Changes in salt marsh sediment microbial community structure in response to petroleum contamination

Traditional and molecular techniques using a variety of indices including bacterial plate counts, MPNs, and DNA-DNA hybridizations were implemented to assess changes in microbial communities in oil-contaminated marine sediments and in sediments undergoing bioremediation with several commercial products. All studies were conducted using sediment microcosms. Microbial DNA was extracted from the sediment using a modified version of direct DNA extraction. With this procedure the efficiency of cell lysis was greater than 95% and the average amount of DNA extracted was 5 to 8 μg/g sediment. However, the extracted DNA was colored (dark red) and required purification by electroelution prior to subsequent molecular analysis. Humic material (>2.0 μg) inhibited the binding of target DNA onto nylon membranes and decreased the efficiency of target DNA detection during hybridizations. Testing of oil-bioremedation products indicated that inorganic nutrient amendment was the most effective treatment for enhancement of oil degradation. Enhanced oil-degradation was associated with increases in the amount of extractable microbial DNA in the sediment, although not with increased viable counts (plate counts, MPN) of oil degraders, or of individual bacterial populations as detected by oligonucleotide probes. In another set of microcosm studies, the proportions of oil-degrading bacteria increased >100 fold in oil amended microcosms and >10 fold in plant detritus or oil + plant detritus amended microcosms compared to that of intact sediments. DNA-hybridizations of extractable DNA from sediments with Bacteria, Archaea and Eukarya oligonucleotide probes indicated little changes in the sediment community resulting from petroleum contamination. In contrast, bacterial community rDNA hybridization similarity indices, indicated that bacterial diversity in oil contaminated sediments decreased after one month. However, after three months the bacterial diversity of oil impacted-sediment increased toward that of the unamended control. Analysis of the bacterial communities by rDNA-hybridization was in agreement with analyses of sediment oil-degrading bacteria by MPN and petroleum degradation by GC/MS. Suggesting that as levels of petroleum contamination decreased in the sediment, the bacterial community structure returned toward the bacterial community of uncontaminated marine sediment.