Detection and enumeration of sulphate-reducing bacteria in estuarine sediments by competitive PCR

The distribution of sulphate-reducing bacteria (SRB) in the sediments of the Colne River estuary, Essex, UK covering different saline concentrations of sediment porewater was investigated by the use of quantitative competitive PCR. Here, we show that a new PCR primer set and a new quantitative method using PCR are useful tools for the detection and the enumeration of SRB in natural environments. A PCR primer set selective for the dissimilatory sulphite reductase gene (dsr) of SRB was designed. PCR amplification using the single set of dsr-specific primers resulted in PCR products of the expected size from all 27 SRB strains tested, including Gram-negative and positive species. Sixty clones derived from sediment DNA using the primers were sequenced and all were closely related with the predicted dsr of SRB. These results indicate that PCR using the newly designed primer set are useful for the selective detection of SRB from a natural sample. This primer set was used to estimate cell numbers by dsr selective competitive PCR using a competitor, which was about 20% shorter than the targeted region of dsr. This procedure was applied to sediment samples from the River Colne estuary, Essex, UK together with simultaneous measurement of in situ rates of sulphate reduction. High densities of SRB ranging from 0.2 - 5.7 × 108 cells ml-1 wet sediment were estimated by the competitive PCR assuming that all SRB have a single copy of dsr. Using these estimates cell specific sulphate reduction rates of 10-17 to 10-15 mol of SO42- cell-1 day-1 were calculated, which is within the range of, or lower than, those previously reported for pure cultures of SRB. Our results show that the newly developed competitive PCR technique targeted to dsr is a powerful tool for rapid and reproducible estimation of SRB numbers in situ and is superior to the use of culture-dependent techniques.

Use of 16S rRNA-targeted oligonucleotide probes to investigate function and phylogeny of sulphate-reducing bacteria and methanogenic archaea in a UK estuary

Sulphate-reducing bacteria (SRB) and methanogenic archaea (MA) are important anaerobic terminal oxidisers of organic matter. However, we have little knowledge about the distribution and types of SRB and MA in the environment or the functional role they play in situ. Here we have utilised sediment slurry microcosms amended with ecologically significant substrates, including acetate and hydrogen, and specific functional inhibitors, to identify the important SRB and MA groups in two contrasting sites on a UK estuary. Substrate and inhibitor additions had significant effects on methane production and on acetate and sulphate consumption in the slurries. By using specific 16S-targeted oligonucleotide probes we were able to link specific SRB and MA groups to the use of the added substrates. Acetate consumption in the freshwater-dominated sediments was mediated by Methanosarcinales under low-sulphate conditions and Desulfobacter under the high-sulphate conditions that simulated a tidal incursion. In the marine-dominated sediments, acetate consumption was linked to Desulfobacter. Addition of trimethylamine, a non-competitive substrate for methanogenesis, led to a large increase in Methanosarcinales signal in marine slurries. Desulfobulbus was linked to non-sulphate-dependent H-2 consumption in the freshwater sediments. The addition of sulphate to freshwater sediments inhibited methane production and reduced signal from probes targeted to Methanosarcinales and Methanomicrobiales, while the addition of molybdate to marine sediments inhibited Desulfobulbus and Desulfobacterium. These data complement our understanding of the ecophysiology of the organisms detected and make a firm connection between the capabilities of species, as observed in the laboratory, to their roles in the environment. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.