油气微生物分子勘探技术与初步应用

许科伟; 郑旭莹; 顾磊; 郭嘉琪; 汤玉平

doi:10.11781/sysydz202205896

油气微生物分子勘探技术与初步应用

doi: 10.11781/sysydz202205896

中国石化石油勘探开发研究院无锡石油地质研究所, 江苏无锡 214126

基金项目:

国家自然科学基金项目“页岩气改造—散失途径与保存条件” 41690133

详细信息

作者简介:
许科伟(1982-), 男, 博士, 高级工程师, 从事油气微生物勘探开发应用研究。E-mail: xukw.syky@sinopec.com

中图分类号: P132⁺.4
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出版历程
- 收稿日期: 2022-01-07
- 修回日期: 2022-07-04
- 刊出日期: 2022-09-28

Molecular microbial prospection for oil and gas and its preliminary application

Wuxi Research Institute of Petroleum Geology, SINOPEC, Wuxi, Jiangsu 214126, China

摘要

摘要: 油气微生物勘探技术因其多解性小、信噪比高、受环境影响小、经济快速的特点，越来越受到勘探家的重视。随着高通量测序、分子生物学技术及生物信息学的快速发展，从微生物群落整体的角度描述特定油气指示微生物的变化，已成为未来微生物勘探技术的重要发展方向。介绍土壤样品采集、DNA提取与分析、判别模型建立和有利区预测等4个油气微生物分子勘探技术关键环节，并针对土壤DNA提取和油气指示微生物检测进行重点阐述。工业级DNA提取技术研究发现，Griffth方法提取并纯化土壤微生物DNA效果最佳。针对大样本量工程，使用高通量移液工作站和商用多孔板DNA抽提试剂盒可大幅提高勘探效率；综合稳定性同位素探针和高通量测序的分子检测技术研究探明了典型含油气区主要油气指示微生物菌种分布规律，高频油指示菌(AMNR族)为节杆菌、分枝杆菌、诺卡氏菌和红螺菌，高频气指示菌为甲基球菌、甲基杆菌、甲基孢囊菌。微生物分子勘探技术在鄂尔多斯盆地杭锦旗地区的初步应用表明，该技术在黄土塬地貌区油气识别精度显著提升，在含油气性评价方面效果良好，为鄂尔多斯盆地北部致密气有利区带筛选提供了参考依据。
- 微生物勘探 /
- 分子生物学 /
- 高通量测序 /
- 稳定性同位素探针 /
- DNA /
- 鄂尔多斯盆地
Abstract: Due to the advantages such as low multi-solution, high signal-to-noise ratio, low environmental impact, high efficiency and low cost, microbial prospection for oil and gas has been accepted for petroleum exploreration. With the rapid development of high-throughput sequencing, molecular biology and bioinformatics, describing the changes of specific oil and gas indicator micro-organisms from the perspective of microbial community as a whole has become an valuable progress of microbial exploration method for the future. In this paper, soil sampling, DNA extraction and analysis, discriminant model development and favorable area prediction are introduced, and soil DNA extraction as well as oil/gas indicator micro-organism detection are emphasized. With the study of industrial-grade DNA extraction technology, Griffth method has been considered as the best effect for the extraction and purification of soil microbial DNA. For projects with a large sample number, high-throughput pipetting workstations and commercial multi-well plate DNA extraction kits can greatly improve working efficiency. Integrated stable isotope probes and the molecular detection of high-throughput sequencing showed that the main hydrocarbon indicator microorganism strains typically imply the expected distribution. High frequency oil indicator bacteria (AMNR) are Arthrobacter, Mycobacterium, Nocardia, and Rhodospirillum, while high frequency gas indicator bacteria include Methylococcus, Methylobacterium, and Methylocystis. The preliminary application in the Hangjinqi area shows that the microbial molecular exploration technology can significantly improve the oil and gas identification accuracy in loess tableland regions, and has a good effect on the evaluation of oil and gas bearing property, which provides a reference for the selection of favorable tight gas regions in the northern part of Ordos Basin.
- microbial prospection for oil and gas /
- molecular biology /
- high-throughput sequencing /
- stable isotope probes /
- DNA /
- Ordos Basin

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图 1 北美Haynesville地区(a)和Eagle Ford地区(b)页岩油气甜点预测准确率^[12]

Figure 1. Prediction accuracy of shale oil and gas sweet spots in Haynesville (a) and Eagle Ford (b), USA

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图 2 油气微生物分子勘探工作流程

Figure 2. Workflow of microbial molecular prospection for oil and gas

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图 3 商用多孔板DNA抽提试剂盒抽提土壤DNA前后对比^[12]

Figure 3. Soil DNA extraction with a commercial multiple well plate DNA extraction kit

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图 4 基于高通量测序和稳定同位素探针的油气指示微生物检测技术框架

Figure 4. Framework for the detection of oil and gas indicator microbial combined with high-throughput sequencing and stable isotope probes

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图 5 鄂尔多斯盆地杭锦旗气田上方土壤DNA-SIP各分层样品(2-12层)中甲烷氧化菌(MOB)的百分比

Figure 5. Percentage of methane-oxidizing bacteria (MOB) in each layer of DNA-SIP samples (2-12 layers) belonging to Hangjinqi gas field soil, Ordos Basin

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图 6 鄂尔多斯盆地杭锦旗地区新召研究区构造位置

Figure 6. Location of Xinzhao research area, Hangjinqi, Ordos Basin

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图 7 鄂尔多斯盆地杭锦旗地区研究区地形地貌

Figure 7. Topography of Hangjinqi area, Ordos Basin

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图 8 鄂尔多斯盆地杭锦旗新召地区油气微生物异常平面分布

Figure 8. Plane distribution of abnormal microbial zones in Xinzhao area, Hangjinqi, Ordos Basin

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表 1 土壤微生物总DNA提取方法

Table 1. Extraction methods of total DNA from soil microorganisms

方法编号	方法类型	方法简称
方法Ⅰ	GRIFFITHS^[13]	GF
方法Ⅱ	GRIFFITHS+纯化	GF-P
方法Ⅲ	Freeze-Thaw^[14]	FT
方法Ⅳ	Freeze-Thaw+纯化	FT-P
方法Ⅴ	eustylos soil DNA kit	KS-1
方法Ⅵ	FastDNA© Spin Kit for Soil kit	KS-2

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表 2 典型含油气区主要油气指示微生物菌种

Table 2. Oil and gas indicator species in typical oil and gas areas

序号	区块	油品类型	地貌	主要油气指示菌种(丰度排列前四)
1	富安	凝析油	农田	Lysobacter、Methylomirabilis、Rhodospirillum、Nocardioides
2	长岭	凝析油	农田、草地	Rhodospirillum、Pseudonocardia、Arthrobacter、Mycobacterium
3	顺北	轻质油	沙漠	Nocardioides、Ralstonia、Pesudomonas、Methylobacillus
4	涠西	轻质油	海洋沉积物	Rhodobacter、Desulfococcus、Desulfuromonas、Desulfobulbus
5	江汉	稠油/轻质油	农田	Lysobacter、Rhodospirillum、Thermomonas、Streptomyces
6	春光	稠油/轻质油	沙漠化区	Nocardioides、Arthrobacter、Rhodospirillum、Streptomyces
7	南阳	稠油	农田	Rhodospirillum、Methylococcus、Arthrobacter、Nocardioides
8	镇泾	稠油	黄土塬	Nocardioides、Rhodospirillum、Arthrobacter、Aeromicrobium
9	沾化	稠油	农田	Arthrobacter、Pseudonocardia、Rhodospirillum、Methylomirabilis
10	玉北	稠油	沙漠	Pseudonocardia、Rhodospirillum、Amycolatopsis、Streptomyces
11	杭锦旗	天然气	草原	Methylocaldum、Methylococcus、Methylobacterium、Rhodospirillum
12	普光	天然气	山地	Methylococcus、Methylobacterium、Rhodospirillum、Nocardioides
13	焦石坝	天然气	山地	Methylococcus、Methylocystis、Methylobacterium、Rhodospirillum
14	涩北	天然气	盐碱地	Methylobacterium、Methylocystis、Nocardioides、Methylococcus

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表 3 鄂尔多斯盆地杭锦旗新召地区土壤物理化学环境参数范围

Table 3. Range of soil physical and chemical environmental parameters in Xinzhao area, Hangjinqi, Ordos Basin

环境参数	均值	范围
含水量/%	7.1	5.0~10.6
pH	8.9	8.8~9.0
NOX^-1-N/(μg·g^-1)	0.96	0.51~1.30
50~100 μm颗粒	24.7	10.5~47.1

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表 4 微生物异常与已知钻井含油气性吻合情况统计

Table 4. Statistics on the coincidence of microbial anomalies with known oil and gas content of wells

井号	综合异常分区	含油气性	化探异常符合情况 (符合率达55%)	微生物异常符合情况 (符合率达61%)	综合异常符合情况 (符合率达72%)
锦25	背景区	气井	不符合	不符合	不符合
锦30	一级异常区	气井	符合	符合	符合
锦59	不确定区	气井	符合	符合	符合
锦60	二级异常区	气井	符合	符合	符合
锦61	不确定区	气井	符合	基本符合	符合
锦62	背景区	气井	不符合	不符合	不符合
锦63	背景区	气井	不符合	不符合	不符合
锦79	二级异常区	气井	符合	不符合	不符合
锦136	不确定区	气井	符合	不符合	基本符合

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