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We examined the potential use of natural-abundance stable carbon isotope ratios of lipids for determining substrate usage by sulfate-reducing bacteria (SRB). Four SRB were grown under autotrophic, mixotrophic, or heterotrophic growth conditions, and the ␦ 13 C values of their individual fatty acids (FA) were determined. The FA were usually 13 C depleted in relation to biomass, with ⌬␦ 13 C (FA ؊ biomass) of ؊4 to ؊17‰; the greatest depletion occurred during heterotrophic growth. The exception was Desulfotomaculum acetoxidans, for which substrate limitation resulted in biomass and FA becoming isotopically heavier than the acetate substrate. The Sulfate-reducing bacteria (SRB) are common and functionally important members of anaerobic microbial communities. SRB populations can be identified and quantified by using molecular and phylogenetic approaches (8,20,25). When combined with measurements of rates of biogeochemical cycling and with an understanding of the physiology of these microbes through isolation and pure-culture studies, these approaches can reveal much about the roles of SRB in a variety of habitats (5,19,23,33). Although the use of stable sulfur isotopes and sensitive measurements of sulfide production have addressed the role of SRB in sulfur transformations (9, 24), little is known about the direct effect of these organisms on carbon flow in microbial communities. It is generally well recognized that SRB are important in reprocessing organic matter and can degrade a wide variety of organic substrates. Several SRB can also grow autotrophically in the absence of organic compounds (4, 29, 38). Molecular genetic and physiological studies can indicate which SRB are present in an environment as well as their potential capabilities, but such studies do not determine which carbon transformations the SRB are actually carrying out in a particular environment. The use of stable-or radioisotope-labeled substrates can reveal flows and rates of transformations (2, 3, 26) but are limited in application, because their use might alter the natural abundance of key substrates and our ability to introduce these substrates into natural environments is limited. However, the study of stable isotopic compositions, at natural abundance, is a potential way to circumvent this problem and to determine the carbon substrates actually used by SRB.We had previously grown four different SRB and determined the isotope fractionation factors for biomass production during autotrophic, mixotrophic, and heterotrophic growth (17). In order to use stable carbon isotopes to assess the mode of growth of SRB in natural environments, we needed an analysis that was more specific than bulk biomass. SRB are typical bacteria in that their membranes are composed primarily of phospholipids with ester-bound fatty acids (PLFA) that can be analyzed as fatty acid methyl esters (FAME). Several genera of SRB possess PLFA with unusual structures that are useful as biomarkers (1, 32, 35), and we have for the first time determined the ␦ 13 C values of individ...

Biogeochemical transformations occurring in the anoxic zones of stratified sedimentary microbial communities can profoundly influence the isotopic and organic signatures preserved in the fossil record. Accordingly, we have determined carbon isotope discrimination that is associated with both heterotrophic and lithotrophic growth of pure cultures of sulfate-reducing bacteria (SRB). For heterotrophic-growth experiments, substrate consumption was monitored to completion. Sealed vessels containing SRB cultures were harvested at different time intervals, and ␦ 13 C values were determined for gaseous CO 2 , organic substrates, and products such as biomass. For three of the four SRB, carbon isotope effects between the substrates, acetate or lactate and CO 2 , and the cell biomass were small, ranging from 0 to 2‰. However, for Desulfotomaculum acetoxidans, the carbon incorporated into biomass was isotopically heavier than the available substrates by 8 to 9‰. SRB grown lithoautotrophically consumed less than 3% of the available CO 2 and exhibited substantial discrimination (calculated as isotope fractionation factors [␣]), as follows: for Desulfobacterium autotrophicum, ␣ values ranged from 1.0100 to 1.0123; for Desulfobacter hydrogenophilus, the ␣ value was 0.0138, and for Desulfotomaculum acetoxidans, the ␣ value was 1.0310. Mixotrophic growth of Desulfovibrio desulfuricans on acetate and CO 2 resulted in biomass with a ␦ 13 C composition intermediate to that of the substrates. The extent of fractionation depended on which enzymatic pathways were used, the direction in which the pathways operated, and the growth rate, but fractionation was not dependent on the growth phase. To the extent that environmental conditions affect the availability of organic substrates (e.g., acetate) and reducing power (e.g., H 2 ), ecological forces can also influence carbon isotope discrimination by SRB.Sulfate-reducing bacteria (SRB) have been identified in a wide variety of anoxic environments, including microbial mats and sediments that have potential for preservation in the fossil record. SRB can account for a substantial fraction of the carbon turnover within cyanobacterial mats and thus contribute to internal biogeochemical carbon cycling, which in turn affects the sedimentary and geochemical features of the mats (5). SRB are metabolically versatile and can degrade a wide variety of organic compounds heterotrophically, while some can also grow autotrophically, fixing inorganic CO 2 into central metabolic intermediates like acetyl coenzyme A (acetyl-CoA) (27). Numerous methods are now available for measuring the impact of these organisms on sulfur transformations in present and ancient microbial communities (13). However, much less is known about carbon recycling by SRB in natural habitats, where multiple substrates are available in low concentrations. One approach for determining which carbon sources are being metabolized by SRB is to track their activities based on their ability to release isotopically distinct products into their s...

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