Peter Casper

DNA-, rRNA- and mRNA-based stable isotope probing of aerobic methanotrophs in lake sediment

A stable isotope probing (SIP) approach was used to study aerobic methane-oxidizing bacteria (methanotrophs) in lake sediment. Oligotrophic Lake Stechlin was chosen because it has a permanently oxic sediment surface. 16S rRNA and the pmoA gene, which encodes a subunit of the methane monooxygenase enzyme, were analysed following the incubation of sediment with (13) CH(4) and the separation of (13) C-labelled DNA and RNA from unlabelled nucleic acids. The incubation with (13) CH(4) was performed over a 4-day time-course and the pmoA genes and transcripts became progressively labelled such that approximately 70% of the pmoA genes and 80% of the transcripts were labelled at 96 h. The labelling of pmoA mRNA was quicker than pmoA genes, demonstrating that mRNA-SIP is more sensitive than DNA-SIP; however, the general rate of pmoA transcript labelling was comparable to that of the pmoA genes, indicating that the incorporation of (13) C into ribonucleic acids of methanotrophs was a gradual process. Labelling of Betaproteobacteria was clearly seen in analyses of 16S rRNA by DNA-SIP and not by RNA-SIP, suggesting that cross-feeding of the (13) C was primarily detected by DNA-SIP. In general, we show that the combination of SIP approaches provided valuable information about the activity and growth of the methanotrophic populations and the cross-feeding of methanotroph metabolites by other microorganisms.

Carbon isotope fractionation during acetoclastic methanogenesis by Methanosaeta concilii in culture and a lake sediment.

ABSTRACT The isotope enrichment factors (ε) in Methanosaeta concilii and in a lake sediment, where acetate was consumed only by Methanosaeta spp., were clearly less negative than the ε usually observed for Methanosarcina spp. The fraction of methane produced from acetate in the sediment, as determined by using stable isotope signatures, was 10 to 15% lower when the appropriate ε of Methanosaeta spp. was used.

Different methods for extracting bacteria from freshwater sediment and a simple method to measure bacterial production in sediment samples

The efficiency of different treatments was tested to extract bacterial cells from freshwater sediment samples. The influence of sonication, density gradient centrifugation, fixation by formalin and centrifugation speed on bacterial recovery was investigated. The method developed by Smith and Azam [Mar. Microb. Food Webs 6 (1992) 107] to measure microbial activity on bacterioplankton (3H-leucine incorporation), was also evaluated in sediment samples. After 1 min of sonication bacterial abundance was reduced by about 47% in diluted sediments with tetrasodium pyrophosphate. With the addition of Percoll after sonication, bacterial counts were not significantly different (P<0.05). Fixation by formalin increased bacterial counts using sonication. However, higher bacterial abundance was estimated in non-sonicated samples. Bacterial abundance in samples centrifuged at 7000xg with and without Percoll was not significantly different (P<0.05). Highest bacterial abundance was obtained after centrifugation at low speed (750xg). Bacterial abundance decreased with higher centrifugation speed (750, 1500 and 3000xg), the difference, however, was not significant. Bacterial production ranged from 0.10 microg C cm(-3) d(-1) in autoclaved sediment to 0. 27 microg C cm(-3) d(-1) in untreated sediment. The radioactivity measured in controls of both untreated and autoclaved sediment was high (70 and 91%, respectively), indicating a high level of leucine adsorption in sediment particles. In contrast, radioactivity in control samples previously centrifuged was markedly lower (6%). Despite the high values of radioactivity in the controls, bacterial production in untreated sediment was significantly higher than in centrifuged sediment (P<0.05).

Methanogenic archaeal community in the sediment of an artificially partitioned acidic bog lake

Abstract The methanogenic archaeal communities in the sediment of two basins of an artificially partitioned acidic bog lake were studied. In the northeast basin, which was separated from a peat bog, a high methane production rate was measured only in the upper layers of the sediment. In contrast, methane production was detected at various depths of the sediment in the southwest basin, which continuously receiving humic acids from the bog. Ten bands were observed in the denaturing gradient gel electrophoresis (DGGE) fingerprint of the 16S rDNA archaeal amplicons from the NE basin top (0-5 cm) sediment layer, which reflected the presence of at least 10 ribotypes. Seventy clones of the 16S rDNA amplicons were obtained from the NE basin top sediment layer, and were grouped into 10 operational taxonomy units (OTUs) according to their positions on the DGGE gel. Seven of these OTUs could be matched with the bands of the community fingerprint. Phylogenetic analyses indicated that the sequences clustered into three groups: five of the OTUs were related to Methanosaeta, four OTUs to Methanomicrobiales and one OTU to Methanobacterium. Among the OTUs, sequences with high similarities (>96%) were retrieved. The sequence data suggested that the diversity of the methanogenic archaeal community was limited. Bands corresponding to those three phylogenetic groups were found in the DGGE fingerprints of both NE and SW basins, which reflected the presence of the same dominant methanogenic archaeal groups in both basins. However, differences in the distribution of the ribotypes that had high 16S rRNA sequence identities were observed in the fingerprints between the two basins at various depths. The microdiversity decreased along the sediment depth in the NE basin, and vice versa in the SW basin. The greater variety of the archaeal ribotypes, apparently, correlated with the higher methanogenesis rate.

Methane in an acidic bog lake: The influence of peat in the catchment on the biogeochemistry of methane

Abstract. Methanogenesis, an anaerobic microbial process in sediments, was investigated in a naturally acidic bog lake, Grosse Fuchskuhle, in northeastern Germany. The lake was artificially divided into four sub-basins: two western basins receiving humic substances from an adjacent Sphagnum bog and two eastern basins isolated from this acidic inflow with installed curtains. Within the northeast (NE) and southwest (SW) basin compartments, one of each group were studied. In the peat influenced western basins the pH-values in the water columns were kept low and increased in the others. The stratification period increased in all four compartments from some weeks to some months, from April to October, with the development of anoxic hypolimnia in all compartments after the installation of the curtains. The layer with the most active methane production moved from a sediment depth of more than 20 cm before separation to close to the surface sediments in all compartments. Methano genic microorganisms were found in the whole sediment core – from surface sediments to a depth of 25 cm. The proportion of methanogens was approximately 15% of total microbial cell numbers, which were approximately 2 × 106 cells/ml sediment. Oligonucleotide probes targeting nearly all families of the phylum Euryarchaeota were tested with fluorescence in-situ hybridization (FISH). In both basins, with and without the influence from peat, oligonucleotide probe (MSMX) targeting Methanosarcinaceae could be detected only as methanogens. The finding of only acetate-using methanogens by FISH indicated acetate as a major methanogenic substrate. Concentration profiles of CH4 as a function of sediment depth were characterized in both basins by high concentrations in the top layers. Methane bubbles were released in the late summer in the eastern sub-basin only, likely from oversaturation in the surface sediments.

Cyanobacterial diversity in the hot spring, pelagic and benthic habitats of a tropical soda lake

Hot springs and saline-alkaline lakes of East Africa are extreme habitats regarding temperature, or salinity and pH, respectively. This study examines whether divergent habitats of Lake Bogoria, Kenya, impacts cyanobacterial community structure. Samples from the hot springs, pelagic zone and sediment were analysed by light microscopy, multilocus 454-amplicons sequencing and metagenomics to compare the cyanobacterial diversity. Most of the phylogenetic lineages of Cyanobacteria occurred exclusively in the Bogoria hot springs suggesting a high degree of endemism. The prevalent phylotypes were mainly members of the Oscillatoriales (Leptolyngbya, Spirulina, Oscillatoria-like and Planktothricoides). The Chroococcales were represented by different clades of Synechococcus but not a single phylotype clustered with any of the lineages described earlier from different continents. In contrast, we found that the pelagic zone and the sediments were inhabited by only a few taxa, dominated by Arthrospira and Anabaenopsis. Arthrospira, the main food base of Lesser Flamingo, was detected in all three habitats by amplicons pyrosequencing, indicating its resilience and key role as a primary producer. Despite the close connection between the three habitats studied, the cyanobacterial communities in the hot springs and lake differed considerably, suggesting that they are unable to adapt to the extreme conditions of the neighbouring habitat.

Vegetation cover of forest, shrub and pasture strongly influences soil bacterial community structure as revealed by 16S rRNA gene T-RFLP analysis

Bacterial community structure is influenced by vegetation, climate and soil chemical properties. To evaluate these influences, terminal restriction fragment length polymorphism (T-RFLP) and cloning of the 16S rRNA gene were used to analyze the soil bacterial communities in different ecosystems in southwestern China. We compared (1) broad-leaved forest, shrub and pastures in a high-plateau region, (2) three broad-leaved forests representing a climate gradient from high-plateau temperate to subtropical and tropical regions and (3) the humus and mineral soil layers of forests, shrub lands and pastures with open and restricted grazing activities, having varied soil carbon and nutrient contents. Principal component analysis of the T-RFLP patterns revealed that soil bacterial communities of the three vegetation types were distinct. The broad-leaved forests in different climates clustered together, and relatively minor differences were observed between the soil layers or the grazing regimes. Acidobacteria dominated the broad-leaved forests (comprising 62% of the total clone sequences), but exhibited lower relative abundances in the soils of shrub (31%) and pasture (23%). Betaproteobacteria was another dominant taxa of shrub land (31%), whereas Alpha- (19%) and Gammaproteobacteria (13%) and Bacteriodetes (16%) were major components of pasture. Vegetation exerted more pronounced influences than climate and soil chemical properties.

Phylogenetic relationship and divergence among planktonic strains of Arthrospira (Oscillatoriales, Cyanobacteria) of African, Asian and American origin deduced by 16S-23S ITS and phycocyanin operon sequences

Dadheech P.K., Ballot A., Casper P., Kotut K., Novelo E., Lemma B., Pröschold T. and Krienitz L. 2010. Phylogenetic relationship and divergence among planktonic strains of Arthrospira (Oscillatoriales, Cyanobacteria) of African, Asian and American origin deduced by 16S–23S ITS and phycocyanin operon sequences. Phycologia 49: 361–372. DOI: 10.2216/09-71.1 Arthrospira comprises multicellular, cylindrical, usually screwlike coiled trichomes and is cultivated commercially. In this study, 33 new strains of Arthrospira isolated from plankton samples collected in Mexico, East Africa and India were investigated and compared with 53 strains or samples of earlier considerations. The study included observations of morphological features and molecular phylogenetic analyses on the basis of nucleotide sequences of internal transcribed spacer (ITS) between 16S rRNA and 23S rRNA genes and partial sequences of beta and alpha subunits including intergenic spacer (cpcBA-IGS) of phycocyanin operon. Morphological traits of Arthrospira such as trichome width, type of coiling and apical cell were not always consistent in culture conditions. It was revealed that Arthrospira phylogeny on the basis of cpcBA-IGS locus was broadly comparable with the ITS region as both phylogenetic trees derived from nucleotide sequences could be divided into two main clusters. Cluster I comprised sequences from American strains mainly, whereas cluster II contained the sequences of the strains originating from Africa and Asia chiefly. Both genetic regions of the strains investigated in the present study coincidently showed a significant sequence divergence among Arthrospira strains from East Africa, India and Mexico indicating possible distinct evolutionary lineages.

Using stable isotope probing to obtain a targeted metatranscriptome of aerobic methanotrophs in lake sediment

In this study, we demonstrate the possibility of obtaining a targeted metatranscriptome from a functional group of microorganisms using a stable isotope probing (SIP) approach. Methanotrophs in lake sediment were labelled using (13)CH4, and both labelled and unlabelled-RNA were isolated and sequenced by 454 pyrosequencing. The unlabelled metatranscriptome had a large diversity of bacterial, archaeal, eukaryotic and viral sequences as expected from a diverse sediment community. In contrast, the labelled-RNA metatranscriptome was dominated by methanotroph sequences, particularly from Methylococcaceae. Transcripts of the methane monooxygenase genes pmoCAB were the most abundant in this metatranscriptome, and the pathway of methane oxidation to CO2 could be traced, as well as many steps in the ribulose monophosphate pathway for carbon assimilation. A high abundance of mRNA transcripts for proteins related to motility was detected, suggesting an importance for methanotrophs in lake sediments. This combination of SIP and metatranscriptomics should be broadly applicable, and will enhance the detection and identification of mRNA from target organisms.

Molecular detection of uncultured cyanobacteria and aminotransferase domains for cyanotoxin production in sediments of different Kenyan lakes

PCR-based denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments was used to identify the cyanobacterial phylotypes in sediments and plankton of saline-alkaline and freshwater lakes of Kenya. The detection of the aminotransferase domain located on modules mcyE and ndaF using specific molecular markers confirmed the presence of potential toxin-producing cyanobacteria. The eight nucleotide sequences obtained from DGGE bands were placed in three divergent cyanobacterial clusters. Five nucleotide sequences were close to members of the genera Anabaenopsis and Umezakia (Nostocales), two sequences fell in the cluster with Arthrospira sp. (Oscillatoriales) and one sequence was related to Chroococcidiopsis sp. (Pleurocapsales). The presence of the latter taxon was demonstrated de novo in the investigated lakes. All nine attained nucleotide sequences of the aminotransferase region belonged to the mcyE module. Five sequences of the aminotransferase domain were included in the cluster having the nucleotide sequence of Anabaena sp. but showed a separate lineage. Other four aminotransferases were placed in the cluster represented by nucleotide sequence of Microcystis aeruginosa. To our knowledge, this is the first report on molecular detection of cyanobacterial phylotypes in sediments of African lakes and aminotransferase domains for cyanotoxin production from sediment samples in general.