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  • Y.J. Chang
  • J.R. Stephen
  • A.P. Richter
  • A.D. Venosa
  • J. Bruggemann
  • S.J. MacNaughton
  • G.A. Kowalchuk
  • J.R. Haines
  • E. Kline
  • D.C. White
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 of these cultures was investigated by PCR of the 16S rDNA gene using multiple primer combinations. Near full-length 16S rDNA clone libraries were generated and screened by restriction analysis prior to sequence analysis. Polymerase 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 to sequences obtained via cloning. All data sets suggested that the seawater culture was dominated by alpha- subgroup proteobacteria, whereas the freshwater culture was dominated by members of the beta- and gamma-proteobacteria. However, the V6-V8 primer pair was deficient in the recovery of Sphingomonas-like 16S rDNA due to a 3' terminal mismatch with the reverse primer. Most 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 dominated by genera containing known hydrocarbon-degrading species. [KEYWORDS: enrichment cultures; DGGE; hydrocarbon degradation; polymerase chain reaction; phylogenetic analysis; 16S rDNA 16s ribosomal-rna; gradient gel-electrophoresis; degrading bacteria; microbial-populations; genus sphingomonas; activated-sludge; ecology; diversity; database; probes]
Original languageEnglish
Pages (from-to)19-31
JournalJournal of Microbiological Methods
Issue number1
StatePublished - 2000

ID: 314683