An efficient enrichment technique for isolation and quantification of indigenous diesel fuel-utilizing bacteria present in freshwater sediments

The toxic effects of petroleum products on the environment, particularly diesel fuel, have been well studied. Equally well researched are the methods for efficient bioremediation of petroleum pollution. Many studies have demonstrated the ability of indigenous microbiota, found in petroleum-polluted environments, to biodegrade diesel fuel. In this study, we quantified the diesel fuel-utilizing bacteria in sediments from two freshwater lakes and then began an enrichment process using a regimen of constant aeration, addition of (NH4)3HPO4 and diesel fuel. The enrichment process was performed in a tabletop bioremediation station based on an Eastern European ship-based bioremediation system. Over a three week period we observed significant increases in diesel fuel-utilizing bacteria, from 1.12 x 105 to 5.40 x 108 cfu/g dry weight in sediments from a lake that has historically been exposed to anthropogenic petroleum hydrocarbons, and from 1.63 x 105 to 5.10 x 109 cfu/g dry weight from another lake with no such exposure. In addition, we identified the predominant diesel fuel-utilizing microbiota using 16S rDNA sequence analysis. These results demonstrate that diesel fuel-utilizing bacteria can be recovered from lake sediments using this technique.