Using Real-Time PCR to Assess Changes in the Hydrocarbon-Degrading Microbial Community in Antarctic Soil During Bioremediation
- 1.3k Downloads
A real-time polymerase chain reaction (PCR) method to quantify the proportion of microorganisms containing alkane monooxygenase was developed and used to follow changes in the microbial community in hydrocarbon-contaminated Antarctic soil during a bioremediation field trial. Assays for the alkB and rpoB genes were validated and found to be both sensitive and reproducible (less than 2% intrarun variation and 25–38% interrun variation). Results from the real-time PCR analysis were compared to analysis of the microbial population by a culture-based technique [most probable number (MPN) counts]. Both types of analysis indicated that fertilizer addition to hydrocarbon-contaminated soil stimulated the indigenous bacterial population within 1 year. The proportion of alkB containing microorganisms was positively correlated to the concentration of n-alkanes in the soil. After the concentration of n-alkanes in the soil decreased, the proportion of alkane-degrading microorganisms decreased, but the proportion of total hydrocarbon-degrading microorganisms increased, indicating another shift in the microbial community structure and ongoing biodegradation.
KeywordsMuch Probable Number rpoB Gene Antarctic Soil alkB Gene Competitive Polymerase Chain Reaction
The authors would like to thank Paul McA. Harvey (Australian Antarctic Division) for carrying out the chemical analysis of the soil samples. This work was supported by AAS grant 1163.
- 6.Dionisi, HM, Harms, G, Layton, AC, Gregory, IR, Parker, J, Hawkins, SA, Robinson, KG, Sayler, GS (2003) Power analysis for real-time PCR quantification of genes in activated sludge and analysis of the variability introduced by DNA extraction. Appl Environ Microbiol 69: 6597–6604PubMedCrossRefGoogle Scholar
- 27.Van Beilen, JB, Mourlane, F, Seeger, MA, Kovac, J, Li, Z, Smits, TH, Fritsche, U, Witholt, B (2003) Cloning of Baeyer–Villiger monooxygenases from Comamonas, Xanthobacter and Rhodococcus using polymerase chain reaction with highly degenerate primers. Environ Microbiol 5: 174–182PubMedCrossRefGoogle Scholar
- 28.Van Beilen, JB, Funhoff, EG, van Loon, A, Just, A, Kaysser, L, Bouza, M, Holtackers, R, Rothlisberger, M, Li, Z, Witholt, B (2006) Cytochrome P450 alkane hydroxylases of the CYP153 family are common in alkane-degrading eubacteria lacking integral membrane alkane hydroxylases. Appl Environ Microbiol 72: 59–65PubMedCrossRefGoogle Scholar