Molecular monitoring of microbial diversity in petroleum-contaminated soil during bioremediation

Petroleum hydrocarbons are the most widespread contaminants in the environment. Several petroleum hydrocarbons are known mutagens or carcinogens for human and other organisms. They are difficult to degrade in the natural environment. Bioremediation is considered a useful method to degrade the pe,troleum hydrocarbons using the oil-degrading bacteria. In this study, cultivation-independent methods were used to study the microbial communities in petroleum contaminated sites. Phylogenetic analysis of bacterial 16S rRNA gene clone libraries from the LY, FD, and YK sites' petroleum-contaminated soil showed that 16 of 47 OTUs (operational taxonomic units) were oil-degrading bacteria belonging to Pseudomonas, Burkholderia, Azoarcus, Xanthoinonas, Bacillus, Comamonas, Nocardioides, Sphingomonas, and Alcaligenes. Proteobacteria was the dominant group (66%) and Pseudonzonas was the most abundant genus (21.6%) in these 3 clone libraries. In KH bioremediation site, there are five different treatments including bioaugmentation and biostimulation. Dynamic changes of bacterial communities in KH petroleum-contaminated site during bioremediation were monitored by PCR-DGGE, which used bacteria universal 16S rRNA gene primers. In the DGGE fingerprinting of KH site, the band at the same position with Pseudomonas markers was found in all samples. Our data indicate that Pseudomonas spp. may be important petroleum hydrocarbon-degradation bacteria in the KH site. The changes in population size of Pseudomonas in KH site during bioremediation were monitored by real-time PCR. In all 5 different bioremediation treatments, the population size of Pseudomonas reached maximum in the 14^th day (6.80*10⁴ - 2.04*10⁵ copy numbers/ng DNA) and decreased to minimum in the 24^th day (7.20*10³ - 1.52* 10⁴ copy numbers/ng DNA). The TPH-d (total petroleum hydrocarbons-diesel) degrading profile of KH site showed that the TPH-d in soil was decreased to 50-60% in the day and was almost degraded in the 24^th day. The results show that the Pseudomonas population size was affected by bioremediation treatment, but the TPH-d degrading rate of different bioremediation group were the same in KH site. The addition of bacteria or biosurfantant did not improve degradation of the TPH-d in KH site. Our findings indicate that the oil-degrading ability of original bacteria in contaminated site should be considered as an important factor for future bioremediation, and monitoring the variation of main oil-degrading bacteria can help us to understand the efficiency of bioremediation process, and enhance bioremediation.