A comparison of stable‐isotope probing of DNA and phospholipid fatty acids to study prokaryotic functional diversity in sulfate‐reducing marine sediment enrichment slurries

Abstract

Summary Marine sediment slurries enriched for anaerobic, sulfate‐reducing prokaryotic communities utilizing glucose and acetate were used to provide the first comparison between stable‐isotope probing (SIP) of phospholipid fatty acids (PLFA) and DNA (16S rRNA and dsrA genes) biomarkers. Different 13 C‐labelled substrates (glucose, acetate and pyruvate) at low concentrations (100 µM) were used over a 7‐day incubation to follow and identify carbon flow into different members of the community. Limited changes in total PLFA and bacterial 16S rRNA gene DGGE profiles over 7 days suggested the presence of a stable bacterial community. A broad range of PLFA were rapidly labelled (within 12 h) in the 13 C‐glucose slurry but this changed with time, suggesting the presence of an active glucose‐utilizing population and later development of another population able to utilize glucose metabolites. The identity of the major glucose‐utilizers was unclear as 13 C‐enriched PLFA were common (16:0, 16:1, 18:1ω7, highest incorporation) and there was little difference between 12 C‐ and 13 C‐DNA 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) profiles. Seemingly glucose, a readily utilizable substrate, resulted in widespread incorporation consistent with the higher extent of 13 C‐incorporation (∼10 times) into PLFA compared with 13 C‐acetate or 13 C‐pyruvate. 13 C‐PLFA in the 13 C‐acetate and 13 C‐pyruvate slurries were similar to each other and to those that developed in the 13 C‐glucose slurry after 4 days. These were more diagnostic, with branched odd‐chain fatty acids ( i 15:0, a 15:0 and 15:1ω6) possibly indicating the presence of Desulfococcus or Desulfosarcina sulfate‐reducing bacteria (SRB) and sequences related to these SRB were in the 13 C‐acetate‐DNA dsrA gene library. The 13 C‐acetate‐DNA 16S rRNA gene library also contained sequences closely related to SRB, but these were the acetate‐utilizing Desulfobacter sp., as well as a broad range of uncultured Bacteria. In contrast, analysis of DGGE bands from 13 C‐DNA demonstrated that the candidate division JS1 and Firmicutes were actively assimilating 13 C‐acetate. Denaturing gradient gel electrophoresis also confirmed the presence of JS1 in the 13 C‐DNA from the 13 C‐glucose slurry. These results demonstrate that JS1, originally found in deep subsurface sediments, is more widely distributed in marine sediments and provides the first indication of its metabolism; incorporation of acetate and glucose (or glucose metabolites) under anaerobic, sulfate‐reducing conditions. Here we demonstrate that PLFA‐ and DNA‐SIP can be used together in a sedimentary system, with low concentrations of 13 C‐substrate and overlapping incubation times (up to 7 days) to provide complementary, although not identical, information on carbon flow and the identity of active members of an anaerobic prokaryotic community.

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