Abstract:We describe a rapid and simple enumeration method for sulphate-reducing bacteria (SRB) from aquatic environments using a quantitative real-time PCR (qPCR) in combination with a rapid DNA isolation method. Enumeration of SRB in the sediment and water samples was performed by quantifying the copy number of the dsrA gene coding for the a-subunit of the dissimilatory sulphite reductase using real-time PCR with the SYBR Green I assay. Using dsrA-specific primers, we demonstrated that quantification of SRB in known numbers of SRB assemblages can be achieved. We compared DNA isolation methods using commercial DNA extraction kits and a published technique. We found two commercial kits were of advantage for extraction of DNA from water or sediment samples, where a large number of samples could be processed at the same time. We showed this newly developed qPCR technique targeting dsrA is rapid, simple and reproducible for the quantification of SRB numbers in situ and is superior to the use of culture-dependent techniques.

Comparison of the diversity of sulfate-reducing bacterial communities in the water column and the surface sediments of a Japanese meromictic lake Abstract The diversity and abundance of sulfate-reducing bacteria (SRB) were investigated in Lake Suigetsu, a meromictic lake in Japan characterized by a permanent oxycline at a depth between 3 and 8 m separating the aerobic freshwater epilimnion from the anaerobic, saline, sulfi dogenic hypolimnion. A quantitative competitive PCR targeting the gene coding for a portion of the α-subunit of dissimilatory sulfi te reductase (dsrA) was used to assess the distribution of the SRB in the stratifi ed water column and the surface sediments. The diversity of the SRB communities was assessed using a sequence analysis of the differing dsrA isomers. The dsrA gene copy numbers of SRB in the hypolimnic waters were from 9.6 × 10 3 to 7.5 × 10 5 copies ml −1 , whereas higher dsrA copy numbers of SRB were observed in surface sediments, ranging from 1.8-8.1 × 10 7 copies ml −1 as estimated by competitive PCR. Phylogenetic analysis of the dsrA sequences retrieved from the surface sediments shows most belong to a deeply branching lineage of diverse dsrA sequences not related to any cultured SRB group. In contrast, dsrA sequences found in the oxycline waters were related to sequences of members of the genera Desulfonema, Desulfosarcina, and Dusulfococcus and to sequences of the incomplete oxidizers from the Desulfobulbaceae family. Diversity and abundance of dsrA genes signifi cantly differed between the samples from the oxycline waters and the surface sediments of Lake Suigetsu, indicating habitat-specifi c SRB communities may contribute to the biogeochemical cycling of carbon and sulfur.

Abstract:The depth distribution of sulphate-reducing bacteria (SRB) in the water column of a meromictic lake, Lake Suigetsu, Fukui, Japan was investigated using quantitative competitive PCR targeting the gene coding for portions of the a-subunit of dissimilatory sulphite reductase (dsrA). The total bacterial cell density (DAPI count) was 5Ϫ13ϫ10 6 cells mL Ϫ1 in the water column of the lake with maximum abundance occurring at the oxic-anoxic interface layer. SRBwere not detected in oxic surface water using competitive PCR. SRB were found in the anoxic waters below the oxycline ranging from 10 4 to 10 5 cells mL Ϫ1 , accounting for 0.3-8.9% of the total bacteria. The SRB cell densities were higher than previously estimated using the most-probable-number (MPN) method. Sequencing of the cloned PCR product of dsrA showed the existence of different SRB groups in the anoxic water. The majority of the dsrA sequences were associated with the Desulfosarcina-Desulfococcus-Desulfonema group and members of the Desulfobulbaceae family.Other dsrA clones belonged to the Desulfomicrobium and Desulfovibrio species as well as to a deeply branched group in the dsrA tree with no representatives from previously isolated SRB groups. These SRB species appear to be important for the sulphur and carbon cycle in the anoxic waters of Lake Suigetsu.

Lake Suigetsu is a typical meromictic lake in Japan characterized by a permanent chemocline at a depth of between 3 and 8 m separating the oxic freshwater mixolimnion from anoxic saline sulfidogenic monimolimnion. Dominant bacterioplankton populations in Lake Suigetsu were investigated using PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The bacterial population was vertically stratified, and temporal shifts in the microbial communities were observed in both the oxic and anoxic layers of Lake Suigetsu during the sampling period. Several dominant DGGE bands were excised and sequenced. In the chemocline, green sulfur bacteria phylogenetically related to the genera Prosthecochloris, Pelodyctyon, and Chlorobium within the phylum Chlorobi were dominant; the colorless sulfur bacteria closely related to the genus Thiomicrospira were detected. These sulfur bacterial groups appear to be important in the biogeochemical cycling of sulfur and/or carbon in Lake Suigetsu. Bacterial sequences affiliated with the Bacteroidetes phylum were frequent among the dominant fragments in the DGGE profiles throughout the water column. Populations possessing a fermentative metabolism exist in Bacteroidetes, suggesting they may contribute to the degradation of organic matter in the anoxic environment of Lake Suigetsu.