Anaerobic conversion of organic wastes and biomass to methane is an important bioenergy strategy, which depends on poorly understood mechanisms of interspecies electron transfer to methanogenic microorganisms. Metatranscriptomic analysis of methanogenic aggregates from a brewery wastewater digester, coupled with fluorescence in situ hybridization with specific 16S rRNA probes, revealed that Methanosaeta species were the most abundant and metabolically active methanogens. Methanogens known to reduce carbon dioxide with H2 or formate as the electron donor were rare. Although Methanosaeta have previously been thought to be restricted to acetate as a substrate for methane production, Methanosaeta in the aggregates had a complete complement of genes for the enzymes necessary for the reduction of carbon to methane, and transcript abundance for these genes was high. Furthermore, Geobacter species, the most abundant bacteria in the aggregates, highly expressed genes for ethanol metabolism and for extracellular electron transfer via electrically conductive pili, suggesting that Geobacter and Methanosaeta species were exchanging electrons via direct interspecies electron transfer (DIET). This possibility was further investigated in defined co-cultures of Geobacter metallireducens and Methanosaeta harundinacea which stoichiometrically converted ethanol to methane. Transcriptomic, radiotracer, and genetic analysis demonstrated that M. harundinacea accepted electrons via DIET for the reduction of carbon dioxide to methane. The discovery that Methanosaeta species, which are abundant in a wide diversity of methanogenic environments, are capable of DIET has important implications not only for the functioning of anaerobic digesters, but also for global methane production.

https://bio.as.uky.edu/sites/default/files/Rotaru%20et%20al.%202014%20EES%20copy.pdf

A new model for electron flow during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane

A replicate long-term experiment was conducted using anaerobic digestion (AD) as a model process to determine the relative role of niche and neutral theory on microbial community assembly, and to link community dynamics to system performance. AD is performed by a complex network of microorganisms and process stability relies entirely on the synergistic interactions between populations belonging to different functional guilds. In this study, three independent replicate anaerobic digesters were seeded with the same diverse inoculum, supplied with a model substrate, α-cellulose, and operated for 362 days at a 10-day hydraulic residence time under mesophilic conditions. Selective pressure imposed by the operational conditions and model substrate caused large reproducible changes in community composition including an overall decrease in richness in the first month of operation, followed by synchronised population dynamics that correlated with changes in reactor performance. This included the synchronised emergence and decline of distinct Ruminococcus phylotypes at day 148, and emergence of a Clostridium and Methanosaeta phylotype at day 178, when performance became stable in all reactors. These data suggest that many dynamic functional niches are predictably filled by phylogenetically coherent populations over long time scales. Neutral theory would predict that a complex community with a high degree of recognised functional redundancy would lead to stochastic changes in populations and community divergence over time. We conclude that deterministic processes may play a larger role in microbial community dynamics than currently appreciated, and under controlled conditions it may be possible to reliably predict community structural and functional changes over time.

/pdf/deterministic-processes-guide-long-term-synchronised-59gvzrkczi.pdf

Deterministic processes guide long-term synchronised population dynamics in replicate anaerobic digesters.

Methane is a key compound in the global carbon cycle that influences both nutrient cycling and the Earth's climate. A limited number of microorganisms control the flux of biologically generated methane, including methane-metabolizing archaea that either produce or consume methane. Methanogenic and methanotrophic archaea belonging to the phylum Euryarchaeota share a genetically similar, interrelated pathway for methane metabolism. The key enzyme in this pathway, the methyl-coenzyme M reductase (Mcr) complex, catalyses the last step in methanogenesis and the first step in methanotrophy. The discovery of mcr and divergent mcr-like genes in new euryarchaeotal lineages and novel archaeal phyla challenges long-held views of the evolutionary origin of this metabolism within the Euryarchaeota. Divergent mcr-like genes have recently been shown to oxidize short-chain alkanes, indicating that these complexes have evolved to metabolize substrates other than methane. In this Review, we examine the diversity, metabolism and evolutionary history of mcr-containing archaea in light of these recent discoveries.

An evolving view of methane metabolism in the Archaea.

The possibility that Methanothrix (formerly Methanosaeta) and Geobacter species cooperate via direct interspecies electron transfer (DIET) in terrestrial methanogenic environments was investigated in rice paddy soils. Genes with high sequence similarity to the gene for the PilA pilin monomer of the electrically conductive pili (e-pili) of Geobacter sulfurreducens accounted for over half of the PilA gene sequences in metagenomic libraries and 42% of the mRNA transcripts in RNA sequencing (RNA-seq) libraries. This abundance of e-pilin genes and transcripts is significant because e-pili can serve as conduits for DIET. Most of the e-pilin genes and transcripts were affiliated with Geobacter species, but sequences most closely related to putative e-pilin genes from genera such as Desulfobacterium, Deferribacter, Geoalkalibacter, and Desulfobacula, were also detected. Approximately 17% of all metagenomic and metatranscriptomic bacterial sequences clustered with Geobacter species, and the finding that Geobacter spp. were actively transcribing growth-related genes indicated that they were metabolically active in the soils. Genes coding for e-pilin were among the most highly transcribed Geobacter genes. In addition, homologs of genes encoding OmcS, a c-type cytochrome associated with the e-pili of G. sulfurreducens and required for DIET, were also highly expressed in the soils. Methanothrix species in the soils highly expressed genes for enzymes involved in the reduction of carbon dioxide to methane. DIET is the only electron donor known to support CO2 reduction in Methanothrix Thus, these results are consistent with a model in which Geobacter species were providing electrons to Methanothrix species for methane production through electrical connections of e-pili.IMPORTANCEMethanothrix species are some of the most important microbial contributors to global methane production, but surprisingly little is known about their physiology and ecology. The possibility that DIET is a source of electrons for Methanothrix in methanogenic rice paddy soils is important because it demonstrates that the contribution that Methanothrix makes to methane production in terrestrial environments may extend beyond the conversion of acetate to methane. Furthermore, defined coculture studies have suggested that when Methanothrix species receive some of their energy from DIET, they grow faster than when acetate is their sole energy source. Thus, Methanothrix growth and metabolism in methanogenic soils may be faster and more robust than generally considered. The results also suggest that the reason that Geobacter species are repeatedly found to be among the most metabolically active microorganisms in methanogenic soils is that they grow syntrophically in cooperation with Methanothrix spp., and possibly other methanogens, via DIET.

Metatranscriptomic Evidence for Direct Interspecies Electron Transfer Between Geobacter and Methanothrix Species in Methanogenic Rice Paddy Soils

A novel mesophilic, hydrogenotrophic methanogen, strain SANAET, was isolated from an anaerobic, propionate-degrading enrichment culture, which was originally established from rice paddy soil. The cells were non-motile, Gram-negative and rod-shaped (1.8-2.4 microm long by 0.3-0.6 microm wide). Growth of strain SANAET was observed at 25-40 degrees C, with an optimum temperature range for growth of 35-37 degrees C. The pH range for growth was 6.5-7.8, with an optimum at pH 7.0. The salinity range for growth was 0-1 g NaCl l(-1) (0-17 mM). The isolate was able to utilize H2/CO2 and formate for growth and methane production. The G+C content of the genomic DNA was 56.6 mol%. Based on comparative 16S rRNA gene sequence analysis, strain SANAET was affiliated with a clone lineage of the Archaea, Rice Cluster I (RC-I), placing it between the orders Methanosarcinales and Methanomicrobiales within the class 'Methanomicrobia'. 16S rRNA gene sequence similarities between strain SANAET and members of Methanosarcinales were in the range 80.0-82.8 %, and those between the strain and members of Methanomicrobiales ranged from 77.5 to 82.4 %. In addition to 16S rRNA gene analysis, sequence analysis of the mcrA gene (encoding the alpha subunit of methyl-coenzyme M reductase, a key enzyme in the methane production pathway) also showed that strain SANAET was affiliated with the RC-I lineage. Here, we propose the name Methanocella paludicola gen. nov., sp. nov. for the isolate, the first of the RC-I lineage. The type strain is SANAET (=JCM 13418T=NBRC 101707T=DSM 17711T). In addition, we also propose the status of order for the RC-I lineage, for which we propose the name Methanocellales ord. nov.

/pdf/methanocella-paludicola-gen-nov-sp-nov-a-methane-producing-3vcuxohobj.pdf

Methanocella paludicola gen. nov., sp. nov., a methane-producing archaeon, the first isolate of the lineage 'Rice Cluster I', and proposal of the new archaeal order Methanocellales ord. nov.

Two methanogenic strains, 8AcT and 6Ac, were isolated from an upflow anaerobic sludge blanket reactor treating beer-manufacture wastewater in Beijing, China. Cells of strains 8AcT and 6Ac were rod-shaped (0·8–1·0×3–5 μm) and non-motile, occurring singly or in pairs; however, at high cell density the cells were arranged in long chains within a common sheath. The two strains used acetate exclusively for growth and methane production. The specific growth rate of strain 8AcT was 0·030 h−1 when growing in acetate (20 mM) at 37 °C. The temperature range for growth was 25–45 °C, with the fastest growth at 34–37 °C. The pH range for growth and methane production was 6·5–9·0, with the fastest growth at pH 7·2–7·6. The G+C content of genomic DNA of strain 8AcT was 55·7 mol%. Phylogenetic analysis based on 16S rRNA gene sequence similarity showed that the novel strains clustered with Methanosaeta species; the 16S rRNA gene sequence similarities between strain 8AcT and Methanosaeta concilii DSM 3013 and ‘Methanosaeta thermophila’ DSM 6194 were 92·5 and 87·3 %, respectively. The sequence similarity levels of mcrA, the gene encoding the α-subunit of methyl-coenzyme M reductase, and of the deduced amino acids of mcrA, between strain 8AcT and Methanosaeta concilii DSM 3671T were 36 and 78·9 %, respectively. Based on the phylogenetic and phenotypic analyses, the novel species Methanosaeta harundinacea sp. nov. is proposed, with strain 8AcT (=JCM 13211T=CGMCC 1.5026T) as the type strain.

Methanosaeta harundinacea sp. nov., a novel acetate-scavenging methanogen isolated from a UASB reactor.

Two methanogenic strains, 8-2T and 4-1, with rod-shaped (0·4–0·5×3–5 μm), non-motile cells, sometimes observed in chains, were isolated from two anaerobic digesters in Beijing, China. The two strains used H2/CO2 and formate for growth and produced methane. The temperature range for growth was 25–50 °C, with fastest growth at 37 °C. The pH ranges for growth and methane production were 6·5–8·0 for strain 8-2T and 6·8–8·6 for strain 4-1, with the fastest growth at pH 7·2 for strain 8-2T and pH 7·5–7·7 for strain 4-1. The G+C content of genomic DNA for strain 8-2T was 38·9 mol%. The similarity levels of the 16S rRNA sequence of strain 8-2T with other species of the genus Methanobacterium ranged from 93·8 to 96·0 %. Based on the phylogenetic analysis and phenotypic characteristics, the novel species Methanobacterium beijingense sp. nov. is proposed, with the type strain 8-2T (=DSM 15999T=CGMCC 1.5011T).

/pdf/methanobacterium-beijingense-sp-nov-a-novel-methanogen-3ikdy70yit.pdf

Methanobacterium beijingense sp. nov., a novel methanogen isolated from anaerobic digesters.

UNLABELLED Methanosaeta harundinacea 6Ac displayed a cell-density dependent cell morphology transition between short rods (3 microm - 5 microm) and filaments ( >200 microm), implying a quorum sensing system in this archaeon. OBJECTIVE This study aimed to confirm that cell morphology related quorum sensing exists in Methanosaeta harundinacea. METHODS By using the bioassay of Agrobacterium tumefaciens NTL4, we determined the presence of acyl homoserine lactones (AHLs) in the spent cultures of Methanosaeta harundinacea 6Ac and other three methanogens. A chemical synthetic AHL, N-(beta-Ketooctanoyl)-L-homoserine lactone was added into the culture of short rods to detect the morphology change. RESULTS We determined that AHLs were produced by Methanosaeta harundinacea, Methanosarcina mazei, Methanothermobacter thermautotrophicus and Methanobacterium formicicum. Addition of N-(beta-Ketooctanoyl)-L-homoserine lactone into the culture of Methanosaeta harundinacea 6Ac stimulated the formation of filamentous cells. CONCLUSION This study indicates that AHL-based quorum sensing may be used by several species of methanogenic Archaea.

Detection of the quorum sensing signals in methanogenic archaea

As the most diverse and numerous life form on Earth, microorganisms play indispensable roles in natural ecological processes. Revealing their diversity patterns across space and through time is of essential importance to better understand the underlying ecological mechanisms controlling the distribution and assembly of microbial communities. ABSTRACT The spatial scaling of biodiversity, such as the taxa-area relationship (TAR) and distance-decay relationship (DDR), is a typical ecological pattern that is followed by both microbes and macrobes in natural ecosystems. Previous studies focusing on microbes mainly aimed to address whether and how different types of microbial taxa differ in spatial scaling patterns, leaving the underlying mechanisms largely untouched. In this study, the spatial scaling of different microbial domains and their associated ecological processes in an intertidal zone were comparatively investigated. The significant spatial scaling of biodiversity could be observed across all microbial domains, including archaea, bacteria, fungi, and protists. Among them, archaea and fungi were found with much stronger DDR slopes than those observed in bacteria and protists. For both TAR and DDR, rare subcommunities were mainly responsible for the observed spatial scaling patterns, except for the DDR of protists and bacteria. This was also evidenced by extending the TAR and DDR diversity metrics to Hill numbers. Further statistical analyses demonstrated that different microbial domains were influenced by different environmental factors and harbored distinct local community assembly processes. Of these, drift was mainly responsible for the compositional variations of bacteria and protists. Archaea were shaped by strong homogeneous selection, whereas fungi were more affected by dispersal limitation. Such differing ecological processes resulted in the domain-level differentiation of microbial spatial scaling. This study links ecological processes with microbial spatial scaling and provides novel mechanistic insights into the diversity patterns of microbes that belong to different trophic levels. IMPORTANCE As the most diverse and numerous life form on Earth, microorganisms play indispensable roles in natural ecological processes. Revealing their diversity patterns across space and through time is of essential importance to better understand the underlying ecological mechanisms controlling the distribution and assembly of microbial communities. However, the diversity patterns and their underlying ecological mechanisms for different microbial domains and/or trophic levels require further exploration. In this study, the spatial scaling of different microbial domains and their associated ecological processes in a mudflat intertidal zone were investigated. The results showed different spatial scaling patterns for different microbial domains. Different ecological processes underlie the domain-level differentiation of microbial spatial scaling. This study links ecological processes with microbial spatial scaling to provide novel mechanistic insights into the diversity patterns of microorganisms that belong to different trophic levels.

Distinct Ecological Processes Mediate Domain-Level Differentiation in Microbial Spatial Scaling

Abstract Internal phosphorus (P) loading from sediments is an important cause of eutrophication, and inorganic-phosphate-solubilizing bacteria (IPB) are known to play a vital role in the process of sediment P release. In this study, 10 IPB strains were isolated from the sediments of a lagoon suffering from macroalgal blooms dominated by Chaetomorpha linum – Rongcheng Swan Lagoon, China – and their taxonomy was determined by 16S rRNA sequence analysis. The phosphate-solubilizing characteristics of the obtained IPB strains were evaluated using a liquid medium, and the ability of IPB strains to dissolve P from sediments and the transformation of inorganic phosphorus (IP) fractions were investigated by inoculating IPB strains into a sediment–water experimental system. The 10 isolated IPB strains belonged to one family, two genera, and four species. All IPB strains in the liquid medium culture dissolved tricalcium phosphate, but the phosphate-solubilizing ability of IPB varied greatly between strains and appeared to be promoted when the strains were grown in mixed-strain cultures. Bacillus megaterium showed the strongest phosphate-solubilizing ability. The IPB inoculation experiment showed a large decrease in the Ca-bound P (HCl-P) concentration of sediments in various IPB inoculation treatments, especially in sediments from the central lake. Our results indicate that, under the action of IPB, there is great potential for the release of HCl-P, which is the primary IP fraction of lagoon sediments, and more attention should be given to the endogenous P pollution in Swan Lagoon Wetland.

Kai Ma

Papers

Methanosaeta harundinacea sp. nov., a novel acetate-scavenging methanogen isolated from a UASB reactor.

Methanobacterium beijingense sp. nov., a novel methanogen isolated from anaerobic digesters.

Detection of the quorum sensing signals in methanogenic archaea

Distinct Ecological Processes Mediate Domain-Level Differentiation in Microbial Spatial Scaling

Isolation of Inorganic-Phosphate-Solubilizing Bacteria and Analysis of their Effect on Phosphorus Release from Lagoon Sediments