Functional Genetic Diversity and Culturability of Petroleum-Degrading Bacteria Isolated From Oil-Contaminated Soils

Abstract

In this study, we compared the culturability of aerobic bacteria isolated from long-term oil-contaminated soils via enrichment and direct-plating methods; bacteria were cultured at 30°C or ambient temperatures. Two soil samples were collected from two sites in the Shengli oilfield located in Dongying, China. One sample (S0) was close to the outlet of an oil-production water treatment plant, and the other sample (S1) was located 500 m downstream of the outlet. In total, 595 bacterial isolates belonging to 56 genera were isolated, distributed in Actinobacteria, Firmicutes, Bacterioidetes, and Proteobacteria. It was interesting that Actinobacteria and Firmicutes were not detected from the 16S rRNA gene clone library. The results suggested the activation of rare species during culture. Using the enrichment method, 239 isolates (31 genera) and 96 (22 genera) isolates were obtained at ambient temperatures and 30°C, respectively, from S0 soil. Using the direct-plating method, 97 isolates (15 genera) and 163 isolates (20 genera) were obtained at ambient temperatures and 30°C, respectively, from two soils. Of the 595 isolates, 244 isolates (41.7% of total isolates) could degrade n-hexadecane. A greater number of alkane-degraders was isolated at ambient temperatures using the enrichment method, suggesting that this method could significantly improve bacterial culturability. Interestingly, the proportion of alkane degrading isolates was lower in the isolates obtained using enrichment method than that obtained using direct-plating methods. Considering the greater species diversity of isolates obtained via the enrichment method, this technique could be used to increase the diversity of the microbial consortia. Furthermore, phenol hydroxylase genes (pheN), medium-chain alkane monooxygenases genes (alkB and CYP153A), and long-chain alkane monooxygenase gene (almA) were detected in 60 isolates (11 genotypes), 91 isolates (27 genotypes) and 93 isolates (24 genotypes), and 34 isolates (14 genotypes), respectively. This study could provide new insights into microbial resources from oil fields or other environments, and this information will be beneficial for bioremediation of petroleum contamination and for other industrial applications.

Keywords: ambient temperature; bacteria; bioremediation; culturability; petroleum degradation.

Figure 1

Composition fractions of isolates cultured in different conditions and from different soil samples on a (A) genus and (B) phylum levels. Bacterial composition obtained from of (C) cultured strains and (D) via the clone library method.

Figure 2

Phylogenetic trees were constructed based on partial amino sequences of (A) phenol hydroxylase and (B) 16S rRNA gene sequence of bacterial strains obtained from the oil-contaminated soils. Tree topology was evaluated by bootstrap analysis based on 1,000 resampling replicates. Bootstrap values (%) are indicated at the nodes; only values > 50 are shown. The strains isolated at ambient temperature were indicated by bolded red letters; isolates cultured at 30°C were indicated by bold letters in blue; numbers in square brackets are the number of isolates within this phylotype. Scale bars represent 0.05 substitutions per site.

Figure 3

Phylogenetic trees were constructed based on the partial amino sequences of the (A) P450 (CYP153A) and (B) 16S rRNA gene sequences of bacterial strains which were obtained from the oil-contaminated soils. The strains isolated at ambient temperature were indicated by bolded red letters; isolates cultured at 30°C were indicated by bold letters in blue; numbers in square brackets are the number of isolates within this phylotype. The scale bar represents 0.1 substitutions per site.

Figure 4

Phylogenetic tree was constructed based on the partial amino sequences of (A) alkane monooxygenase (AlkB) and (B) 16S rRNA gene sequences of bacterial strains obtained from oil-contaminated soils. The scale bar represents 0.1 substitutions per site. Acinetobacter venetianus 6A2 has the same 16S rRNA as Acinetobacter venetianus RAG-1 (Throne-Holst et al., 2006).

Figure 5

Phylogenetic trees were constructed based on the partial amino sequence of (A) alkane monooxygenase (AlmA) and (B) the 16S rRNA gene sequences of bacterial strains obtained from the oil-contaminated soils. The scale bar represents 0.5 and 0.05 substitutions per site.