Genetic Diversity Of Waste Drilling Mud Degrading Bacteria And Determination Of Two New Degrading Bacterial Species

Petroleum and nature gas were very important fossil energy source for the economic development of china; however, a large amount of drilling mud was generated in the process of oil and gas exploitation. Due to its complex composition and high organic pollutant, drilling mud was difficult to degradation in natural, which coursed serious pollution for emissions in environment. At present, since the peculiarities of low cost, non-polluting and high efficiency, microbial remediation had received much concern and been widely applied in degrading the waste drilling mud.In this paper, thirty six waste drilling mud degrading bacteria isolated from three different muds were selected as materials. Using BOX-PCR and 16S ?DBA PCR-RFLP, the genetic diversity of these bacterial were determined, and the phylogeny of the representative strains was constructed by 16S rRNA gene sequence analysis of 15 representative strains. The dominant strains were screened from the representative degrading bacteria to test their degradation characteristics and primary metabolites. And then, the phylogenetic position of two potential novel species of were determined based on morphological, physiological and biochemical characteristics, whole-cell fatty acid components, the similarity and phylogenetic analysis of 16S rRNA and gyrB gene sequences. The results were as the follows:(1) The genetic diversity of waste drilling mud degrading bacteria was very rich. Analysis of BOX-PCR divided 36 bacteria into five genetic groups, and 16S rRNA gene PCR-RFLP analysis revealed that the 36 strains were divided into 18 genetic types, which revealed the genetic diversity was existed among the waste drilling mud degrading bacteria. Based on the BOX-PCR analysis,15 representative strains were selected, and the 16S rRNA gene sequences were analyzed. The results showed that the degrading strains belonging to genus of Bacillus, Halomonas, Pseudomonas, Sinobaca, Belliella and Rhizobium, and Bacillus was the predominant one.(2) The representative bacterial strains could effectively remove the COD and diesel in the waste mud under laboratory conditions, and strain JH2 was the best one. The removal efficiency of COD and diesel was 60.5% and 50% after 7 days' culture, respectively. The results illustrated that, along with the increase of quantity inoculated, the COD removal rate of drilling mud by JH2 was increased, and when the inoculation amount was 10%. the removal rate to drilling mud achieved the highest.The results also showed that JH2 had good tolerance to the environment pH value..JH2 could degrade the COD and diesel in drilling mud effectively at both pH7.0 and pH9.3 condition. JH2 could use diesel as sole carbon source to grow. The GC-MS analysis results showed that JH2 was able to degrade straight-chain paraffin of C10-C27 and some branched paraffins in diesel.(3) The polyphasic taxonomy technology was used to determine the taxonomic position of JHZ4~T and JH2~T.Strain JHZ4~T was gram-positive, motile with flagella. and rod-shaped, Its major fatty acid (>10%) was anteiso-C15:0, C-11:0 iso 3OH and iso-C16:0. The (G+C) mol% content was 50.4%(Tm). The 16S rRNA gene sequence similarity between strain JHZ4~T and Paenibacillus amylolyticus DSM11747T, Paenibacillus xylanexedens DSM 21292T and Paenibacillus tundrae DSM 21291T was 98.6%,98.2% and 98.1%, respectively. However, strain JHZ4~T only shared 69.8-88.5% gyrB gene sequence similarity with that of all strains belongs to Paenibacillus. Furthermore, the DNA-DNA relatedness between strain JHZ4~T and Paenibacillus amylolyticus DSM117471, Paenibacillus xylanexedens DSM 21292T and Paenibacillus tundrae DSM 21291T was 32.4%±1.6,39.3%±0.6 and 4.9%±0.8, respectively. All the results above suggested that strain JHZ4~T represents a novel species of the genus Paenibacillus, and the name Paenibacillus qionglaiensis sp. nov. was proposed, the type strain was JHZ4~T.JH2~T was Gram-positive, non-spore-forming and aerobic coccus. The major fatty acid (>10%) was anteiso-C15:0 and anteiso-C17:0, The (G+C) mol% content was 49%(Tm). The 16S rRNA gene and gyrB gene sequence similarity between strain JH2~T and type strain Sinobaca qinghaiensis DSM 170081 was 98.9%, and 87.8%. Futhermore, the DNA-DNA relatedness between strain JH2~T and Sinobaca qinghaiensis DSM 17008T was 40.3%. Hence, strain JH2~T represents a novel species of the genus sinobaca, and was proposed the name Sinobaca bifengensis sp. nov., the type strain was JH2~T.