Comparison of DNA extraction methods on different sample matrices within the same terrestrial ecosystem.

Metataxonomic studies of ecosystem microbiotas require the simultaneous processing of samples with contrasting physical and biochemical traits. However, there are no published studies of comparisons of different DNA extraction kits to characterize the microbiotas of the main components of terrestrial ecosystems. Here, and to our knowledge for the first time, five DNA extraction kits were used to investigate the composition and diversity of the microbiota of a subset of samples typically studied in terrestrial ecosystems such as bulk soil, rhizosphere soil, invertebrate taxa and mammalian feces. DNA extraction kit was associated with changes in the relative abundance of hundreds of ASVs, in the same samples, resulting in significant differences in alpha and beta diversity estimates of their microbiotas. Importantly, the impact of DNA extraction kit on sample diversity varies according to sample type, with mammalian feces and soil samples showing the most and least consistent diversity estimates across DNA extraction kits, respectively. We show that the MACHEREY-NAGEL NucleoSpin® Soil kit was associated with the highest alpha diversity estimates, providing the highest contribution to the overall sample diversity, as indicated by comparisons with computationally assembled reference communities, and is recommended to be used for any large-scale microbiota study of terrestrial ecosystems.

Microbial perspective of inhibited carbon turnover in Tangel humus of the Northern Limestone Alps.

Tangel humus primarily occurs in montane and subalpine zones of the calcareous Alps that exhibit low temperatures and high precipitation sums. This humus form is characterized by inhibited carbon turnover and accumulated organic matter, leading to the typical thick organic layers. However, the reason for this accumulation of organic matter is still unclear, and knowledge about the microbial community within Tangel humus is lacking. Therefore, we investigated the prokaryotic and fungal communities along with the physical and chemical properties within a depth gradient (0-10, 10-20, 20-30, 30-40, 40-50 cm) of a Tangel humus located in the Northern Limestone Alps. We hypothesized that humus properties and microbial activity, biomass, and diversity differ along the depth gradient and that microbial key players refer to certain humus depths. Our results give the first comprehensive information about microbiota within the Tangel humus and establish a microbial zonation of the humus. Microbial activity, biomass, as well as microbial alpha diversity significantly decreased with increasing depths. We identified microbial biomarkers for both, the top and the deepest depth, indicating different, microbial habitats. The microbial characterization together with the established nutrient deficiencies in the deeper depths might explain reduced C-turnover and Tangel humus formation.

Mock community as an in situ positive control for amplicon sequencing of microbiotas from the same ecosystem.

Metataxonomy has become the standard for characterizing the diversity and composition of microbial communities associated with multicellular organisms and their environment. Currently available protocols for metataxonomy assume a uniform DNA extraction, amplification and sequencing efficiency for all sample types and taxa. It has been suggested that the addition of a mock community (MC) to biological samples before the DNA extraction step could aid identification of technical biases during processing and support direct comparisons of microbiota composition, but the impact of MC on diversity estimates of samples is unknown. Here, large and small aliquots of pulverized bovine fecal samples were extracted with no, low or high doses of MC, characterized using standard Illumina technology for metataxonomics, and analysed with custom bioinformatic pipelines. We demonstrated that sample diversity estimates were distorted only if MC dose was high compared to sample mass (i.e. when MC > 10% of sample reads). We also showed that MC was an informative in situ positive control, permitting an estimation of the sample 16S copy number, and detecting sample outliers. We tested this approach on a range of sample types from a terrestrial ecosystem, including rhizosphere soil, whole invertebrates, and wild vertebrate fecal samples, and discuss possible clinical applications.

Lignin intermediates lead to phenyl acid formation and microbial community shifts in meso- and thermophilic batch reactors.

BACKGROUND: Lignin intermediates resulting from lignocellulose degradation have been suspected to hinder anaerobic mineralisation of organic materials to biogas. Phenyl acids like phenylacetate (PAA) are early detectable intermediates during anaerobic digestion (AD) of aromatic compounds. Studying the phenyl acid formation dynamics and concomitant microbial community shifts can help to understand the microbial interdependencies during AD of aromatic compounds and may be beneficial to counteract disturbances. RESULTS: The length of the aliphatic side chain and chemical structure of the benzene side group(s) had an influence on the methanogenic system. PAA, phenylpropionate (PPA), and phenylbutyrate (PBA) accumulations showed that the respective lignin intermediate was degraded but that there were metabolic restrictions as the phenyl acids were not effectively processed. Metagenomic analyses confirmed that mesophilic genera like Fastidiosipila or Syntrophomonas and thermophilic genera like Lactobacillus, Bacillus, Geobacillus, and Tissierella are associated with phenyl acid formation. Acetoclastic methanogenesis was prevalent in mesophilic samples at low and medium overload conditions, whereas Methanoculleus spp. dominated at high overload conditions when methane production was restricted. In medium carbon load reactors under thermophilic conditions, syntrophic acetate oxidation (SAO)-induced hydrogenotrophic methanogenesis was the most important process despite the fact that acetoclastic methanogenesis would thermodynamically be more favourable. As acetoclastic methanogens were restricted at medium and high overload conditions, syntrophic acetate oxidising bacteria and their hydrogenotrophic partners could step in for acetate consumption. CONCLUSIONS: PAA, PPA, and PBA were early indicators for upcoming process failures. Acetoclastic methanogens were one of the first microorganisms to be impaired by aromatic compounds, and shifts to syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis occurred in thermophilic reactors. Previously assumed associations of specific meso- and thermophilic genera with anaerobic phenyl acid formation could be confirmed.

Carbon-dependent growth, community structure and methane oxidation performance of a soil-derived methanotrophic mixed culture.

Soil-borne methane-oxidizing microorganisms act as a terrestrial methane (CH4) sink and are potentially useful in decreasing global CH4 emissions. Understanding the ecophysiology of methanotrophs is crucial for a thorough description of global carbon cycling. Here, we report the in situ balance of soils from abandoned landfills, meadows and wetlands, their capacities to produce and oxidize CH4 at laboratory-scale and the isolation of a soil-borne methanotrophic-heterotrophic mixed culture that was used for carbon (C1 and C2) feeding experiments. We showed that even with similar soil properties, the in situ CH4 balance depends on land-use. Different soils had different potentials to adapt to increased CH4 availability, leading to the highest CH4 oxidation capacities for landfill and wetland soils. The most efficient mixed culture isolated from the landfill was dominated by the methanotrophs Methylobacter sp. and Methylosinus sp., which were accompanied by Variovorax sp. and Pseudomonas sp. and remained active in oxidizing CH4 when supplied with additional C-sources. The ratios between type I and type II methanotrophs and between methanotrophic and heterotrophic bacteria changed when C-sources were altered. A significant effect of the application of the mixed culture on the CH4 oxidation of soils was established but the extent varied depending on soil type.

Microbial community composition in the rhizosphere of Larix decidua under different light regimes with additional focus on methane cycling microorganisms.

Microbial community and diversity in the rhizosphere is strongly influenced by biotic and/or abiotic factors, like root exudates, nutrient availability, edaphon and climate. Here we report on the microbial diversity within the rhizosphere of Larix decidua, a dominant tree species in the Alps, as compared with the microbiome within the surrounding soil. We describe how increased light intensity influenced the rhizobiome and put emphasize on methane cycling microorganisms. Microbial taxa were classified into 26 bacterial, 4 archaeal and 6 fungal phyla revealing significant differences between bulk and rhizosphere soils. The dominant prokaryotic phyla were Proteobacteria, Acidobacteria, Actinobacteria (both, rhizosphere and bulk soil) and Bacteroidetes (rhizosphere soil only) and dominant fungal phyla in both fractions included Ascomycota and Basidiomycota. The rhizosphere community was indicated by Suillus sp., plant growth-promoting bacteria and Candidatus Saccharibacteria. Predicted genes in membrane transport and carbohydrate metabolism were significantly more abundant in rhizosphere soils while genes connected with energy metabolisms and cell motility increased in bulk soils. Dominant methanotrophic microorganisms were Upland Soil Cluster (USC) α methanotrophs, Methylogaea spp. and Methylosinus spp., while most methanogens belonged to Methanomassiliicoccales. The overall abundance of methanotrophs distinctly increased in the rhizosphere but to a very different species-specific extent. The increased light intensity only led to minor changes in the rhizobiome, nevertheless a couple of indicator species (e.g. Pseudomonas sp.) for intensified light conditions were established.

Soil-Derived Inocula Enhance Methane Production and Counteract Common Process Failures During Anaerobic Digestion.

Although soil-borne methanogens are known to be highly diverse and adapted to extreme environments, their application as potential (anaerobic) inocula to improve anaerobic digestion has not been investigated until now. The present study aimed at evaluating if soil-derived communities can be beneficial for biogas (methane, CH4) production and endure unfavorable conditions commonly associated with digestion failure. Nine study sites were chosen and tested for suitability as inoculation sources to improve biogas production via in situ measurements (CH4 fluxes, physical and chemical soil properties, and abundance of methanogens) and during a series of anaerobic digestions with (a) combinations of both sterile or unsterile soil and diluted fermenter sludge, and (b) pH-, acetate-, propionate-, and ammonium-induced disturbance. Amplicon sequencing was performed to assess key microbial communities pivotal for successful biogas production. Four out of nine tested soil inocula exerted sufficient methanogenic activity and repeatedly allowed satisfactory CH4/biogas production even under deteriorated conditions. Remarkably, the significantly highest CH4 production was observed using unsterile soil combined with sterile sludge, which coincided with both a higher relative abundance of methanogens and predicted genes involved in CH4 metabolism in these variants. Different bacterial and archaeal community patterns depending on the soil/sludge combinations and disturbance variations were established and these patterns significantly impacted CH4 production. Methanosarcina spp. seemed to play a key role in CH4 formation and prevailed even under stressed conditions. Overall, the results provided evidence that soil-borne methanogens can be effective in enhancing digestion performance and stability and, thus, harbor vast potential for further exploitation.

Microbial community dynamics in mesophilic and thermophilic batch reactors under methanogenic, phenyl acid-forming conditions.

BACKGROUND: Proteinaceous wastes exhibit high theoretical methane yields and their residues are considered valuable fertilisers. The routine anaerobic degradation of proteins often raises problems like high aromatic compound concentrations caused by the entry of aromatic amino acids into the system. A profound investigation of the consequences of aromatic compound exposure on various microorganisms, which cascade-like and interdependently degrade complex molecules to biogas, is still pending. RESULTS: In mesophilic samples, methane was predominantly produced via acetoclastic methanogenesis. The highest positive correlation was observed between phenylacetate (PAA) and Psychrobacter spp. and between phenylpropionate (PPA) and Haloimpatiens spp. Moreover, Syntrophus spp. negatively correlated with PAA (Spearman's rank correlations coefficient (rs) = - 0.46, p < 0.05) and PPA concentrations (rs = - 0.44, p < 0.05) and was also associated with anaerobic benzene ring cleavage. In thermophilic samples, acetate was predominantly oxidised by Tepidanaerobacter spp. or Syntrophaceticus spp. in syntrophic association with a hydrogenotrophic methanogen. The genera Sedimentibacter and Syntrophaceticus correlated positively with both PAA and PPA concentrations. Moreover, Sedimentibacter spp., Tepidanaerobacter spp., Acetomicrobium spp., and Sporanaerobacter spp. were significant LEfSe (linear discriminant analysis effect size) biomarkers for high meso- as well as thermophilic phenyl acid concentrations. Direct negative effects of phenyl acids on methanogenic properties could not be proven. CONCLUSIONS: Anaerobic phenyl acid formation is not restricted to specific microbial taxa, but rather done by various meso- and thermophilic bacteria. The cleavage of the highly inert benzene ring is possible in methanogenic batch reactors-at least in mesophilic fermentation processes. The results indicated that phenyl acids rather affect microorganisms engaged in preceding degradation steps than the ones involved in methanogenesis.

Effect of sulfate addition on carbon flow and microbial community composition during thermophilic digestion of cellulose.

Substrates with high sulfate levels pose problems for biogas production as they allow sulfate reducing bacteria to compete with syntrophic and methanogenic members of the community. In addition, the end product of sulfate reduction, hydrogen sulfide, is toxic and corrosive. Here we show how sulfate addition affects physiological processes in a thermophilic methanogenic system by analyzing the carbon flow and the microbial community with quantitative PCR and amplicon sequencing of the 16s rRNA gene. A sulfate addition of 0.5 to 3 g/L caused a decline in methane production by 73-92%, while higher sulfate concentrations had no additional inhibitory effect. Generally, sulfate addition induced a shift in the composition of the microbial community towards a higher dominance of Firmicutes and decreasing abundances of Bacteroidetes and Euryarchaeota. The abundance of methanogens (e.g., Methanoculleus and Methanosarcina) was reduced, while sulfate reducing bacteria (especially Candidatus Desulforudis and Desulfotomaculum) increased significantly in presence of sulfate. The sulfate addition had a significant impact on the carbon flow within the system, shifting the end product from methane and carbon dioxide to acetate and carbon dioxide. Interestingly, methane production quickly resumed, when sulfate was no longer present in the system. Despite the strong impact of sulfate addition on the carbon flow and the microbial community structure during thermophilic biogas production, short-term process disturbances caused by unexpected introduction of sulfate may be overcome due to the high resilience of the engaged microorganisms.

pH and Phosphate Induced Shifts in Carbon Flow and Microbial Community during Thermophilic Anaerobic Digestion.

pH is a central environmental factor influencing CH4 production from organic substrates, as every member of the complex microbial community has specific pH requirements. Here, we show how varying pH conditions (5.0-8.5, phosphate buffered) and the application of a phosphate buffer per se induce shifts in the microbial community composition and the carbon flow during nine weeks of thermophilic batch digestion. Beside monitoring the methane production as well as volatile fatty acid concentrations, amplicon sequencing of the 16S rRNA gene was conducted. The presence of 100 mM phosphate resulted in reduced CH4 production during the initial phase of the incubation, which was characterized by a shift in the dominant methanogenic genera from a mixed Methanosarcina and Methanoculleus to a pure Methanoculleus system. In buffered samples, acetate strongly accumulated in the beginning of the batch digestion and subsequently served as a substrate for methanogens. Methanogenesis was permanently inhibited at pH values ≤5.5, with the maximum CH4 production occurring at pH 7.5. Adaptations of the microbial community to the pH variations included shifts in the archaeal and bacterial composition, as less competitive organisms with a broad pH range were able to occupy metabolic niches at unfavorable pH conditions.

Microbial and Phenyl Acid Dynamics during the Start-up Phase of Anaerobic Straw Degradation in Meso- and Thermophilic Batch Reactors.

Aromatic compounds like phenyl acids derived from lignocellulose degradation have been suspected to negatively influence biogas production processes. However, results on this topic are still inconclusive. To study phenyl acid formation in batch reactors during the start-up phase of anaerobic degradation, different amounts of straw from grain were mixed with mesophilic and thermophilic sludge, respectively. Molecular biological parameters were assessed using next-generation sequencing and qPCR analyses. Metagenomic predictions were done via the program, piphillin. Methane production, concentrations of phenylacetate, phenylpropionate, phenylbutyrate, and volatile fatty acids were monitored chromatographically. Methanosarcina spp. was the dominant methanogen when high straw loads were effectively degraded, and thus confirmed its robustness towards overload conditions. Several microorganisms correlated negatively with phenyl acids; however, a negative effect, specifically on methanogens, could not be proven. A cascade-like increase/decrease from phenylacetate to phenylpropionate, and then to phenylbutyrate could be observed when methanogenesis was highly active. Due to these results, phenylacetate was shown to be an early sign for overload conditions, whereas an increase in phenylbutyrate possibly indicated a switch from degradation of easily available to more complex substrates. These dynamics during the start-up phase might be relevant for biogas plant operators using complex organic wastes for energy exploitation.

Spiking a Silty-Sand Reference Soil with Bacterial DNA: Limits and Pitfalls in the Discrimination of Live and Dead Cells When Applying Ethidium Monoazide (EMA) Treatment.

In the present study, EMA (ethidium monoazide) treatment was applied to a silty-sand reference soil prior to DNA extraction to enable a differentiation between dead and living cells. For this purpose, a reference soil was spiked with Listeria monocytogenes cells or cell equivalents, respectively. With the purpose of evaluating optimum treatment conditions, different EMA concentrations have been tested. However, the results remained largely inconclusive. Furthermore, varied dark incubation periods allowing EMA to penetrate dead cells did not allow the selective removal of DNA from membrane-compromised cells in downstream analyses. In contrast to undiluted soil, an effect of EMA treatment during DNA extraction could be observed when using a 1:10 dilution of the reference soil; however, the effect has not been sufficiently selective to act on heat-treated cells only. Although the application of EMA to soil requires further evaluation, the procedure harbors future potential for improving DNA-based approaches in microbial ecology studies.

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions.

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

Microbial Diversity in Bulk and Rhizosphere Soil of Ranunculus glacialis Along a High-Alpine Altitudinal Gradient.

Serving as "natural laboratories", altitudinal gradients can be used to study changes in the distribution of microorganisms in response to changing environmental conditions that typically occur over short geographical distances. Besides, rhizosphere zones of plants are known to be hot-spots for microbial diversity and to contain different microbial communities when compared with surrounding bulk soil. To discriminate the effects of altitude and plants, we investigated the microbial communities in the rhizosphere of Ranunculus glacialis and bulk soil along a high-alpine altitudinal gradient (2,600-3,400 m a.s.l.). The research area of this study was Mount (Mt.) "Schrankogel" in the Central Alps of Tyrol (Austria). Our results point to significantly different microbial diversities and community compositions in the different altitudinal belts. In the case of prokaryotes, environmental parameters could explain 41% of the total variation of soil communities, with pH and temperature being the strongest influencing factors. Comparing the effects derived from fraction (bulk vs. rhizosphere soil) and environmental factors, the effects of the roots of R. glacialis accounted for about one third of the explained variation. Fungal communities on the other hand were nearly exclusively influenced by environmental parameters accounting for 37.4% of the total variation. Both, for altitudinal zones as well as for bulk and rhizosphere fractions a couple of very specific biomarker taxa could be identified. Generally, the patterns of abundance of several taxa did not follow a steady increased or decreased trend along the altitudinal gradient but in many cases a maximal or minimal occurrence was established at mid-altitudes (3,000-3,100 m). This mid-altitudinal zone is a transition zone (the so-called alpine-nival ecotone) between the (lower) alpine grassland/tundra zone and the (upper) sparsely vegetated nival zone and was shown to correspond with the summer snow line. Climate change and the associated increase in temperature will shift this transition zone and thus, might also shift the described microbial patterns and biomarkers.

Benthic Diatom Communities in an Alpine River Impacted by Waste Water Treatment Effluents as Revealed Using DNA Metabarcoding.

Freshwater ecosystems are continuously affected by anthropogenic pressure. One of the main sources of contamination comes from wastewater treatment plant (WWTP) effluents that contain wide range of micro- and macropollutants. Chemical composition, toxicity levels and impact of treated effluents (TEs) on the recipient aquatic ecosystems may strongly differ depending on the wastewater origin. Compared to urban TEs, hospital ones may contain more active pharmaceutical substances. Benthic diatoms are relevant ecological indicators because of their high species and ecological diversity and rapid response to human pressure. They are routinely used for water quality monitoring. However, there is a knowledge gap on diatom communities' development and behavior in treated wastewater in relation to prevailing micro- and macropollutants. In this study, we aim to (1) investigate the response of diatom communities to urban and hospital TEs, and (2) evaluate TEs effect on communities in the recipient river. Environmental biofilms were colonized in TEs and the recipient river up- and downstream from the WWTP output to study benthic diatoms using DNA metabarcoding combined with high-throughput sequencing (HTS). In parallel, concentrations of nutrients, pharmaceuticals and seasonal conditions were recorded. Diatom metabarcoding showed that benthic communities differed strongly in their diversity and structure depending on the habitat. TE sites were generally dominated by few genera with polysaprobic preferences belonging to the motile guild, while river sites favored diverse communities from oligotrophic and oligosaprobic groups. Seasonal changes were visible to lower extent. To categorize parameters important for diatom changes we performed redundancy analysis which suggested that communities within TE sites were associated to higher concentrations of beta-blockers and non-steroidal anti-inflammatory drugs in urban effluents vs. antibiotics and orthophosphate in hospital effluents. Furthermore, indicator species analysis showed that 27% of OTUs detected in river downstream communities were indicator for urban or hospital TE sites and were absent in the river upstream. Finally, biological diatom index (BDI) calculated to evaluate the ecological status of the recipient river suggested water quality decrease linked to the release of TEs. Thus, in-depth assessment of diatom community composition using DNA metabarcoding is proposed as a promising technique to highlight the disturbing effect of pollutants in Alpine rivers.

Formation of phenylacetic acid and phenylpropionic acid under different overload conditions during mesophilic and thermophilic anaerobic digestion.

BACKGROUND: Substrate spectra for anaerobic digestion have been broadened in the past decade, inter alia, due to the application of different pretreatment strategies and now include materials rich in lignocellulose, protein, and/or fat. The application of these substrates, however, also entails risks regarding the formation of undesired by-products, among which phenolic compounds are known to accumulate under unfavorable digestion conditions. METHODS: Different states of overload were simulated in batch experiments while reviewing the generation of phenyl acids out of different lab-use substrates in order to evaluate the impact on biogas and methane production as well as some additional process performance parameters under defined laboratory conditions. Investigations were conducted under both mesophilic and thermophilic conditions. RESULTS: It could be shown that the tested input materials led to the formation of phenyl acids in a substrate-dependent manner with the formation itself being less temperature driven. Once formed, the formation of phenyl acids turned out to be a reversible process. CONCLUSIONS: Although a mandatory negative impact of phenyl acids per se on the anaerobic digestion process in general and the methanogenesis process in particular could not be proven, phenyl acids, however, seem to play an important role in the microbial response to overloaded biogas systems.

Larix decidua and additional light affect the methane balance of forest soil and the abundance of methanogenic and methanotrophic microorganisms.

Due to the activity of methane-oxidizing bacteria, forest soils are usually net sinks for the greenhouse gas methane (CH4). Despite several hints that CH4 balances might be influenced by vegetation, there are only few investigations dealing with this connection. Therefore, we studied this soil-plant-microbe interaction by using mesocosm experiments with forest soil and Larix decidua, a common coniferous tree species within the Alps. Gas measurements showed that the presence of L. decidua significantly reduced CH4 oxidation of the forest soil by ∼10% (-0.95 µmol m-2 h-1 for soil vs -0.85 µmol m-2 h-1 for soil plus L. decidua) leading to an increased net CH4 balance. Increased light intensity was used to intensify the influence of the plant on the soil's CH4 balance. The increase in light intensity strengthened the effect of the plant and led to a greater reduction of CH4 oxidation. Besides, we examined the impact of L. decidua and light on the abundance of methanogens and methanotrophs in the rhizosphere as compared with bulk soil. The abundance of both methane-oxidizing bacteria and methanogenic archaea was significantly increased in the rhizosphere compared with bulk soil but no significant response of methanogens and methanotrophs upon light exposure was established.

Hydrogenotrophic Methanogenesis and Autotrophic Growth of Methanosarcina thermophila.

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.

The influence of cattle grazing on methane fluxes and engaged microbial communities in alpine forest soils.

Recent dynamics and uncertainties in global methane budgets necessitate a dissemination of current knowledge on the controls of sources and sinks of atmospheric methane. Forest soils are considered to be efficient methane sinks; however, as they are microbially mediated they are sensitive to anthropogenic influences and tend to switch from being sinks to being methane sources. With regard to global changes in land use, the present study aimed at (i) investigating the influence of grazing on flux rates of methane in forest soils, (ii) deducing possible (a)biotic factors regulating these fluxes, and (iii) gaining an insight into the complex interactions between methane-cycling microorganisms and ecosystem functioning. Here we show that extensive grazing significantly mitigated the soil's sink strength for atmospheric methane through alterations of both microbial activity and community composition. In situ flux measurements revealed that all native, non-grazed areas were net methane consumers, while the adjacent, grazed areas were net methane producers. Whereas neither parent material nor soil properties including moisture and organic matter showed any correlation to the ascertained fluxes, significantly higher archaeal abundances at the grazed study sites indicated that small inputs of methanogens associated with cattle grazing may be sufficient to sustainably increase methane emissions.

Biological Pretreatment Strategies for Second-Generation Lignocellulosic Resources to Enhance Biogas Production.

With regard to social and environmental sustainability, second-generation biofuel and biogas production from lignocellulosic material provides considerable potential, since lignocellulose represents an inexhaustible, ubiquitous natural resource, and is therefore one important step towards independence from fossil fuel combustion. However, the highly heterogeneous structure and recalcitrant nature of lignocellulose restricts its commercial utilization in biogas plants. Improvements therefore rely on effective pretreatment methods to overcome structural impediments, thus facilitating the accessibility and digestibility of (ligno)cellulosic substrates during anaerobic digestion. While chemical and physical pretreatment strategies exhibit inherent drawbacks including the formation of inhibitory products, biological pretreatment is increasingly being advocated as an environmentally friendly process with low energy input, low disposal costs, and milder operating conditions. Nevertheless, the promising potential of biological pretreatment techniques is not yet fully exploited. Hence, we intended to provide a detailed insight into currently applied pretreatment techniques, with a special focus on biological ones for downstream processing of lignocellulosic biomass in anaerobic digestion.