Marc Llirós

High archaeal richness in the water column of a freshwater sulfurous karstic lake along an interannual study

We surveyed the archaeal assemblage in a stratified sulfurous lake (Lake Vilar, Banyoles, Spain) over 5 consecutive years to detect potential seasonal and interannual trends in the free-living planktonic Archaea composition. The combination of different primer pairs and nested PCR steps revealed an unexpectedly rich archaeal community. Overall, 140 samples were analyzed, yielding 169 different 16S rRNA gene sequences spread over 14 Crenarchaeota (109 sequences) and six Euryarchaeota phylogenetic clusters. Most of the Crenarchaeota (98% of the total crenarchaeotal sequences) affiliated within the Miscellaneous Crenarchaeota Group (MCG) and were related to both marine and freshwater phylotypes. Euryarchaeota mainly grouped within the Deep Hydrothermal Vent Euryarchaeota (DHVE) cluster (80% of the euryarchaeotal sequences) and the remaining 20% distributed into three less abundant taxa, most of them composed of soil and sediment clones. The largest fraction of phylotypes from the two archaeal kingdoms (79% of the Crenarchaeota and 54% of the Euryarchaeota) was retrieved from the anoxic hypolimnion, indicating that these cold and sulfide-rich waters constitute an unexplored source of archaeal richness. The taxon rank-frequency distribution showed two abundant taxa (MCG and DHVE) that persisted in the water column through seasons, plus several rare ones that were only detected occasionally. Differences in richness distribution and seasonality were observed, but no clear correlations were obtained when multivariate statistical analyses were carried out.

Vertical distribution of ammonia-oxidizing crenarchaeota and methanogens in the epipelagic waters of Lake Kivu (Rwanda-Democratic Republic of the Congo)

ABSTRACT Four stratified basins in Lake Kivu (Rwanda-Democratic Republic of the Congo) were sampled in March 2007 to investigate the abundance, distribution, and potential biogeochemical role of planktonic archaea. We used fluorescence in situ hybridization with catalyzed-reported deposition microscopic counts (CARD-FISH), denaturing gradient gel electrophoresis (DGGE) fingerprinting, and quantitative PCR (qPCR) of signature genes for ammonia-oxidizing archaea (16S rRNA for marine Crenarchaeota group 1.1a [MCG1] and ammonia monooxygenase subunit A [amoA]). Abundance of archaea ranged from 1 to 4.5% of total DAPI (4′,6-diamidino-2-phenylindole) counts with maximal concentrations at the oxic-anoxic transition zone (∼50-m depth). Phylogenetic analysis of the archaeal planktonic community revealed a higher level of richness of crenarchaeal 16S rRNA gene sequences (21 of the 28 operational taxonomic units [OTUs] identified [75%]) over euryarchaeotal ones (7 OTUs). Sequences affiliated with the kingdom Euryarchaeota were mainly recovered from the anoxic water compartment and mostly grouped into methanogenic lineages (Methanosarcinales and Methanocellales). In turn, crenarchaeal phylotypes were recovered throughout the sampled epipelagic waters (0- to 100-m depth), with clear phylogenetic segregation along the transition from oxic to anoxic water masses. Thus, whereas in the anoxic hypolimnion crenarchaeotal OTUs were mainly assigned to the miscellaneous crenarchaeotic group, the OTUs from the oxic-anoxic transition and above belonged to Crenarchaeota groups 1.1a and 1.1b, two lineages containing most of the ammonia-oxidizing representatives known so far. The concomitant vertical distribution of both nitrite and nitrate maxima and the copy numbers of both MCG1 16S rRNA and amoA genes suggest the potential implication of Crenarchaeota in nitrification processes occurring in the epilimnetic waters of the lake.

Contribution of deep dark fixation processes to overall CO2 incorporation and large vertical changes of microbial populations in stratified karstic lakes

We carried out a detailed study in five stratified lakes in the karstic regions of NE Spain along a redox gradient combining vertical profiles of inorganic carbon dioxide fixation and analysis of microbial (bacteria and archaea) community composition determined by 16S rRNA gene fingerprinting (DGGE), microscopic counts, and pigment analysis. High rates of non-photosynthetic (i.e., “dark”) inorganic carbon incorporation were detected mostly at deeper layers after short-term in situ incubations at noon. Significant contribution of dark CO2 incorporation was observed at the whole lake level for the single time sampling, ranging between 4 and 19% of total carbon fixation measured, and up to 31% in the case of a meromictic basin. Good agreement was found between vertical patterns in redox conditions and the different microbial diversity descriptors (DGGE band sequencing, microscopic analysis, and pigment data), showing large vertical changes in microbial community composition covering a wide range of phylogenetic diversity. Cyanobacteria, Alpha and Beta-Proteobacteria, Actinobacteria, Flavobacteria and Flectobacillaceae were the most frequently recovered groups in the DGGE from oxygenated water masses. In anoxic waters, we found Beta-Proteobacteria mostly of the Rhodoferax group, Gamma-Proteobacteria (Chromatiaceae), Delta-Proteobacteria related to different sulfate reducing bacteria, Chlorobiaceae, and anaerobic Bacteroidetes spread among the Bacteroidales, Flavobacteriales and Saprospiraceae. However, as a whole, we did not find any significant correlation between dark fixation rates and either nutrient distribution and microbial community composition in the study lakes. All of this suggests that (1) different physiologies and ecologies are simultaneously contributing to the process (2) more sensitive methods are needed and more specific compounds measured and (3) some of the non-specialist microbial populations detected may carry out carbon dioxide assimilation in the dark under in situ conditions.

Active bacteria and archaea cells fixing bicarbonate in the dark along the water column of a stratified eutrophic lagoon

We studied the carbon dioxide fixation activity in a stratified hypereutrophic karstic lagoon using a combination of fingerprinting techniques targeting bacterial and archaeal 16S rRNA genes, functional gene cloning [the acetyl-CoA carboxylase (accC)], and isotopic labelling ((14)C-bicarbonate) coupled to single-cell analyses [microautoradiography combined with catalyzed reported deposition-FISH (MAR-CARD-FISH)]. The microbial planktonic community was dominated by bacteria with maximal abundances of archaea just below the oxic/anoxic transition zone (7% of total cells). In situ incubations with radiolabelled bicarbonate showed maximal photoassimilation activity in the oxic epilimnion, whereas dark CO(2) fixation was consistently observed throughout the water column, with a maximum at the oxic/anoxic interface (8.6 mg C m(-3) h(-1)). The contributions of light and dark carbon fixation activities in the whole water column were 69% and 31% of the total C incorporated, respectively. MAR-CARD-FISH incubations corroborated these results and revealed that the highest fraction of bacterial and archaeal cells actively uptaking bicarbonate in the light was found at the surface. The bacterial community was mainly composed of green sulfur bacteria (Chlorobi) and members of the Betaproteobacteria and the Bacteroidetes. The archaeal assemblage was composed of phylotypes of the Miscellaneous Crenarchaeotic Group and a few methanogens. Clone libraries of the accC gene showed an absolute dominance of bacterial carboxylases. Our results suggest that the dark carbon fixation activity measured was mainly related to CO(2) incorporation by heterotrophs rather than to the activity of true chemoautotrophs.

Maintenance of previously uncultured freshwater archaea from anoxic waters under laboratory conditions

Culture conditions for the maintenance of previously uncultured members of the Archaea thriving in anoxic water layers of stratified freshwater lakes are described. The proposed enrichment conditions, based on the use of defined medium composition and the maintenance of anoxia, have been proven effective for the maintenance of the archaeal community with virtually no changes over time for periods up to 6xa0months as revealed by a PCR-DGGE analysis. Phylotypes belonging to groups poorly represented in culture collections such as the Deep-Sea Hydrothermal Vent Euryarchaeota (DHVE) and the Miscellaneous Crenarchaeotic Group (MCG) were maintained and selectively enriched when compared to the correspondent indigenous planktonic archaeal community.