Detection of hydrocarbon degrading thermophilic bacteria using molecular biology techniques and possible uses in accelerated hydrocarbon bioremediation

During the past two years we have established that thermophilic bacteria belonging to the genus Geobacillus exist in large numbers in otherwise cool soil environments. These thermophiles have a growth range of 40-80°C and can be found in soils in high numbers (10⁴ colony forming units (CFU)/g). These thermophilic geobacilli have a rapid growth rate with doubling times between 20-30 minutes at 65-70°C on nutrient broth and most have a wide rang ability to degrade hydrocarbons including aromatic molecules. This suggested that they could play a role in the attenuation of hydrocarbon-contaminated soils. To assess their activity in situ, molecular biology techniques were employed firstly though designing a taxon specific forward PCR primer (THERM) to detect the geobacilli and secondly through targeting the alkane monooxygenase (alkB) gene expression. A reverse transcriptase PCR experiment established that the alkane monooxygenase activity in pure cultures of Geobacillus are only inducible when they are grown at temperatures > 40°C in the presence of alkane. Similarly soil microcosms spiked with alkane only gave the alkB RT PCR product when incubated at temperatures above 40°C. Mixed sequences of the alkB gene fragment were detected in soil and appear to belong to numerous thermophilic rather than mesophilic bacteria. We believe these thermophilic geobacilli may have a significant role in hydrocarbon biodegradation at elevated temperatures and may be utilized for bioremediation of hydrocarboncontaminated soils.