palm kernels. Owing to its high discharge temperatures, the thermophilic anaerobic digestion (AD) of POME could be advantageous as treatment at thermophilic temperatures can reduce loads for cooling the wastewater. In this study, the effects of stepwise temperature increments of 5 °C from 55 °C to 70 °C on the AD of POME were investigated in a continuous stirred tank reactor (CSTR) operated under high organic loading rates (OLRs). The process performance and microbial community structure at each temperature interval were evaluated. It was observed that the methane production rates of the CSTR increased with increasing OLRs up to values of 13.7 g/L d, 25.7 g/L d, and 26.5 g/L d at operating temperatures of 55 °C, 60 °C, and 65 °C, respectively. As a result of the increasing OLRs, the maximum rate of methane production increased from 3.8 L/L d at 55 °C to 4.4 L/L d at 60 °C and to 3.8 L/L d at 65 °C. The microbial community structure analysis showed that there were notable reductions in the gene copy number of the bacterial domain and the Methanosarcinales order with increasing temperatures from 55 °C to 60 °C and to 65 °C, whereas hydrogenotrophic methanogens, especially the genus Methanobacterium, in the order Methanobacteriales became dominant at 60 °C and 65 °C. Thus, the methanogenesis pathway was suggested to be a combination of acetoclastic and hydrogenotrophic methanogenesis at 55 °C and 60 °C with an increased contribution of hydrogenotrophic methanogenesis at 60 °C, whereas methane was mainly generated via hydrogenotrophic methanogenesis at 65 °C. The thermophilic AD of POME at 60 °C was found to be promising because the methane content in the biogas and the methane production rates were optimal, with an average methane content of approximately 73%.

Hydrogen as a Co-electron Donor for Chain Elongation With Complex Communities

Electron donor scarcity is seen as one of the major issues limiting economic production of medium-chain carboxylates from waste streams. Previous studies suggest that co-fermentation of hydrogen in microbial communities that realize chain elongation relieves this limitation. To better understand how hydrogen co-feeding can support chain elongation, we enriched three different microbial communities from anaerobic reactors (A, B, and C with ascending levels of diversity) for their ability to produce medium-chain carboxylates from conventional electron donors (lactate or ethanol) or from hydrogen. In the presence of abundant acetate and CO2, the effects of different abiotic parameters (pH values in acidic to neutral range, initial acetate concentration, and presence of chemical methanogenesis inhibitors) were tested along with the enrichment. The presence of hydrogen facilitated production of butyrate by all communities and improved production of i-butyrate and caproate by the two most diverse communities (B and C), accompanied by consumption of acetate, hydrogen, and lactate/ethanol (when available). Under optimal conditions, hydrogen increased the selectivity of conventional electron donors to caproate from 0.23 ± 0.01 mol e–/mol e– to 0.67 ± 0.15 mol e–/mol e– with a peak caproate concentration of 4.0 g L–1. As a trade-off, the best-performing communities also showed hydrogenotrophic methanogenesis activity by Methanobacterium even at high concentrations of undissociated acetic acid of 2.9 g L–1 and at low pH of 4.8. According to 16S rRNA amplicon sequencing, the suspected caproate producers were assigned to the family Anaerovoracaceae (Peptostreptococcales) and the genera Megasphaera (99.8% similarity to M. elsdenii), Caproiciproducens, and Clostridium sensu stricto 12 (97–100% similarity to C. luticellarii). Non-methanogenic hydrogen consumption correlated to the abundance of Clostridium sensu stricto 12 taxa (p < 0.01). If a robust methanogenesis inhibition strategy can be found, hydrogen co-feeding along with conventional electron donors can greatly improve selectivity to caproate in complex communities. The lessons learned can help design continuous hydrogen-aided chain elongation bioprocesses.

Alternating wet-dry cycles rather than sulfate fertilization control pathways of methanogenesis and methane turnover in rice straw-amended paddy soil

<p>Alternate wet-drying (AWD) and sulfate fertilization have been considered as effective management practices for lowering CH<sub>4</sub> emissions from paddy soils. However, the effects of management practices on in situ belowground CH<sub>4</sub> turnover (production and oxidation) are not yet fully understood. Here, soil CO<sub>2</sub> and CH<sub>4</sub> concentrations and their C isotope compositions were measured at three rice growing stages in straw-amended paddy soils with and without sulfate fertilization under continuously flooded conditions and two wet-dry-cycles. CH<sub>4</sub> concentration reached 51.0 mg C L<sup>-1</sup> at flowering stage under flooded conditions, while it decreased to 0.04 mg C L<sup>-1</sup> under AWD. Relative enrichment of δ<sup>13</sup>C in CH<sub>4</sub> and depletion of δ<sup>13</sup>C in CO<sub>2</sub> under AWD indicated CH<sub>4</sub> oxidation. Sulfate addition had no significant effect on CH<sub>4</sub> concentration. The ample substrate supply might have prevented sulfate-reducing bacteria from out-competing methanogenic archaea and could therefore explain the absence of a fall in CH<sub>4</sub> production. The δ<sup>13</sup>C-CO<sub>2</sub> enrichment over time (7 ‰ and 5‰ with and without sulfate fertilizer, respectively) under flooded conditions likely indicates an increasing contribution of hydrogenotrophic methanogenesis to CH<sub>4</sub> production with ongoing rice growth. Overall, the results showed that AWD could more efficiently reduce CH<sub>4</sub> production than sulfate fertilization in rice-straw-amended paddy soils.</p><p> </p>