Physiological characteristics and primary identification of a methane-fed bacterium from a natural gasfield in China
Introduction
Natural gas is an important source of energy, methane accounts for about 90% of its content. In the region of natural gas reservoir, light hydrocarbons seep and rise to the ground from subterranean oil and gas deposits (Saunders et al., 1999; Prasanna et al., 2010; Spulber et al., 2010). Some bacteria grow with light hydrocarbons as the carbon source in the soil of oil and gas reservoirs (Sleat and Vonderdick, 1990; Voordouw, 2011; Liu et al., 2011). Therefore, the detecting of the anomalous distribution and the characteristic of special types of bacteria in soil can be used to forecast the existence of oil and gas deposits (Larter et al., 2003; Rasheed et al., 2009). As a representative of these special bacteria, the methanotrophs can be used as the indicator bacteria in microbial prospecting of oil and gas (MPOG) (Kim et al., 2004; Wendlandt et al., 2010; Zhang et al., 2010). Additionally, methanotrophs are widely existed in nature (Liebner and Wagner, 2007; Overland et al., 2010; Kip et al., 2010). However, a number of non-methane-oxidizing bacteria are generally engaged in methane oxidation (Nakamura et al., 2007; Alperin and Hoehler, 2009). They play important roles in the carbon cycle (Delsontro et al., 2010). Until now, MPOG techniques involving the isolation of hydrocarbon-oxidizing bacteria (Sleat and Vonderdick, 1990) and molecular biology techniques (Zhang et al., 2010), but the second method is costly and indirectly. And the traditional isolation of bacteria can provide a direct study of MPOG. The types of methane-feeding bacteria are different from methanotrophs greatly. As they all can use methane as the carbon source. However, methanotrophs only survive with methane as the sole carbon source. But methane-feeding bacteria can grow with other carbon sources.
The main objectives of this study were to isolate a methane-feeding bacterium and analyze its characteristics, then identify the bacterium and construct the phylogenetic tree. The existence of the methane-feeding bacterium proves not only methanotrophs but also methane-feeding bacterium survive in soil above the natural gas reservoir. It confirms the theory and applies microbial prospecting of oil and gas (MPOG).
Section snippets
Sample collection
Soil samples were collected from a natural gasfield (38.86°N, 117.46°E) in China. Sampling sites were located above the groundwater level to the depth of 1.0 m. Soil samples were collected in sterile sealed bags and returned to the laboratory immediately.
Reagents and instruments
Pure methane gas was purchased from Beijing God Bless Shun Gas Company at purity of 99.99%. Taq enzyme, primer synthesis and other biochemical reagents were purchased from Takara Biotechnology (Dalian) Company. All other reagents were purchased
Isolation and morphological observation
In accordance with the enrichment, separation and purification methods, the strain was isolated and tentatively named Q3. Observation of colony morphology showed that it was round, slightly rough edges and central protuberance, its size was about 0.5–1 μm. In addition, the colony was light yellow. In the liquid medium, the bacteria attached to the wall displayed white and slightly yellow color. Under the light microscope, the bacteria were spherical, its sizes were 0.4–0.6 μm to 1–2 μm and
Conclusions
A methane-feeding bacterium Q3 was isolated from soil of a natural gasfield in China. The analysis based on 16S rRNA gene sequence showed that it is most similar to P. mexicana and was named Pseudoxanthomonas sp. Q3. Strain Q3 could grow with methane as the sole carbon source, and reduced to 60% in 120 h of incubation. However, other hydrocarbons-oxidizing bacteria may also exist for light hydrocarbons. In conclusion, culture-dependent microbial technique is based on the fact that
Acknowledgments
Special thanks go to Xiaowu Huang who helped improve this manuscript. This study was sponsored by the Petrotech Junior Innovation Fund of China (code: 07E1025).
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