Complexity of temperature dependence in methanogenic microbial environments.

There is virtually no environmental process that is not dependent on temperature. This includes the microbial processes that result in the production of CH4, an important greenhouse gas. Microbial CH4 production is the result of a combination of many different microorganisms and microbial processes, which together achieve the mineralization of organic matter to CO2 and CH4. Temperature dependence applies to each individual step and each individual microbe. This review will discuss the different aspects of temperature dependence including temperature affecting the kinetics and thermodynamics of the various microbial processes, affecting the pathways of organic matter degradation and CH4 production, and affecting the composition of the microbial communities involved. For example, it was found that increasing temperature results in a change of the methanogenic pathway with increasing contribution from mainly acetate to mainly H2/CO2 as immediate CH4 precursor, and with replacement of aceticlastic methanogenic archaea by thermophilic syntrophic acetate-oxidizing bacteria plus thermophilic hydrogenotrophic methanogenic archaea. This shift is consistent with reaction energetics, but it is not obligatory, since high temperature environments exist in which acetate is consumed by thermophilic aceticlastic archaea. Many studies have shown that CH4 production rates increase with temperature displaying a temperature optimum and a characteristic apparent activation energy (Ea). Interestingly, CH4 release from defined microbial cultures, from environmental samples and from wetland field sites all show similar Ea values around 100 kJ mol-1 indicating that CH4 production rates are limited by the methanogenic archaea rather than by hydrolysis of organic matter. Hence, the final rather than the initial step controls the methanogenic degradation of organic matter, which apparently is rarely in steady state.

Winter drainage and film mulching cultivation mitigated CH4 emission by regulating the function and structure of methanogenic archaeal and fermenting bacterial communities in paddy soil.

Winter flooding of harvested rice fields is a typical cropping system in mountainous areas, which emits considerable amounts of CH4. Plastic film mulching cultivation is recognized as an important rice cultivation practice in paddy field for water-saving irrigation. However, the effects of these managements on CH4 emissions in paddy soil and the underlying microbial mechanism are unclear. A field experiment was carried out with the application of winter drainage followed by traditional rice cultivation (WD), winter drainage followed by plastic film mulching cultivation (MC), as well as winter flooding followed by traditional rice cultivation (WF) as control in hilly paddy fields. We investigated the CH4 emissions, functional (CH4 production rate, 13C isotope) and structural (abundance, structure) responses of soil methanogenic archaeal and fermenting bacterial communities during rice season. Shifting the fields from WF into WD and MC substantially mitigated CH4 emissions by 62.3% and 59.2%, respectively, paralleled with the enhancement of soil Eh and the reductions of soil DOC content. Compared with WF, WD and MC both significantly decreased CH4 production rates and the copy numbers of mcrA gene. Moreover, an increasing contribution of hydrogenotrophic methanogenesis (from 30.7% to 50.0%) to total CH4 production was observed during the conversion from WF to MC under an anaerobic incubation, paralleled with the decreased acetate content and increased δ13C values of acetate-methyl and total acetate. The communities of methanogenic archaea and fermenting bacteria strongly responded to the shift from WF to WD, while MC only showed significant effects on the methanogenic archaeal communities. Compared with WF, WD and MC significantly increased the relative abundance of Methanothrix, Methanosarcina and Methanocella, while those of Methanoregula, Massilia and Geobacter were decreased. The co-occurrence networks showed that WD and MC induced the loss of mixed methanogenic fermentation modules, indicating the decrease in functional biodiversity and redundancy of fermenting bacterial and methanogenic archaeal communities.The findings suggest that WD and MC approach mitigate CH4 emission by regulating the function and structure of methanogenic archaeal and fermenting bacterial communities in paddy soil, which represent the effective management strategies considering the water availability and CH4 mitigation in paddy-field agriculture.

Reconstructing Genomes of Carbon Monoxide Oxidisers in Volcanic Deposits Including Members of the Class Ktedonobacteria.

Microorganisms can potentially colonise volcanic rocks using the chemical energy in reduced gases such as methane, hydrogen (H2) and carbon monoxide (CO). In this study, we analysed soil metagenomes from Chilean volcanic soils, representing three different successional stages with ages of 380, 269 and 63 years, respectively. A total of 19 metagenome-assembled genomes (MAGs) were retrieved from all stages with a higher number observed in the youngest soil (1640: 2 MAGs, 1751: 1 MAG, 1957: 16 MAGs). Genomic similarity indices showed that several MAGs had amino-acid identity (AAI) values >50% to the phyla Actinobacteria, Acidobacteria, Gemmatimonadetes, Proteobacteria and Chloroflexi. Three MAGs from the youngest site (1957) belonged to the class Ktedonobacteria (Chloroflexi). Complete cellular functions of all the MAGs were characterised, including carbon fixation, terpenoid backbone biosynthesis, formate oxidation and CO oxidation. All 19 environmental genomes contained at least one gene encoding a putative carbon monoxide dehydrogenase (CODH). Three MAGs had form I coxL operon (encoding the large subunit CO-dehydrogenase). One of these MAGs (MAG-1957-2.1, Ktedonobacterales) was highly abundant in the youngest soil. MAG-1957-2.1 also contained genes encoding a [NiFe]-hydrogenase and hyp genes encoding accessory enzymes and proteins. Little is known about the Ktedonobacterales through cultivated isolates, but some species can utilise H2 and CO for growth. Our results strongly suggest that the remote volcanic sites in Chile represent a natural habitat for Ktedonobacteria and they may use reduced gases for growth.

Methane Production in Soil Environments-Anaerobic Biogeochemistry and Microbial Life between Flooding and Desiccation.

Flooding and desiccation of soil environments mainly affect the availability of water and oxygen. While water is necessary for all life, oxygen is required for aerobic microorganisms. In the absence of O2, anaerobic processes such as CH4 production prevail. There is a substantial theoretical knowledge of the biogeochemistry and microbiology of processes in the absence of O2. Noteworthy are processes involved in the sequential degradation of organic matter coupled with the sequential reduction of electron acceptors, and, finally, the formation of CH4. These processes follow basic thermodynamic and kinetic principles, but also require the presence of microorganisms as catalysts. Meanwhile, there is a lot of empirical data that combines the observation of process function with the structure of microbial communities. While most of these observations confirmed existing theoretical knowledge, some resulted in new information. One important example was the observation that methanogens, which have been believed to be strictly anaerobic, can tolerate O2 to quite some extent and thus survive desiccation of flooded soil environments amazingly well. Another example is the strong indication of the importance of redox-active soil organic carbon compounds, which may affect the rates and pathways of CH4 production. It is noteworthy that drainage and aeration turns flooded soils, not generally, into sinks for atmospheric CH4, probably due to the peculiarities of the resident methanotrophic bacteria.

Competition Between Chemolithotrophic Acetogenesis and Hydrogenotrophic Methanogenesis for Exogenous H2/CO2 in Anaerobically Digested Sludge: Impact of Temperature.

Anaerobic digestion is a widely applied technology for sewage sludge treatment. Hydrogen and CO2 are important degradation products, which serve as substrates for both hydrogenotrophic methanogenesis and chemolithotrophic acetogenesis. In order to understand the competition between these processes for H2/CO2, sludge samples were incubated under H2/CO2 headspace at different temperatures, and analyzed with respect to turnover of H2, CO2, CH4 and acetate including their δ13C values. At 15°C, 13C-depleted acetate (δ13C of -41 to -43‰) and transient acetate accumulation were observed under H2/CO2, and CH4 accumulated with δ13C values increasing from -53 to -33‰. The copy numbers of the fhs gene, which is characteristic for acetogenic bacteria, were at 15°C one order of magnitude higher in the H2/CO2 incubations than the N2 control. At 30°C, however, acetate did not accumulate in the H2/CO2 incubation and the δ13C of CH4 was very low (-100 to -77‰). At 50°C, isotopically enriched acetate was transiently formed and subsequently consumed followed by the production of 13C-depleted CH4. Collectively, the results indicate a high contribution of chemolithotrophic acetogenesis to H2/CO2 utilization at 15°C and 50°C, while H2/CO2 was mainly consumed by hydrogenotrophic methanogenesis at 30°C. Fermentative production and methanogenic consumption of acetate were active at 50°C.

Temperature-Dependent Network Modules of Soil Methanogenic Bacterial and Archaeal Communities.

Temperature is an important factor regulating the production of the greenhouse gas CH4. Structure and function of the methanogenic microbial communities are often drastically different upon incubation at 45°C versus 25°C or 35°C, but are also different in different soils. However, the extent of taxonomic redundancy within each functional group and the existence of different temperature-dependent microbial community network modules are unknown. Therefore, we investigated paddy soils from Italy and the Philippines and a desert soil from Utah (United States), which all expressed CH4 production upon flooding and exhibited structural and functional differences upon incubation at three different temperatures. We continued incubation of the pre-incubated soils (Liu et al., 2018) by changing the temperature in a factorial manner. We determined composition, abundance and function of the methanogenic archaeal and bacterial communities using HiSeq Illumina sequencing, qPCR and analysis of activity and stable isotope fractionation, respectively. Heatmap analysis of operational taxonomic units (OTU) from the different incubations gave detailed insights into the community structures and their putative functions. Network analysis showed that the microbial communities in the different soils were all organized within modules distinct for the three incubation temperatures. The diversity of Bacteria and Archaea was always lower at 45°C than at 25 or 35°C. A shift from 45°C to lower temperatures did not recover archaeal diversity, but nevertheless resulted in the establishment of structures and functions that were largely typical for soil at moderate temperatures. At 25 and 35°C and after shifting to one of these temperatures, CH4 was always produced by a combination of acetoclastic and hydrogenotrophic methanogenesis being consistent with the presence of acetoclastic (Methanosarcinaceae, Methanotrichaceae) and hydrogenotrophic (Methanobacteriales, Methanocellales, Methanosarcinaceae) methanogens. At 45°C, however, or after shifting from moderate temperatures to 45°C, only the Philippines soil maintained such combination, while the other soils were devoid of acetoclastic methanogens and consumed acetate instead by syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis. Syntrophic acetate oxidation was apparently achieved by Thermoanaerobacteraceae, which were especially abundant in Italian paddy soil and Utah desert soil when incubated at 45°C. Other bacterial taxa were also differently abundant at 45°C versus moderate temperatures, as seen by the formation of specific network modules. However, the archaeal OTUs with putative function in acetoclastic or hydrogenotrophic methanogenesis as well as the bacterial OTUs were usually not identical across the different soils and incubation conditions, and if they were, they suggested the existence of mesophilic and thermophilic ecotypes within the same OTUs. Overall, methanogenic function was determined by the bacterial and/or archaeal community structures, which in turn were to quite some extent determined by the incubation temperature, albeit largely individually in each soil. There was quite some functional redundancy as seen by different taxonomic community structures in the different soils and at the different temperatures.

Upland Soil Cluster Gamma dominates methanotrophic communities in upland grassland soils.

Aerobic methanotrophs in upland soils consume atmospheric methane, serving as a critical counterbalance to global warming; however, the biogeographic distribution patterns of their abundance and community composition are poorly understood, especial at a large scale. In this study, soils were sampled from 30 grasslands across >2000 km on the Qinghai-Tibetan Plateau to determine the distribution patterns of methanotrophs and their driving factors at a regional scale. Methanotroph abundance and community composition were analyzed using quantitative PCR and Illumina Miseq sequencing of pmoA genes, respectively. The pmoA gene copies ranged from 8.2 × 105 to 1.1 × 108 per gram dry soil. Among the 30 grassland soil samples, Upland Soil Cluster Gamma (USCγ) dominated the methanotroph communities in 26 samples. Jasper Ridge Cluster (JR3) was the most dominant methanotrophic cluster in two samples; while Methylocystis, cluster FWs, and Methylobacter were abundant in other two wet soil samples. Interestingly, reanalyzing the pmoA genes sequencing data from existing publications suggested that USCγ was also the main methanotrophic cluster in grassland soils in other regions, especially when their mean annual precipitation was <500 mm. Canonical Analysis of Principal Coordinates including all soil samples indicated that the methanotrophic community composition was significantly correlated with local environmental factors, among which mean annual precipitation and pH showed the strongest correlations. Variance partitioning analysis showed that environmental factors and spatial distance were significant factors affecting the community structure of methanotrophs, and environmental properties were more important factors. Collectively, these findings indicate that atmospheric methane may be mainly oxidized by USCγ in upland soils. They also highlight the key role of water availability and pH in determining the abundance and community profiles of grassland soil methanotrophs.

Structure, function and resilience to desiccation of methanogenic microbial communities in temporarily inundated soils of the Amazon rainforest (Cunia Reserve, Rondonia).

The floodplain of the Amazon River is a large source for the greenhouse gas methane, but the soil microbial communities and processes involved are little known. We studied the structure and function of the methanogenic microbial communities in soils across different inundation regimes in the Cunia Reserve, encompassing nonflooded forest soil (dry forest), occasionally flooded Igapo soils (dry Igapo), long time flooded Igapo soils (wet Igapo) and sediments from Igarape streams (Igarape). We also investigated a Transect (four sites) from the water shoreline into the dry forest. The potential and resilience of the CH4 production process were studied in the original soil samples upon anaerobic incubation and again after artificial desiccation and rewetting. Bacterial and archaeal 16S rRNA genes and methanogenic mcrA were always present in the soils, except in dry forest soils where mcrA increased only upon anaerobic incubation. NMDS analysis showed a clear effect of desiccation and rewetting treatments on both bacterial and archaeal communities. However, the effects of the different sites were less pronounced, with the exception of Igarape. After anaerobic incubation, methanogenic taxa became more abundant among the Archaea, while there was only little change among the Bacteria. Contribution of hydrogenotrophic methanogenesis was usually around 40%. After desiccation and rewetting, we found that Firmicutes, Methanocellales and Methanosarcinaceae became the dominant taxa, but rates and pathways of CH4 production stayed similar. Such change was also observed in soils from the Transects. The results indicate that microbial community structures of Amazonian soils will in general be strongly affected by flooding and drainage events, while differences between specific field sites will be comparatively minor.

Fermentative Spirochaetes mediate necromass recycling in anoxic hydrocarbon-contaminated habitats.

Spirochaetes are frequently detected in anoxic hydrocarbon- and organohalide-polluted groundwater, but their role in such ecosystems has remained unclear. To address this, we studied a sulfate-reducing, naphthalene-degrading enrichment culture, mainly comprising the sulfate reducer Desulfobacterium N47 and the rod-shaped Spirochete Rectinema cohabitans HM. Genome sequencing and proteome analysis suggested that the Spirochete is an obligate fermenter that catabolizes proteins and carbohydrates, resulting in acetate, ethanol, and molecular hydrogen (H2) production. Physiological experiments inferred that hydrogen is an important link between the two bacteria in the enrichment culture, with H2 derived from fermentation by R. cohabitans used as reductant for sulfate reduction by Desulfobacterium N47. Differential proteomics and physiological experiments showed that R. cohabitans utilizes biomass (proteins and carbohydrates) released from dead cells of Desulfobacterium N47. Further comparative and community genome analyses indicated that other Rectinema phylotypes are widespread in contaminated environments and may perform a hydrogenogenic fermentative lifestyle similar to R. cohabitans. Together, these findings indicate that environmental Spirochaetes scavenge detrital biomass and in turn drive necromass recycling at anoxic hydrocarbon-contaminated sites and potentially other habitats.

Hydrogenotrophic methanogenesis is the dominant methanogenic pathway in neotropical tank bromeliad wetlands.

Several thousands of tank bromeliads per hectare of neotropical forest create a unique wetland ecosystem that emits substantial amounts of CH4 . Tank bromeliads growing in the forest canopy (functional type-II tank bromeliads) were found to emit more CH4 than tank bromeliads growing on the forest floor (functional type-I tank bromeliads) but the reasons for this difference and the underlying microbial CH4 -cycling processes have not been studied. Therefore, we characterized archaeal communities in bromeliad tanks of the two different functional types in a neotropical montane forest of southern Ecuador using terminal-restriction fragment length polymorphism (T-RFLP) and performed tank-slurry incubations to measure CH4 production potential, stable carbon isotope fractionation and pathway of CH4 formation. The archaeal community composition was dominated by methanogens and differed between bromeliad functional types. Hydrogenotrophic Methanomicrobiales were the dominant methanogens and hydrogenotrophic methanogenesis was the dominant methanogenic pathway among all bromeliads. The relative abundance of aceticlastic Methanosaetaceae and the relative contribution of aceticlastic methanogenesis increased in type-I tank bromeliads probably due to more oxic conditions in type-I than in type-II bromeliads leading to the previously observed lower in situ CH4 emissions from type-I tank bromeliads but to higher CH4 production potentials in type-I tank bromeliad slurries.

Identification of Syntrophobacteraceae as major acetate-degrading sulfate reducing bacteria in Italian paddy soil.

Methane is an important greenhouse gas and acetate is the most important intermediate (average 70%) of the carbon flow to CH4 in paddy fields. Sulfate (e.g., gypsum) application can reduce CH4 emissions up to 70%. However, the effect of gypsum application on acetate degradation and the microbial communities involved are unclear. Therefore, we studied acetate-dependent sulfate reduction in anoxic microcosms of Italian rice paddy soil, combining profiling of 16S rRNA and dissimilatory sulfite reductase (dsrB) genes and transcripts and rRNA based stable isotope probing (SIP) analysis. Methane production was completely inhibited by gypsum in the absence of exogenous acetate. Amended acetate (either 13 C labelled or non-labelled) was stoichiometrically coupled to sulfate reduction or CH4 production. With methyl fluoride in the presence of sulfate, added propionate and butyrate were incompletely oxidized to acetate, which transiently accumulated. After the depletion of propionate and butyrate the accumulated acetate was rapidly consumed. The relative abundance of dsrB and 16S rRNA genes and transcripts from Syntrophobacteraceae (Desulfovirga spp., Syntrophobacter spp. and unclassified Syntrophobacteraceae) increased upon addition of gypsum and acetate. Simultaneously, Syntrophobacteraceae affiliated species were significantly labelled with 13 C. In addition, minor groups like Desulforhabdus spp., Desulfobacca spp. and Desulfotomaculum spp. substantially incorporated 13 C into their nucleic acids. The relative abundance of Desulfovibrio spp. slightly increased upon gypsum amendments. However, 13 C labelling of Desulfovibrio spp. was only moderate. In summary, Syntrophobacteraceae affiliated species were identified as the major acetotrophic sulfate reducers (SRB) in Italian paddy soil. The identification of these SRB as dominant acetate degraders well explained the scenarios of competition between SRB and acetoclastic methanogens as observed in rice paddy soil.

Extensive processing of sediment pore water dissolved organic matter during anoxic incubation as observed by high-field mass spectrometry (FTICR-MS).

Dissolved organic matter (DOM) contained in lake sediments is a carbon source for many microbial degradation processes, including aerobic and anaerobic mineralization. During anaerobic degradation, DOM is partially consumed and transformed into new molecules while the greenhouse gases methane (CH4) and carbon dioxide (CO2) are produced. In this study, we used ultrahigh resolution mass spectrometry to trace differences in the composition of solid-phase extractable (PPL resin) pore water DOM (SPE-DOM) isolated from surface sediments of three boreal lakes before and after 40 days of anoxic incubation, with concomitant determination of CH4 and CO2 evolution. CH4 and CO2 production detected by gas chromatography varied considerably among replicates and accounted for fractions of ∼2-4 × 10-4 of sedimentary organic carbon for CO2 and ∼0.8-2.4 × 10-5 for CH4. In contrast, the relative changes of key bulk parameters during incubation, such as relative proportions of molecular series, elemental ratios, average mass and unsaturation, were regularly in the percent range (1-3% for compounds decreasing and 4-10% for compounds increasing), i.e. several orders of magnitude higher than mineralization alone. Computation of the average carbon oxidation state in CHO molecules of lake pore water DOM revealed rather non-selective large scale transformations of organic matter during incubation, with depletion of highly oxidized and highly reduced CHO molecules, and formation of rather non-labile fulvic acid type molecules. In general, proportions of CHO compounds slightly decreased. Nearly saturated CHO and CHOS lipid-like substances declined during incubation: these rather commonplace molecules were less specific indicators of lake sediment alteration than the particular compounds, such as certain oxygenated aromatics and carboxyl-rich alicyclic acids (CRAM) found more abundant after incubation. There was a remarkable general increase in many CHNO compounds during incubation across all lakes. Differences in DOM transformation between lakes corresponded with lake size and water residence time. While in the small lake Svarttjärn, CRAM increased during incubation, lignin-and tannin-like compounds were enriched in the large lake Bisen, suggesting selective preservation of these rather non-labile aromatic compounds rather than recent synthesis. SPE-DOM after incubation may represent freshly synthesized compounds, leftover bulk DOM which is primarily composed of intrinsically refractory molecules and/or microbial metabolites which were not consumed in our experiments. In spite of a low fraction of the total DOM being mineralized to CO2 and CH4, the more pronounced change in molecular DOM composition during the incubation indicates that diagenetic modification of organic matter can be substantial compared to complete mineralization.

Determination of Resistant Starch Assimilating Bacteria in Fecal Samples of Mice by In vitro RNA-Based Stable Isotope Probing.

The impact of the intestinal microbiota on human health is becoming increasingly appreciated in recent years. In consequence, and fueled by major technological advances, the composition of the intestinal microbiota in health and disease has been intensively studied by high throughput sequencing approaches. Observations linking dysbiosis of the intestinal microbiota with a number of serious medical conditions including chronic inflammatory disorders and allergic diseases suggest that restoration of the composition and activity of the intestinal microbiota may be a treatment option at least for some of these diseases. One possibility to shape the intestinal microbiota is the administration of prebiotic carbohydrates such as resistant starch (RS). In the present study, we aim at establishing RNA-based stable isotope probing (RNA-SIP) to identify bacterial populations that are involved in the assimilation of RS using anaerobic in vitro fermentation of murine fecal material with stable [U13C] isotope-labeled potato starch. Total RNA from these incubations was extracted, processed by gradient ultracentrifugation and fractionated by density. 16S rRNA gene sequences were amplified from reverse transcribed RNA of high and low density fractions suspected to contain labeled and unlabeled RNA, respectively. Phylogenetic analysis of the obtained sequences revealed a distinct subset of the intestinal microbiota involved in starch metabolism. The results suggest Bacteroidetes, in particular genera affiliated with Prevotellaceae, as well as members of the Ruminococcacea family to be primary assimilators of resistant starch due to a significantly higher relative abundance in higher density fractions in RNA samples isolated after 2 h of incubation. Using high performance liquid chromatography coupled to isotope ratio mass spectrometry (HPLC-IRMS) analysis, some stable isotope label was recovered from acetate, propionate and butyrate. Here, we demonstrate the suitability of RNA-SIP to link specific groups of microorganisms with fermentation of a specific substrate. The application of RNA-SIP in future in vivo studies will help to better understand the mechanisms behind functionality of a prebiotic carbohydrate and its impact on an intestinal ecosystem with potential implications for human health.

Methane emission from feather moss stands.

Data from remote sensing and Eddy towers indicate that forests are not always net sinks for atmospheric CH4 . However, studies describing specific sources within forests and functional analysis of microorganisms on sites with CH4 turnover are scarce. Feather moss stands were considered to be net sinks for carbon dioxide, but received little attention to their role in CH4 cycling. Therefore, we investigated methanogenic rates and pathways together with the methanogenic microbial community composition in feather moss stands from temperate and boreal forests. Potential rates of CH4 emission from intact moss stands (n = 60) under aerobic conditions ranged between 19 and 133 pmol CH4 h-1 gdw-1 . Temperature and water content positively influenced CH4 emission. Methanogenic potentials determined under N2 atmosphere in darkness ranged between 22 and 157 pmol CH4 h-1 gdw-1 . Methane production was strongly inhibited by bromoethane sulfonate or chloroform, showing that CH4 was of microbial origin. The moss samples tested contained fluorescent microbial cells and between 104 and 105 copies per gram dry weight moss of the mcrA gene coding for a subunit of the methyl CoM reductase. Archaeal 16S rRNA and mcrA gene sequences in the moss stands were characteristic for the archaeal families Methanobacteriaceae and Methanosarcinaceae. The potential methanogenic rates were similar in incubations with and without methyl fluoride, indicating that the CH4 was produced by the hydrogenotrophic rather than aceticlastic pathway. Consistently, the CH4 produced was depleted in 13 C in comparison with the moss biomass carbon and acetate accumulated to rather high concentrations (3-62 mM). The δ13 C of acetate was similar to that of the moss biomass, indicating acetate production by fermentation. Our study showed that the feather moss stands contained active methanogenic microbial communities producing CH4 by hydrogenotrophic methanogenesis and causing net emission of CH4 under ambient conditions, albeit at low rates.

Response of Methanogenic Microbial Communities to Desiccation Stress in Flooded and Rain-Fed Paddy Soil from Thailand.

Rice paddies in central Thailand are flooded either by irrigation (irrigated rice) or by rain (rain-fed rice). The paddy soils and their microbial communities thus experience permanent or arbitrary submergence, respectively. Since methane production depends on anaerobic conditions, we hypothesized that structure and function of the methanogenic microbial communities are different in irrigated and rain-fed paddies and react differently upon desiccation stress. We determined rates and relative proportions of hydrogenotrophic and aceticlastic methanogenesis before and after short-term drying of soil samples from replicate fields. The methanogenic pathway was determined by analyzing concentrations and δ13C of organic carbon and of CH4 and CO2 produced in the presence and absence of methyl fluoride, an inhibitor of aceticlastic methanogenesis. We also determined the abundance (qPCR) of genes and transcripts of bacterial 16S rRNA, archaeal 16S rRNA and methanogenic mcrA (coding for a subunit of the methyl coenzyme M reductase) and the composition of these microbial communities by T-RFLP fingerprinting and/or Illumina deep sequencing. The abundances of genes and transcripts were similar in irrigated and rain-fed paddy soil. They also did not change much upon desiccation and rewetting, except the transcripts of mcrA, which increased by more than two orders of magnitude. In parallel, rates of CH4 production also increased, in rain-fed soil more than in irrigated soil. The contribution of hydrogenotrophic methanogenesis increased in rain-fed soil and became similar to that in irrigated soil. However, the relative microbial community composition on higher taxonomic levels was similar between irrigated and rain-fed soil. On the other hand, desiccation and subsequent anaerobic reincubation resulted in systematic changes in the composition of microbial communities for both Archaea and Bacteria. It is noteworthy that differences in the community composition were mostly detected on the level of operational taxonomic units (OTUs; 97% sequence similarity). The treatments resulted in change of the relative abundance of several archaeal OTUs. Some OTUs of Methanobacterium, Methanosaeta, Methanosarcina, Methanocella and Methanomassiliicoccus increased, while some of Methanolinea and Methanosaeta decreased. Bacterial OTUs within Firmicutes, Cyanobacteria, Planctomycetes and Deltaproteobacteria increased, while OTUs within other proteobacterial classes decreased.

RNA-Based Stable Isotope Probing Suggests Allobaculum spp. as Particularly Active Glucose Assimilators in a Complex Murine Microbiota Cultured In Vitro.

RNA-based stable isotope probing (RNA-SIP) and metabolic profiling were used to detect actively glucose-consuming bacteria in a complex microbial community obtained from a murine model system. A faeces-derived microbiota was incubated under anaerobic conditions for 0, 2, and 4 h with 40 mM [U13C]glucose. Isopycnic density gradient ultracentrifugation and fractionation of isolated RNA into labeled and unlabeled fractions followed by 16S rRNA sequencing showed a quick adaptation of the bacterial community in response to the added sugar, which was dominated by unclassified Lachnospiraceae species. Inspection of distinct fractions of isotope-labeled RNA revealed Allobaculum spp. as particularly active glucose utilizers in the system, as the corresponding RNA showed significantly higher proportions among the labeled RNA. With time, the labeled sugar was used by a wider spectrum of faecal bacteria. Metabolic profiling indicated rapid fermentation of [U13C]glucose, with lactate, acetate, and propionate being the principal 13C-labeled fermentation products, and suggested that "cross-feeding" occurred in the system. RNA-SIP combined with metabolic profiling of 13C-labeled products allowed insights into the microbial assimilation of a general model substrate, demonstrating the appropriateness of this technology to study assimilation processes of nutritionally more relevant substrates, for example, prebiotic carbohydrates, in the gut microbiota of mice as a model system.

Syntrophobacteraceae-affiliated species are major propionate-degrading sulfate reducers in paddy soil.

Methane is an important greenhouse gas and propionate is next to acetate the main intermediate (average 23%) of the carbon flow to CH4 in paddy fields. Sulfate (e.g., gypsum) application can reduce CH4 emissions up to 70%. However, the effect of gypsum application on propionate degradation and the microbial communities involved are not well understood. Therefore, we studied propionate-dependent sulfate reduction in anoxic microcosms of paddy soils from Italy and the Philippines, combining 16S rRNA and dissimilatory sulfite reductase (dsrB) gene profiling and co-occurrence network analysis. Sulfate was stoichiometrically reduced in treatments with propionate addition, while CH4 production was partially suppressed. Methane production but not sulfate reduction were suppressed and acetate accumulated after addition of methyl fluoride or fluoroacetate. With methyl fluoride in the presence of sulfate, the accumulated acetate was consumed after the depletion of propionate. Simultaneously, the relative abundances of Syntrophobacteraceae and Desulfovibrionaceae were significantly enhanced, while fluoroacetate repressed Desulfobulbaceae in both soils. Syntrophobacter 16S rRNA and dsrB gene copy numbers were also remarkably increased with gypsum amendment. Network analysis of both 16S rRNA and dsrB genes illustrated a strong co-occurrence of operational taxonomic units belonging to Syntrophobacteraceae, Desulfovibrionaceae and Desulfobulbaceae. In summary, Syntrophobacteraceae affiliated species were identified as the major propionate-dependent sulfate reducers in paddy soil. They (together with Desulfobulbaceae) oxidized propionate directly to acetate and CO2 , or coupled the oxidation syntrophically to H2 /formate-utilizing Desulfovibrionaceae. The transiently accumulating acetate was preferentially consumed by acetoclastic Methanosarcinaceae.

Salinity Affects the Composition of the Aerobic Methanotroph Community in Alkaline Lake Sediments from the Tibetan Plateau.

Lakes are widely distributed on the Tibetan Plateau, which plays an important role in natural methane emission. Aerobic methanotrophs in lake sediments reduce the amount of methane released into the atmosphere. However, no study to date has analyzed the methanotroph community composition and their driving factors in sediments of these high-altitude lakes (>4000 m). To provide new insights on this aspect, the abundance and composition in the sediments of six high-altitude alkaline lakes (including both freshwater and saline lakes) on the Tibetan Plateau were studied. The quantitative PCR, terminal restriction fragment length polymorphism, and 454-pyrosequencing methods were used to target the pmoA genes. The pmoA gene copies ranged 104-106 per gram fresh sediment. Type I methanotrophs predominated in Tibetan lake sediments, with Methylobacter and uncultivated type Ib methanotrophs being dominant in freshwater lakes and Methylomicrobium in saline lakes. Combining the pmoA-pyrosequencing data from Tibetan lakes with other published pmoA-sequencing data from lake sediments of other regions, a significant salinity and alkalinity effect (P = 0.001) was detected, especially salinity, which explained ∼25% of methanotroph community variability. The main effect was Methylomicrobium being dominant (up to 100%) in saline lakes only. In freshwater lakes, however, methanotroph composition was relatively diverse, including Methylobacter, Methylocystis, and uncultured type Ib clusters. This study provides the first methanotroph data for high-altitude lake sediments (>4000 m) and shows that salinity is a driving factor for the community composition of aerobic methanotrophs.

Stable carbon isotope fractionation as tracer of carbon cycling in anoxic soil ecosystems.

While the structure of microbial communities can nowadays be determined by applying molecular analytical tools to soil samples, microbial function can usually only be determined by physiological experiments requiring incubation of samples. However, analysis of stable isotope fractionation might be able to analyse microbial function without incubation in soil samples. We describe the limitations of diagnosing and quantifying carbon flux pathways in soil by using the determination of stable carbon isotope composition in soil compounds and emphasize the importance of determining stable isotope fractionation factors for defined biochemical pathways. Fractionation factors are sufficiently different for some central biochemical pathways in anaerobic degradation of organic carbon. Thus, it is possible to quantify the relative contribution of CH4 production by hydrogenotrophic or aceticlastic methanogenic pathways, and of acetate formation by chemolithotrophic (acetyl-CoA synthase) or heterotrophic (fermentation) pathways. In addition, stable isotope analysis may allow the differentiation between different organic substrates used for degradation, for example, the relative contribution of root exudation versus soil organic matter degradation, provided the different substrates are sufficiently distinct in their isotopic compositions (e.g., mixture of C3 and C4 plants) and the carbon conversion pathways display only small fractionation factors or are identical for the different substrates.

Structure and function of methanogenic microbial communities in sediments of Amazonian lakes with different water types.

Tropical lake sediments are a significant source for the greenhouse gas methane. We studied function (pathway, rate) and structure (abundance, taxonomic composition) of the microbial communities (Bacteria, Archaea) leading to methane formation together with the main physicochemical characteristics in the sediments of four clear water, six white water and three black water lakes of the Amazon River system. Concentrations of sulfate and ferric iron, pH and δ13 C of organic carbon were usually higher, while concentrations of carbon, nitrogen and rates of CH4 production were generally lower in white water versus clear water or black water sediments. Copy numbers of bacterial and especially archaeal ribosomal RNA genes also tended to be relatively lower in white water sediments. Hydrogenotrophic methanogenesis contributed 58 ± 16% to total CH4 production in all systems. Network analysis identified six communities, of which four were comprised mostly of bacteria found in all sediment types, while two were mostly in clear water sediment. Terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing showed that the compositions of the communities differed between the different sediment systems, statistically related to the particular physicochemical conditions and to CH4 production rates. Among the archaea, clear water, white water, and black water sediments contained relatively more Methanomicrobiales, Methanosarcinaceae and Methanocellales, respectively, while Methanosaetaceae were common in all systems. Proteobacteria, Deltaproteobacteria (Myxococcales, Syntrophobacterales, sulfate reducers) in particular, Acidobacteria and Firmicutes were the most abundant bacterial phyla in all sediment systems. Among the other important bacterial phyla, clear water sediments contained relatively more Alphaproteobacteria and Planctomycetes, whereas white water sediments contained relatively more Betaproteobacteria, Firmicutes, Actinobacteria, and Chloroflexi than the respective other sediment systems. The data showed communities of bacteria common to all sediment types, but also revealed microbial groups that were significantly different between the sediment types, which also differed in physicochemical conditions. Our study showed that function of the microbial communities may be understood on the basis of their structures, which in turn are determined by environmental heterogeneity.