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The prototypical representatives of the Euryarchaeota —the methanogens—are oxygen sensitive and are thought to occur only in highly reduced, anoxic environments. However, we found methanogens of the genera Methanosarcina and Methanocella to be present in many types of upland soils (including dryland soils) sampled globally. These methanogens could be readily activated by incubating the soils as slurry under anoxic conditions, as seen by rapid methane production within a few weeks, without any additional carbon source. Analysis of the archaeal 16S ribosomal RNA gene community profile in the incubated samples through terminal restriction fragment length polymorphism and quantification through quantitative PCR indicated dominance of Methanosarcina , whose gene copy numbers also correlated with methane production rates. Analysis of the δ 13 C of the methane further supported this, as the dominant methanogenic pathway was in most cases aceticlastic, which Methanocella cannot perform. Sequences of the key methanogenic enzyme methyl coenzyme M reductase retrieved from the soil samples before incubation confirmed that Methanosarcina and Methanocella are the dominant methanogens, though some sequences of Methanobrevibacter and Methanobacterium were also detected. The global occurrence of only two active methanogenic archaea supports the hypothesis that these are autochthonous members of the upland soil biome and are well adapted to their environment.

Syntrophic oxidization of acetate and propionate are both critical steps of methanogenesis during thermophilic anaerobic digestion. However, knowledge on syntrophic acetate-oxidizing bacteria (SAOB) and syntrophic propionate-oxidizing bacteria (SPOB) is limited because of the difficulty in pure culture isolation due to symbiotic relationship. In this study, two thermophilic acetate-fed anaerobic chemostats, ATL (dilution rate of 0.025 day −1 ) and ATH (0.05 day −1 ) and one thermophilic propionate-fed anaerobic chemostat PTL (0.025 day −1 ) were constructed, AOB and POB in these chemostats were studied via microbial community analysis and DNA stable-isotope probing (SIP). The results showed that, in addition to Tepidanaerobacter , a known SAOB, species of Thauera , Thermodesulfovibrio , Anaerobaculum , Ruminiclostridium , Comamonas , and uncultured bacteria belonging to Lentimicrobiaceae , o_MBA03, Thermoanaerobacteraceae , Anaerolineaceae , Clostridiales , and Ruminococcaceae were determined to be potential AOB in chemostats. Pelotomaculum was the key SPOB detected in the propionate-fed chemostat. Based on the intense fluorescence of coenzyme F 420 , majority of Methanosarcina cells in acetate-fed chemostats were involved in hydrogenotrophic methanogenesis, suggesting the existence of highly active SAOB among the detected AOB. In the propionate-fed chemostat, most of the species detected as AOB were similar to those detected in the acetate-fed chemostats, suggesting the contribution of the syntrophic acetate oxidization pathway for methane generation. These results revealed the existence of previously unknown AOB with high diversity in thermophilic chemostats and suggested that methanogenesis from acetate via the syntrophic oxidization pathway is relevant for thermophilic anaerobic digestion.

Microbially induced corrosion of metallic iron (Fe 0 )-containing structures is an environmental and economic hazard. Methanogens are abundant in low-sulfide environments and yet their specific role in Fe 0 corrosion is poorly understood. In this study, Sporomusa and Methanosarcina  dominated enrichments from Baltic Sea methanogenic sediments that were established with Fe 0 as the sole electron donor and CO 2 as the electron acceptor. The Baltic- Sporomusa was phylogenetically affiliated to the electroactive acetogen S. silvacetica . Baltic- Sporomusa adjusted rapidly to growth on H 2 . On Fe 0 , spent filtrate enhanced growth of this acetogen suggesting that it was using endogenous enzymes to retrieve electrons and produce acetate. Previous studies have proposed that acetate produced by acetogens can feed commensal acetoclastic methanogens such as Methanosarcina . However, Baltic-methanogens could not generate methane from acetate, plus the decrease or absence of acetogens stimulated their growth. The decrease in numbers of Sporomusa was concurrent with an upsurge in Methanosarcina and increased methane production, suggesting that methanogens compete with acetogens for electrons from Fe 0 . Furthermore, Baltic-methanogens were unable to use H 2 (1.5 atm) for methanogenesis and were inhibited by spent filtrate additions, indicating that enzymatically produced H 2 is not a favorable electron donor. We hypothesize that Baltic-methanogens retrieve electrons from Fe 0 via a yet enigmatic direct electron uptake mechanism.

Syntrophic bacteria drive the anaerobic degradation of certain fermentation products (e.g., butyrate, ethanol, propionate) to intermediary substrates (e.g., H 2 , formate, acetate) that yield methane at the ecosystem level. However, little is known about the in situ activities and identities of these syntrophs in peatlands, ecosystems that produce significant quantities of methane. The consumption of butyrate, ethanol or propionate by anoxic peat slurries at 5 and 15 °C yielded methane and CO 2 as the sole accumulating products, indicating that the intermediates H 2 , formate and acetate were scavenged effectively by syntrophic methanogenic consortia. 16S rRNA stable isotope probing identified novel species/strains of Pelobacter and Syntrophomonas that syntrophically oxidized ethanol and butyrate, respectively. Propionate was syntrophically oxidized by novel species of Syntrophobacter and Smithella , genera that use different propionate-oxidizing pathways. Taxa not known for a syntrophic metabolism may have been involved in the oxidation of butyrate ( Telmatospirillum -related) and propionate (unclassified Bacteroidetes and unclassified Fibrobacteres ). Gibbs free energies (Δ G s) for syntrophic oxidations of ethanol and butyrate were more favorable than Δ G s for syntrophic oxidation of propionate. As a result of the thermodynamic constraints, acetate transiently accumulated in ethanol and butyrate treatments but not in propionate treatments. Aceticlastic methanogens ( Methanosarcina , Methanosaeta ) appeared to outnumber hydrogenotrophic methanogens ( Methanocella , Methanoregula ), reinforcing the likely importance of aceticlastic methanogenesis to the overall production of methane. Δ G s for acetogenesis from H 2 to CO 2 approximated to −20 kJ mol −1 when acetate concentrations were low, indicating that acetogens may have contributed to the flow of carbon and reductant towards methane.

Methanogenesis is traditionally thought to occur only in highly reduced, anoxic environments. Wetland and rice field soils are well known sources for atmospheric methane, while aerated soils are considered sinks. Although methanogens have been detected in low numbers in some aerated, and even in desert soils, it remains unclear whether they are active under natural oxic conditions, such as in biological soil crusts (BSCs) of arid regions. To answer this question we carried out a factorial experiment using microcosms under simulated natural conditions. The BSC on top of an arid soil was incubated under moist conditions in all possible combinations of flooding and drainage, light and dark, air and nitrogen headspace. In the light, oxygen was produced by photosynthesis. Methane production was detected in all microcosms, but rates were much lower when oxygen was present. In addition, the δ(13)C of the methane differed between the oxic/oxygenic and anoxic microcosms. While under anoxic conditions methane was mainly produced from acetate, it was almost entirely produced from H(2)/CO(2) under oxic/oxygenic conditions. Only two genera of methanogens were identified in the BSC-Methanosarcina and Methanocella; their abundance and activity in transcribing the mcrA gene (coding for methyl-CoM reductase) was higher under anoxic than oxic/oxygenic conditions, respectively. Both methanogens also actively transcribed the oxygen detoxifying gene catalase. Since methanotrophs were not detectable in the BSC, all the methane produced was released into the atmosphere. Our findings point to a formerly unknown participation of desert soils in the global methane cycle.

Background Mangrove wetlands are critical hotspots of methane emissions, yet the role of naturally occurring minerals in shaping their microbial communities and methanogenic processes is poorly understood. Magnetite, a common iron mineral in soils and sediments, has been reported to enhance aceticlastic methanogenesis and facilitate syntrophic methanogenesis. In this study, we integrated multi-omic profiling with cultivation-based approaches to investigate the impact of magnetite on methanogenesis of microbial consortia derived from mangrove sediments, using lactate as a substrate. Results Across five serial transfers, mangrove microbial consortia converted lactate to propionate and acetate, which were subsequently degraded into methane. Magnetite addition significantly stimulated methane production, leading to notable changes in community structure, particularly for aceticlastic methanogens, with Methanosarcina predominating in the magnetite-amended cultures and Methanothrix in controls. Four Methanosarcina strains T3, T4, T13, and MeOH were subsequently isolated from magnetite-amended cultures. Combined analyses of metagenome-assembled genomes and the genomes of these isolates revealed that the enriched Methanosarcina in magnetite-amended cultures belonged to type II deficient in hydrogenotrophic methanogenesis pathway. Metatranscriptomic analyses suggested that magnetite addition stimulated aceticlastic methanogenesis of type II Methanosarcina and hydrogenotrophic methanogenesis of Methanomicrobiales in the consortia. Furthermore, pure culture experiments confirmed that magnetite stimulated aceticlastic methanogenesis by Methanosarcina sp. T3, although its gene expression patterns differed from those observed in the microbial consortia. Additionally, Methanofastidiosales , an uncultured archaeal lineage possessing H 2 -dependent methylotrophic methanogenesis, was detected in all transfers. Conclusions Our findings demonstrate that magnetite alters methanogenic consortia in mangrove sediments, selectively stimulating aceticlastic methanogenesis of type II Methanosarcina and modulating hydrogenotrophic activity in Methanomicrobiales . By integrating multi-omics analyses with pure culture validation, we demonstrate, for the first time, that magnetite directly enhances the aceticlastic methanogenesis of type II non-hydrogenotrophic Methanosarcina . This study provides new insights into the influence of magnetite on complex microbial consortia, offers a deeper understanding of the physiology of type II non-hydrogenotrophic Methanosarcina , and advances knowledge of mineral-mediated regulation of methanogenic networks in anoxic environments. EY3FCQq_vcoj-4BzkaV9_7 Video Abstract

Although Methanosarcinales are versatile concerning their methanogenic substrates, the ability of Methanosarcina thermophila to use carbon dioxide (CO2) for catabolic and anabolic metabolism was not proven until now. Here, we show that M. thermophila used CO2 to perform hydrogenotrophic methanogenesis in the presence as well as in the absence of methanol. During incubation with hydrogen, the methanogen utilized the substrates methanol and CO2 consecutively, resulting in a biphasic methane production. Growth exclusively from CO2 occurred slowly but reproducibly with concomitant production of biomass, verified by DNA quantification. Besides verification through multiple transfers into fresh medium, the identity of the culture was confirmed by 16s RNA sequencing, and the incorporation of carbon atoms from 13CO2 into 13CH4 molecules was measured to validate the obtained data. New insights into the physiology of M. thermophila can serve as reference for genomic analyses to link genes with metabolic features in uncultured organisms.

Methanogenic archaea are genotypically and phenotypically diverse organisms that are integral to carbon cycling in anaerobic environments. Owing to their genetic tractability and ability to be readily cultivated, Methanosarcina spp. have become a powerful model system for understanding methanogen biology at the cellular systems level. However, relatively little is known of how genotypic and phenotypic variation is partitioned in Methanosarcina populations inhabiting natural environments and the possible ecological and evolutionary implications of such variation. Here, we have identified how genomic and phenotypic diversity is partitioned within and between Methanosarcina mazei populations obtained from two different sediment environments in the Columbia River Estuary (Oregon, USA). Population genomic analysis of 56 M. mazei isolates averaging <1% nucleotide divergence revealed two distinct clades, which we refer to as ‘mazei-T’ and ‘mazei-WC’. Genomic analyses showed that these clades differed in gene content and fixation of allelic variants, which point to potential differences in primary metabolism and also interactions with foreign genetic elements. This hypothesis of niche partitioning was supported by laboratory growth experiments that revealed significant differences in trimethylamine utilization. These findings improve our understanding of the ecologically relevant scales of genomic variation in natural systems and demonstrate interactions between genetic and ecological diversity in these easily cultivable and genetically tractable model methanogens.

A better understand the ecology of microbes and their role in the global ecosystem could be achieved if traditional ecological theories can be applied to microbes. In ecology organisms are defined as specialists or generalists according to the breadth of their niche. Spatial distribution is often used as a proxy measure of niche breadth; generalists have broad niches and a wide spatial distribution and specialists a narrow niche and spatial distribution. Previous studies suggest that microbial distribution patterns are contrary to this idea; a microbial generalist genus (Desulfobulbus) has a limited spatial distribution while a specialist genus (Methanosaeta) has a cosmopolitan distribution. Therefore, we hypothesise that this counter-intuitive distribution within generalist and specialist microbial genera is a common microbial characteristic. Using molecular fingerprinting the distribution of four microbial genera, two generalists, Desulfobulbus and the methanogenic archaea Methanosarcina , and two specialists, Methanosaeta and the sulfate-reducing bacteria Desulfobacter were analysed in sediment samples from along a UK estuary. Detected genotypes of both generalist genera showed a distinct spatial distribution, significantly correlated with geographic distance between sites. Genotypes of both specialist genera showed no significant differential spatial distribution. These data support the hypothesis that the spatial distribution of specialist and generalist microbes does not match that seen with specialist and generalist large organisms. It may be that generalist microbes, while having a wider potential niche, are constrained, possibly by intrageneric competition, to exploit only a small part of that potential niche while specialists, with far fewer constraints to their niche, are more capable of filling their potential niche more effectively, perhaps by avoiding intrageneric competition. We suggest that these counter-intuitive distribution patterns may be a common feature of microbes in general and represent a distinct microbial principle in ecology, which is a real challenge if we are to develop a truly inclusive ecology.

Degradation of biomass in the absence of exogenous electron acceptors via anaerobic digestion involves a syntrophic association of a plethora of anaerobic microorganisms. The commercial application of this process is the large-scale production of biogas from renewable feedstock as an alternative to fossil fuels. After hydrolysis of polymers, monomers are fermented to short-chain fatty acids and alcohols, which are further oxidized to acetate. Carbon dioxide, molecular hydrogen (H 2 ), and acetate generated during the process are converted to methane by methanogenic archaea. Since many of the metabolic pathways as well as the syntrophic interactions and dependencies during anaerobic digestion involve formation, utilization, or transfer of H 2 , its metabolism and the methanogenic population were assessed in various samples from three commercial biogas plants. Addition of H 2 significantly increased the rate of methane formation, which suggested that hydrogenotrophic methanogenesis is not a rate-limiting step during biogas formation. Methanoculleus and Methanosarcina appeared to numerically dominate the archaeal population of the three digesters, but their proportion and the Bacteria -to- Archaea ratio did not correlate with the methane productivity. Instead, hydrogenase activity in cell-free extracts from digester sludge correlated with methane productivity in a positive fashion. Since most microorganisms involved in biogas formation contain this activity, it approximates the overall anaerobic metabolic activity and may, thus, be suitable for monitoring biogas reactor performance.