典型油气藏区域环境因素差异对甲烷氧化菌丰度与群落结构分布的影响

Effects of environmental factors on the abundance and distribution of methane-oxidizing bacteria in typical oil and gas reservoirs

摘要:

【目的】甲烷氧化菌(methane-oxidizing bacteria, MOB)是油气微生物勘探中重要的指标微生物，其丰度与群落结构分布受到地理位置和多种环境因素的影响。本研究以采集的7个典型油气藏区域的土壤样品为研究对象，探究油气藏区域环境因素差异对甲烷氧化菌丰度与群落结构分布的影响。【方法】对采集自7个典型油气藏区域土壤样品进行pmoA基因实时荧光定量PCR (real-time fluorescence quantitative PCR, qPCR)、细菌16S rRNA基因测序和甲烷氧化菌功能基因pmoA测序，结合环境因子，比较甲烷氧化菌丰度差异，分析环境因子对甲烷氧化菌分布的影响。【结果】土壤样品的理化性质测定发现江汉盆地样品的含水量最高，约22.8%，硝态氮平均含量最高位于玉北油田，达到31.96 μg/g干重土壤，春光油田出现最高的SO₄^2- (6 425.0 mg/g干重土壤)及Cl^- (1 617.0 mg/g干重土壤)浓度。qPCR分析发现pmoA基因丰度仅为土壤样本总细菌丰度的0.77%，表明甲烷氧化菌绝对数量较少。16S rRNA基因测序发现type I型甲基八叠球菌属(Methylosarcina)、甲基嗜热菌属(Methylocaldum)、甲基球菌属(Methylococcus)与type II型的甲基胞囊菌属(Methylocystis) 4种主要的甲烷氧化菌，甲烷氧化菌中的优势菌属的相对丰度极低，最高仅为0.124%。甲烷氧化菌功能基因pmoA测序发现type II型的甲基胞囊菌属、甲基弯曲菌属(Methylosinus)为主要优势pmoA基因型。环境因子与甲烷氧化菌丰度相关性分析表明甲烷氧化菌绝对丰度与铵态氮、pH、颗粒大小、硫酸根和氯离子显著相关，而环境因子与甲烷氧化菌的相对丰度的相关性分析仅发现与颗粒大小、总氮、总磷、金属离子(Al、Fe、K、Ca、Mg、Mn、Zn和Cu)显著相关。甲烷氧化菌pmoA功能基因组成判别相关分析(discriminant correlation analysis, DCA)表明含水量(土壤湿度)、pH值、<2 μm的土壤颗粒、总氮及钙离子对甲烷氧化菌pmoA功能基因组成影响具有显著性。【结论】本研究通过对来自7个典型油气藏区域的土壤样品的甲烷氧化菌的绝对丰度与相对丰度进行分析，发现基于不同的测量方法，甲烷氧化菌的丰度存在较大差异，16S rRNA基因测序无法较为全面地反映土壤样本中优势甲烷氧化菌的群落结构，不同地理位置的甲烷氧化菌指示群落存在差异，在全国尺度下油气区及背景点均无特有的甲烷氧化菌指示群落。对环境因子与甲烷氧化菌相关性分析发现，基于不同的甲烷氧化菌丰度指标，环境因子的显著影响具有较大差别。

Abstract:

[Objective] Methane-oxidizing bacteria (MOB) are crucial indicators in the microbial exploration of oil and gas reservoirs, while their diversity and distribution are influenced by geographical location and environmental factors. This study aims to explore the effects of environmental variations on the abundance and distribution of MOB in the soil samples collected from seven representative areas in typical oil and gas reservoirs. [Methods] Soil samples were subjected to real-time fluorescence quantitative PCR (qPCR) for pmoA and sequencing of bacterial 16S rRNA gene and pmoA. The abundance of MOB was compared among different samples, on the basis of which the impacts of environmental factors on the distribution of MOB were analyzed. [Results] The highest water content (approximately 22.8%) was detected in the samples from the Jianghan Basin. The average content of nitrate nitrogen was highest in the Yubei oil field, reaching approximately 31.96 μg/g. The Chunguang oil field showcased the highest concentrations of SO₄^2- (6 425.0 mg/g) and Cl^- (1 617.0 mg/g). The qPCR results revealed that the pmoA in MOB accounted for only 0.77% of that in total soil bacteria, indicating the low absolute abundance of MOB in the soil. The 16S rRNA gene sequencing identified three type I MOB genera (Methylosarcina, Methylocaldum, and Methylococcus) and one type II MOB genera (Methylocystis). However, the dominant genera in the MOB had extremely low relative abundance, with the maximum of 0.124%. Sequencing of pmoA revealed that Methylocystis and Methylosinus, two genera of type II MOB, were dominant. The absolute abundance of MOB showed significant correlations with ammonium nitrogen, pH, particle size, SO₄^2-, and Cl^-. The relative abundance of MOB had significant correlations only with particle size, total nitrogen, total phosphorus, and metal ions (Al, Fe, K, Ca, Mg, Mn, Zn, and Cu). The discriminant correlation analysis (DCA) indicated that soil moisture, pH, soil particles<2 μm, total nitrogen, and Ca²⁺ significantly influenced the composition of the functional gene pmoA in MOB. [Conclusion] This study analyzed the absolute and relative abundance of MOB in soil samples from seven typical oil and gas reservoirs. The MOB abundance showed significant differences depending on the measurement method used. The 16S rRNA gene sequencing fails to comprehensively reflect the community structure of MOB in the soil samples. Different geographical locations showed variations in MOB communities, and no specific indicator communities were detected for oil and gas reservoirs or background sites on a national scale. The correlation analysis between environmental factors and MOB revealed that the environmental factors had different effects on the absolute and relative abundance of MOB.