Field-based and laboratory stable isotope probing surveys of the identities of both aerobic and anaerobic benzene-metabolizing microorganisms in freshwater sediment

Laboratory incubations of coal-tar waste-contaminated sediment microbial communities under relatively controlled physiological conditions were used to interpret results of a field-based stable isotope probing (SIP) assay. Biodegradation activity of C-13-benzene was examined by GC/MS determination of net (CO2)-C-13 production and by GC headspace analysis of benzene loss. Key experimental variables were: the site of the assays (laboratory serum-bottle incubations and in situ field sediments), benzene concentration (10, 36 or 200 p.p.m. in laboratory assays), and physiological conditions (anaerobic with or without sulfate or nitrate additions versus aerobic headspace or the uncontrolled field). In anaerobic laboratory incubations of benzene at 10 p.p.m., greater than 60% of the substrate was eliminated within 15 days. During anaerobic incubations of 200 p.p.m. benzene (70 days), 0.9% benzene mineralization occurred. When benzene (36 p.p.m.) was added to sediment with air in the serum-bottle headspace, 14% of the initial C-13 was mineralized to (CO2)-C-13 in 2.5 days. In the field experiment (178 mu g C-13-benzene dosed to undisturbed sediments), net (CO2)-C-13 production reached 0.3% within 8.5 h. After isopycnic separation of C-13 (heavy)-labelled DNA from the above biodegradation assays, sequencing of C-13-DNA clone libraries revealed a broad diversity of taxa involved in benzene metabolism and distinctive libraries for each biodegradation treatment. Perhaps most importantly, in the field SIP experiment the clone libraries produced were dominated by Pelomonas (betaproteobacteria) sequences similar to those found in the anaerobic 10 p.p.m. benzene laboratory experiment. These data indicate that the physiological conditions that prevail and govern in situ biodegradation of pollutants in the field may be interpreted by knowing the physiological preferences of potentially active populations.