The diversity of methanogenic archaea in enrichment cultures established from the sediments of Lonar Lake (India), a soda lake having pH approximately 10, was investigated using 16S rDNA molecular phylogenetic approach. Methanogenic enrichment cultures were developed in a medium that simulated conditions of soda lake with three different substrates viz., H(2):CO(2), sodium acetate, and trimethylamine (TMA), at alkaline pH. Archaeal 16S rRNA clone libraries were generated from enrichment cultures and 13 RFLP groups were obtained. Representative sequence analysis of each RFLP group indicated that the majority of the 16S rRNA gene sequences were phylogenetically affiliated with uncultured Archaea. Some of the groups may belong to new archaeal genera or families. Three RFLP groups were related to Methanoculleus sp, while two related to Methanocalculus sp. 16S rRNA gene sequences found in Lonar Lake were different from sequences reported from other soda lakes and more similar to those of oil reservoirs, palm oil waste treatment digesters, and paddy fields. In culture-based studies, three isolates were obtained. Two of these were related to Methanoculleus sp. IIE1 and one to Methanocalculus sp. 01F97C. These results clearly show that the Lonar Lake ecosystem harbors unexplored methanogens.
The phylogenetic diversity of bacterial communities in microbial mats of two different seasons from saline and hyperalkaline Lonar Lake was investigated using 16S rRNA gene library analysis. Arthrospira (Cyanobacteria) related clones (>80% of total clones) dominated libraries of both the seasons. Clear differences were found in both the seasons as the operational taxonomic units (OTUs) related to Fusibacter (LAI-1 and LAI-59) and Tindallia magadiensis found in post-monsoon were not found in the pre-monsoon library. Likewise, OTUs related to Planococcus rifietensis (LAII-67), Bordetella hinzii (LAII-2) and Methylobacterium variabile found in the pre-monsoon were not found in post-monsoon. The study was extended to identify methanotrophs in the surface mats. Libraries constructed with type I and type II methanotroph specific 16S rRNA gene primers showed the presence of clones (LAMI-99 and LAMII-2) closely related to Methylomicrobium buryaticum and Beijerinckiaceae family members. Denaturing gradient gel electrophoresis (DGGE) fingerprinting based on protein-coding genes (pmoA and mxaF) further confirmed the detection of Methylomicrobium sp. Hence, we report here for the first time the detection of putative methanotrophs in surface mats of Lonar Lake. The finding of clones related to organisms with interesting functional attributes such as assimilation of C 1 compounds (LAII-25, LAMI-39, LAMI-99 and LAMII-2), non-sulfur photosynthetic bacteria (LAMII-43) and clones distantly affiliated to organisms of heavily polluted environments (LAI-59 and LAMII-52), is of significant note. These preliminary results would direct future studies on the functional dynamics of microbial mat associated food web chain in the extreme environment.
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