In this study, the taxonomic and functional diversity of methanogenic archaea in two parallel 120 l fermenters operated at different temperatures and fed with maize silage was estimated by mcrA metabarcoding analysis using two typical primer pairs (ML and MLA) amplifying part of the functional methyl coenzyme M reductase (mcrA) gene. The alpha diversity indices showed that the ML primer pair detected a higher Operational Taxonomic Unit (OTU) abundance compared to the MLA primer pair and methanogen diversity was significantly lower in the 60 °C fermenters. The beta diversity analysis showed the methanogenic community clustered together at 50 °C and 40° and was statistically different from the 60 °C community. Similar, to alpha diversity, beta diversity was also significantly different between primer pairs. At all temperatures analysed, the primer pairs showed a different abundance of the different methanogenic OTUs, e.g. more OTUs relative to Methanoculleus sp. with the ML primer pair, and more OTUs corresponding to Methanobacterium sp. with the MLA primer pair. Moreover, OTUs corresponding to Methanosphaera sp. and Methanobrevibacter sp. were found only by using ML primer pair, while the MLA primer pair detected sequences corresponding to Methanothrix sp.

• Klíčová slova
• Anaerobic digestion, Biogas, Methanogenic Archaea, Next-Generation Sequencing, Temperature, mcrA primers,
• MeSH
• Archaea genetika   metabolismus   MeSH
• biodiverzita MeSH
• biopaliva * MeSH
• bioreaktory MeSH
• DNA archebakterií genetika   MeSH
• Euryarchaeota MeSH
• fermentace * MeSH
• fylogeneze MeSH
• methan MeSH
• oxidoreduktasy genetika   MeSH
• teplota * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biopaliva * MeSH
• DNA archebakterií MeSH
• methan MeSH
• methyl coenzyme M reductase MeSH Prohlížeč
• oxidoreduktasy MeSH

Biological methanogenesis is linked to permanent water logged systems, e.g., rice field soils or lake sediments. In these systems the methanogenic community as well as the pathway of methane formation are well-described. By contrast, the methanogenic potential of river sediments is so far not well-investigated. Therefore, we analyzed (a) the methanogenic potential (incubation experiments), (b) the pathway of methane production (stable carbon isotopes and inhibitor studies), and (c) the methanogenic community composition (terminal restriction length polymorphism of mcrA) in depth profiles of sediment cores of River Sitka, Czech Republic. We found two depth-related distinct maxima for the methanogenic potentials (a) The pathway of methane production was dominated by hydrogenotrophic methanogenesis (b) The methanogenic community composition was similar in all depth layers (c) The main TRFs were representative for Methanosarcina, Methanosaeta, Methanobacterium, and Methanomicrobium species. The isotopic signals of acetate indicated a relative high contribution of chemolithotrophic acetogenesis to the acetate pool.

• Klíčová slova
• T-RFLP, depth profile, isotope fractionation, mcrA, methane production potential, methyl fluoride, river sediment, stable carbon isotope,
• Publikační typ
• časopisecké články MeSH

Studies on methanogenesis from freshwater sediments have so far primarily focused on lake sediments. To expand our knowledge on the community composition of methanogenic archaea in river sediments, we studied the abundance and diversity of methanogenic archaea at two localities along a vertical profile (top 50 cm) obtained from sediment samples from Sitka stream (the Czech Republic). In this study, we compare two sites which previously have been shown to have a 10-fold different methane emission. Archaeal and methanogen abundance were analyzed by real-time PCR and T-RFLP. Our results show that the absolute numbers for the methanogenic community (qPCR) are relatively stable along a vertical profile as well as for both study sites. This was also true for the archaeal community and for the three major methanogenic orders in our samples (Methanosarcinales, Methanomicrobiales, and Methanobacteriales). However, the underlying community structure (T-RFLP) reveals different community compositions of the methanogens for both locations as well as for different depth layers and over different sampling times. In general, our data confirm that Methanosarcinales together with Methanomicrobiales are the two dominant methanogenic orders in river sediments, while members of Methanobacteriales contribute a smaller community and Methanocellales are only rarely present in this sediment. Our results show that the previously observed 10-fold difference in methane emission of the two sites could not be explained by molecular methods alone.

• Klíčová slova
• qPCR, T-RFLP, depth profile, mcrA, methanogen,
• MeSH
• Archaea klasifikace   genetika   metabolismus   MeSH
• biodiverzita * MeSH
• DNA archebakterií genetika   MeSH
• geologické sedimenty mikrobiologie   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• methan metabolismus   MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• řeky mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• DNA archebakterií MeSH
• methan MeSH

In the present study, we tested the hypothesis that animal treading associated with a high input of organic matter would favour methanogenesis in soils used as overwintering pasture. Hence, methane emissions and methanogen populations were examined at sections with different degree of cattle impact in a Farm in South Bohemia, Czech Republic. In spring, methane emission positively corresponded to the gradient of animal impact. Applying phospholipid etherlipid analysis, the highest archaeal biomass was found in section severe impact (SI), followed by moderate impact (MI) and no impact. The same trend was observed for the methanogens as showed by real-time quantitative PCR analyses of methyl coenzyme M reductase (mcrA) genes. The detection of monounsaturated isoprenoid side chain hydrocarbons (i20:1) indicated the presence of acetoclastic methanogens in the cattle-impacted sites. This result was corroborated by the phylogenetic analysis of mcrA gene sequences obtained from section SI, which showed that 33% of the analysed clones belonged to the genus Methanosarcina. The majority of the sequenced clones (41%) showed close affiliations with uncultured rumen archaeons. This leads to the assumption that a substantial part of the methanogenic community in plot SI derived from the grazing cattle itself. Compared to the spring sampling, in autumn, a significant reduction in archaeal biomass and number of copies of mcrA genes was observed mainly for section MI. It can be concluded that after 5 months without cattle impact, the severely impact section maintained its methane production potential, whereas the methane production potential under moderate impact returned to background values.

• MeSH
• archeální geny MeSH
• biomasa MeSH
• chov zvířat * MeSH
• Euryarchaeota genetika   metabolismus   MeSH
• fylogeneze MeSH
• genetická variace MeSH
• methan metabolismus   MeSH
• půdní mikrobiologie * MeSH
• roční období MeSH
• skot fyziologie   MeSH
• zvířata MeSH
• Check Tag
• skot fyziologie   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• methan MeSH

Methanogenic archaea produce methane as a metabolic product under anoxic conditions and they play a crucial role in the global methane cycle. In this study molecular diversity of methanogenic archaea in the hyporheic sediment of the lowland stream Sitka (Olomouc, Czech Republic) was analyzed by PCR amplification, cloning and sequencing analysis of the methyl coenzyme M reductase alpha subunit (mcrA) gene. Sequencing analysis of 60 clones revealed 24 different mcrA phylotypes from hyporheic sedimentary layers to a depth of 50 cm. Phylotypes were affiliated with Methanomicrobiales, Methanosarcinales and Methanobacteriales orders. Only one phylotype remains unclassified. The majority of the phylotypes showed higher affiliation with uncultured methanogens than with known methanogenic species. The presence of relatively rich assemblage of methanogenic archaea confirmed that methanogens may be an important component of hyporheic microbial communities and may affect CH4 cycling in rivers.

• MeSH
• Archaea genetika   MeSH
• archeální geny MeSH
• fylogeneze MeSH
• genová knihovna MeSH
• geologické sedimenty mikrobiologie   MeSH
• methan metabolismus   MeSH
• mikrobiologie životního prostředí MeSH
• molekulární sekvence - údaje MeSH
• řeky mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• methan MeSH

Wetlands are the largest natural source of terrestrial CH4 emissions. Afforestation can enhance soil CH4 oxidation and decrease methanogenesis, yet the driving mechanisms leading to these effects remain unclear. We analyzed the structures of communities of methanogenic and methanotrophic microbes, quantification of mcrA and pmoA genes, the soil microbial metagenome, soil properties and CH4 fluxes in afforested and non-afforested areas in the marshland of the Yangtze River. Compared to the non-afforested land use types, net CH4 emission decreased from bare land, natural vegetation and 5-year forest plantation and transitioned to net CH4 sinks in the 10- and 20-year forest plantations. Both abundances of mcrA and pmoA genes decreased significantly with increasing plantation age. By combining random forest analysis and structural equation modeling, our results provide evidence for an important role of the abundance of functional genes related to methane production in explaining the net CH4 flux in this ecosystem. The structures of methanogenic and methanotrophic microbial communities were of lower importance as explanatory factors than functional genes in terms of in situ CH4 flux. We also found a substantial interaction between functional genes and soil properties in the control of CH4 flux, particularly soil particle size. Our study provides empirical evidence that microbial community function has more explanatory power than taxonomic microbial community structure with respect to in situ CH4 fluxes. This suggests that focusing on gene abundances obtained, e.g., through metagenomics or quantitative/digital PCR could be more effective than community profiling in predicting CH4 fluxes, and such data should be considered for ecosystem modeling.

• Klíčová slova
• CH4 flux, methanogens, methanotrophs, soil metagenome, soil particle size composition,
• Publikační typ
• časopisecké články MeSH

In order to assess the activity of metabolic pathways during anaerobic biogas production, it is necessary to isolate total RNA from the anaerobic sludge. mRNA activity profiling complements the quantification of excreted metabolites for a comprehensive anaerobic digestion model (ADM1). Four non-commercial total RNA extraction protocols were examined to extract total RNA from suspended solids of anaerobic sludge. The most suitable protocol was identified and optimised. In relation to total RNA extraction efficiency, total RNA purity and RNA integrity, the best homogenisation method was a combined method of nitrogen grinding and bead beating. When bead beating or nitrogen grinding was used alone for homogenisation, total RNA extraction efficiency was lower than when both homogenisation methods were applied. Depending on the homogenisation method, the whole RNA extraction procedure takes approximately 2 to 3 h, which is as fast as when using commercial available soil RNA extraction kits. The proposed method is rapid in extracting total RNA from a biocoenosis present in an anaerobic sludge environment. Furthermore, we could apply any of the extracted homogenization methods for reverse transcription and subsequent PCR amplification of the gene for the methyl coenzyme M reductase alpha subunit (mcrA/mrtA).

• MeSH
• anaerobióza MeSH
• časové faktory MeSH
• mikrobiologické techniky metody   MeSH
• molekulární biologie metody   MeSH
• odběr biologického vzorku metody   MeSH
• odpadní vody mikrobiologie   MeSH
• RNA genetika   izolace a purifikace   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• odpadní vody MeSH
• RNA MeSH

Substantial amounts of topsoil organic matter (OM) in Arctic Cryosols have been translocated by the process of cryoturbation into deeper soil horizons (cryoOM), reducing its decomposition. Recent Arctic warming deepens the Cryosols´ active layer, making more topsoil and cryoOM carbon accessible for microbial transformation. To quantify bacteria, archaea and selected microbial groups (methanogens - mcrA gene and diazotrophs - nifH gene) and to investigate bacterial and archaeal diversity, we collected 83 soil samples from four different soil horizons of three distinct tundra types located in Qikiqtaruk (Hershel Island, Western Canada). In general, the abundance of bacteria and diazotrophs decreased from topsoil to permafrost, but not for cryoOM. No such difference was observed for archaea and methanogens. CryoOM was enriched with oligotrophic (slow-growing microorganism) taxa capable of recalcitrant OM degradation. We found distinct microbial patterns in each tundra type: topsoil from wet-polygonal tundra had the lowest abundance of bacteria and diazotrophs, but the highest abundance of methanogens. Wet-polygonal tundra, therefore, represented a hotspot for methanogenesis. Oligotrophic and copiotrophic (fast-growing microorganism) genera of methanogens and diazotrophs were distinctly distributed in topsoil and cryoOM, resulting in different rates of nitrogen flux into these horizons affecting OM vulnerability and potential CO2 and CH4 release.

• Klíčová slova
• arctic, climate change, gene abundance, microbial community, permafrost, vegetation,
• MeSH
• mikrobiota * MeSH
• ostrovy MeSH
• permafrost * MeSH
• půda MeSH
• půdní mikrobiologie MeSH
• tundra MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Arktida MeSH
• Kanada MeSH
• ostrovy MeSH
• Názvy látek
• půda MeSH

Chloroform is one of the common disinfection byproducts, which is not susceptible to degradation and poses great health concern. In this study, the chloroform removal efficiencies and contributions of sorption, microbial degradation, plant uptake, and volatilization were evaluated in six model constructed wetlands (CWs). The highest chloroform removal efficiency was achieved in litter-added CWs (99%), followed by planted (46-54%) and unplanted CWs (39%). Mass balance study revealed that sorption (73.5-81.2%) and microbial degradation (17.6-26.2%) were the main chloroform removal processes in litter-added CWs, and that sorption (53.6-66.1%) and plant uptake (25.3-36.2%) were the primary contributors to chloroform removal in planted CWs. Around 60% of chloroform got accumulated in the roots after plant uptake, and both transpiration and gas-phase transport were expected to be the drivers for the plant uptake. Sulfate-reducing bacteria and methanogens were found to be the key microorganisms for chloroform biodegradation through cometabolic dechlorination, and positive correlations were observed between functional genes (dsrA, mcrA) and biodegradation rates. Overall, this study suggests that wetland is an efficient ecosystem for sustainable chloroform removal, and that plant and litter can enhance the removal performance through root uptake and microbial degradation stimulation, respectively.

• MeSH
• adsorpce MeSH
• Bacteria genetika   metabolismus   MeSH
• bakteriální geny MeSH
• biodegradace MeSH
• biotransformace MeSH
• chemické látky znečišťující vodu analýza   MeSH
• chloroform chemie   MeSH
• čištění vody * MeSH
• kinetika MeSH
• mokřady * MeSH
• poločas MeSH
• volatilizace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chemické látky znečišťující vodu MeSH
• chloroform MeSH

Methanogenic archaeal communities existing in freshwater sediments are responsible for approximately 50 % of the total global emission of methane. This process contributes significantly to global warming and, hence, necessitates interventional control measures to limit its emission. Unfortunately, the diversity and functional interactions of methanogenic populations occurring in these habitats are yet to be fully characterized. Considering several disadvantages of conventional culture-based methodologies, in recent years, impetus is given to molecular biology approaches to determine the community structure of freshwater sedimentary methanogenic archaea. 16S rRNA and methyl coenzyme M reductase (mcrA) gene-based cloning techniques are the first choice for this purpose. In addition, electrophoresis-based (denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, and terminal restriction fragment length polymorphism) and quantitative real-time polymerase chain reaction techniques have also found extensive applications. These techniques are highly sensitive, rapid, and reliable as compared to traditional culture-dependent approaches. Molecular diversity studies revealed the dominance of the orders Methanomicrobiales and Methanosarcinales of methanogens in freshwater sediments. The present review discusses in detail the status of the diversity of methanogens and the molecular approaches applied in this area of research.

• MeSH
• Archaea klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• biodiverzita * MeSH
• genetická variace MeSH
• genetické techniky * MeSH
• geologické sedimenty mikrobiologie   MeSH
• methan metabolismus   MeSH
• sladká voda mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• methan MeSH