Phylogenetic analysis of aerobic freshwater and marine enrichment culturesefficient in hydrocarbon degradation: effect of profiling method

Aerobically grown enrichment cultures derived from hydrocarbon-contaminated seawater and freshwater sediments were generated by growth on crude oil as sole carbon source. Both cultures displayed a high rate of degradation for a wide range of hydrocarbon compounds. The bacterial species composition o f these cultures was investigated by PCR of the 16S rDNA gene using multipl e primer combinations. Near full-length 16S rDNA clone libraries were gener ated and screened by restriction analysis prior to sequence analysis. Polym erase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was carried out using two other PCR primer sets targeting either the V3 or V6- V8 regions, and sequences derived from prominent DGGE bands were compared t o sequences obtained via cloning. All data sets suggested that the seawater culture was dominated by alpha-subgroup proteobacteria, whereas the freshw ater culture was dominated by members of the beta- and gamma-proteobacteria . However, the V6-V8 primer pair was deficient in the recovery of Sphingomo nas-like 16S rDNA due to a 3' terminal mismatch with the reverse primer. Mo st 16S rDNA sequences recovered from the marine enrichment were not closely related to genera containing known oil-degrading organisms, although some were detected. All methods suggested that the freshwater enrichment was dom inated by genera containing known hydrocarbon-degrading species. (C) 2000 E lsevier Science B.V. All rights reserved.