Methanogenic archaea diversity in hyporheic sediments of a small lowland stream.

Abundance and diversity of methanogenic archaea were studied at five localities along a longitudinal profile of a Sitka stream (Czech Republic). Samples of hyporheic sediments were collected from two sediment depths (0-25 cm and 25-50 cm) by freeze-core method. Methanogen community was analyzed by fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) and sequencing method. The proportion of methanogens to the DAPI-stained cells varied among all localities and depths with an average value 2.08 × 10(5) per g of dry sediment with the range from 0.37 to 4.96 × 10(5) cells per g of dry sediment. A total of 73 bands were detected at 19 different positions on the DGGE gel and the highest methanogen diversity was found at the downstream located sites. There was no relationship between methanogen diversity and sediment depth. Cluster analysis of DGGE image showed three main clusters consisting of localities that differed in the number and similarity of the DGGE bands. Sequencing analysis of representative DGGE bands revealed phylotypes affiliated with members belonging to the orders Methanosarcinales, Methanomicrobiales and Methanocellales. The knowledge about occurrence and diversity of methanogenic archaea in freshwater ecosystems are essential for methane dynamics in river sediments and can contribute to the understanding of global warming process.

Dominance of Methanosarcinales phylotypes and depth-wise distribution of methanogenic community in fresh water sediments of Sitka stream from Czech Republic.

The variation in the diversity of methanogens in sediment depths from Sitka stream was studied by constructing a 16S rRNA gene library using methanogen-specific primers and a denaturing gradient gel electrophoresis (DGGE)-based approach. A total of nine different phylotypes from the 16S rRNA library were obtained, and all of them were clustered within the order Methanosarcinales. These nine phylotypes likely represent nine new species and at least 5-6 new genera. Similarly, DGGE analysis revealed an increase in the diversity of methanogens with an increase in sediment depth. These results suggest that Methanosarcinales phylotypes might be the dominant methanogens in the sediment from Sitka stream, and the diversity of methanogens increases as the depth increases. Results of the present study will help in making effective strategies to monitor the dominant methanogen phylotypes and methane emissions in the environment.

Molecular diversity and tools for deciphering the methanogen community structure and diversity in freshwater sediments.

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.

Testing the applicability of tagging the Great crested newt (Triturus cristatus) using passive integrated transponders.

Tracking individual animals with small-sized passive integrated transponder tags (PIT tags) has become a popular and widespread method, one which can be used for investigating life history traits, including dispersal patterns of small protected animals such as newts. In this study, we tested the applicability of PIT tag usage for individual marking with the Great crested newt (Triturus cristatus) as a model amphibian species, and to test the detection of the newts in nature using a passive telemetry system. Clove oil was used as an anaesthetic before surgery. We implanted PIT tags under the skin of 140 newts. The survival rate of newts was 98.57%. X-ray images were taken to check the exact positions of the PIT tags. Since approximately 15.71% of the newts were capable of expelling the tag from their bodies, tag loss has to be accounted for in future behavioural studies dealing with newts and other amphibians potentially capable of frequent tag expulsion. Lastly, we detected by passive telemetry 97 individuals out of 100 released into a natural breeding pond. Males had higher activity (13 detected males vs 7 females per hour) than females, thus males could be detected if present with more certainty. The result of the movement behaviour showed that e.g. the male of T. cristatus in a breeding pond can travel up to 20 m in 78 seconds. In summary, this promising method could allow the automatic data collection of marked newts in aquatic as well as in terrestrial biotopes, providing data on their dispersal, diurnal activity and movement behaviour.

Species-specific habitat preferences do not shape the structure of a crested newt hybrid zone (Triturus cristatus x T. carnifex).

Reproductive isolation barriers maintain the integrity of species by preventing interspecific gene flow. They involve temporal, habitat or behavioral isolation acting before fertilization, and postzygotic isolation manifested as hybrid mortality or sterility. One of the approaches of how to study reproductive isolation barriers is through the analysis of hybrid zones. In this paper, we describe the structure of a hybrid zone between two crested newt species (Triturus cristatus and T. carnifex) in the southern part of the Czech Republic using morphological, microsatellite, and mitochondrial (mtDNA) markers. Specifically, we tested the hypothesis that the structure of the hybrid zone is maintained by species-specific habitat preferences. Comparing the genetic structure of populations with geographical and ecological parameters, we found that the hybrid zone was structured primarily geographically, with T. cristatus-like populations occurring in the northeast and T. carnifex-like populations in the southwest. Despite T. cristatus tending to occur in deeper ponds and T. carnifex on localities with more shading, the effect of both ecological parameters on the structure of the zone was minimal. Next, we corroborated that T. carnifex individuals and some hybrids possess mtDNA of T. dobrogicus, whose nuclear background was not detected in the studied hybrid zone. Hybridization between T. carnifex and T. dobrogicus (resulting in unidirectional mtDNA introgression) had to predate subsequent formation of the hybrid zone between T. cristatus and T. carnifex. Populations of crested newts in the southern part of the Czech Republic thus represent a genetic mosaic of nuclear and mitochondrial genomes of three species.

The interplay between total mercury, methylmercury and dissolved organic matter in fluvial systems: A latitudinal study across Europe.

Large-scale studies are needed to identify the drivers of total mercury (THg) and monomethyl-mercury (MeHg) concentrations in aquatic ecosystems. Studies attempting to link dissolved organic matter (DOM) to levels of THg or MeHg are few and geographically constrained. Additionally, stream and river systems have been understudied as compared to lakes. Hence, the aim of this study was to examine the influence of DOM concentration and composition, morphological descriptors, land uses and water chemistry on THg and MeHg concentrations and the percentage of THg as MeHg (%MeHg) in 29 streams across Europe spanning from 41°N to 64 °N. THg concentrations (0.06-2.78 ng L-1) were highest in streams characterized by DOM with a high terrestrial soil signature and low nutrient content. MeHg concentrations (7.8-159 pg L-1) varied non-systematically across systems. Relationships between DOM bulk characteristics and THg and MeHg suggest that while soil derived DOM inputs control THg concentrations, autochthonous DOM (aquatically produced) and the availability of electron acceptors for Hg methylating microorganisms (e.g. sulfate) drive %MeHg and potentially MeHg concentration. Overall, these results highlight the large spatial variability in THg and MeHg concentrations at the European scale, and underscore the importance of DOM composition on mercury cycling in fluvial systems.

Is the methanogenic community reflecting the methane emissions of river sediments?-comparison of two study sites.

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.

Effect of weir impoundments on methane dynamics in a river.

We measured CH4 concentration, CH4 oxidation in the water column and total CH4 emissions to the atmosphere (diffusion and ebullition) in three weir impoundments and river reaches between them, in order to understand their role in river methane (CH4) dynamics. Sediment samples were also collected to determine CH4 consumption and production potentials together with the contribution of individual methanogenic pathways. The CH4 surface water concentration increased 7.5 times in the 16km long river stretch. Microbial CH4 oxidation in the water column reached values ranging from 51 to 403nmoll-1d-1 and substantially contributed to the CH4 removal from surface water, together with CH4 emissions. The total CH4 emissions to the atmosphere varied between 0.8 and 207.1mmolCH4m-2d-1 with the highest values observed upstream of the weirs (mean 68.5±29.9mmolCH4m-2d-1). Most of the CH4 was transported through the air-water interface by ebullition upstream of the weirs, while the ebullition accounted for 95.8±2.0% of the total CH4 emissions. Both CH4 production and oxidation potential of sediments were higher upstream of the weirs compared to downstream of the weirs. The contribution of hydrogenotrophic methanogenesis to total CH4 sediment production was 36.7-89.4% and prevailed upstream of the weirs. Our findings indicate that weirs might influence river CH4 dynamics, especially by increased CH4 production and consumption by sediments, followed by increasing CH4 emissions to the atmosphere.

Methane production potentials, pathways, and communities of methanogens in vertical sediment profiles of river Sitka.

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.

Determination of biodegradable dissolved organic carbon in waters: comparison of batch methods.

An effect of different types of bacterial inocula upon the final biodegradable dissolved organic carbon (BDOC) result was investigated in samples of both low and high BDOC concentrations. Stream water and leaf leachate samples were incubated either with free, suspended bacteria or with bacteria attached to the stream sediment particles or attached to artificial substrata. The time course of dissolved organic carbon (DOC) decomposition was observed using absorbance analysis of DOC. BDOC determination by means of commonly used suspended bacteria as the inoculum made for an underestimation of BDOC between 5% and 25%, compared with attached bacterial community (biofilm). The reason for these findings could be the higher microbial diversity, higher metabolic activity of attached bacteria and abiotic adsorption of organic molecules to inorganic support and biofilm matrix surfaces. Adsorbed DOC is easily hydrolyzed and utilized by biofilm bacteria.

Molecular identification of the occurrence of magnetotactic bacteria in fresh water sediments (Czech Republic).

Magnetotactic bacteria (MTB) are of considerable interest because of their importance in the manufacture of various bioinspired materials. In order to find out the status of magnetotactic bacteria at three different sediment in Czech Republic, samples collected from both standing and running freshwaters were subjected to molecular diversity analysis by using 16S rRNA gene approach. Total community DNA from sediment sample was isolated and used for PCR, cloning and sequence analysis. Of the 24 analyzed sequences, six clones are closely related to Magnetobacterium sp. affiliated with the Nitrospira phylum which showed the dominance of Magnetobacterium phylotypes in the sample. This study will provide useful insight about the community structure of MTB in this particular geographical region. However more detailed and specific studies are warranted in order to properly assess the community structure of MTB's in fresh water sediments.

The phylogenetic structure of microbial biofilms and free-living bacteria in a small stream.

The phylogenetic composition, bacterial biomass, and biovolume of both planktonic and biofilm communities were studied in a low-order Bystrice stream near Olomouc City, in the Czech Republic. The aim of the study was to compare the microbial communities colonizing different biofilm substrata (stream aggregates, stream sediment, underwater tree roots, stream stones, and aquatic macrophytes) to those of free-living bacteria. The phylogenetic composition was analyzed using fluorescence in situ hybridization for main phylogenetic groups. All phylogenetic groups studied were detected in all sample types. The stream stone was the substratum where nearly all phylogenetic groups were the most abundant, while the lowest proportion to the DAPI-stained cells was found for free-living bacteria. The probe specific for the domain Bacteria detected 20.6 to 45.8 % of DAPI-stained cells while the probe specific for the domain Archaea detected 4.3 to 17.9 %. The most abundant group of Proteobacteria was Alphaproteobacteria with a mean of 14.2 %, and the least abundant was Betaproteobacteria with a mean of 11.4 %. The average value of the Cytophaga-Flavobacteria group was 10.5 %. Total cell numbers and bacterial biomass were highest in sediment and root biofilm. The value of cell biovolume was highest in stone biofilm and lowest in sediment. Overall, this study revealed relevant differences in phylogenetic composition, bacterial biomass, and biovolume between different stream biofilms and free-living bacteria.

Identification of methanogenic archaea in the hyporheic sediment of Sitka stream.

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.

Molecular identification of the occurrence of magnetotactic bacteria in fresh water sediments (Czech Republic)

Magnetotactic bacteria (MTB) are of considerable interest because of their importance in the manufacture of various bioinspired materials. In order to find out the status of magnetotactic bacteria at three different sediment in Czech Republic, samples collected from both standing and running freshwaters were subjected to molecular diversity analysis by using 16S rRNA gene approach. Total community DNA from sediment sample was isolated and used for PCR, cloning and sequence analysis. Of the 24 analyzed sequences, six clones are closely related to Magnetobacterium sp. affiliated with the Nitrospira phylum which showed the dominance of Magnetobacterium phylotypes in the sample. This study will provide useful insight about the community structure of MTB in this particular geographical region. However more detailed and specific studies are warranted in order to properly assess the community structure of MTB's in fresh water sediments.