生物压裂液驱提页岩油内源复合菌群的筛选与条件优化

doi:10.19586/j.2095-2341.2024.0143

生物技术进展 ›› 2025, Vol. 15 ›› Issue (2): 305-313.DOI: 10.19586/j.2095-2341.2024.0143

• 研究论文 • 上一篇

生物压裂液驱提页岩油内源复合菌群的筛选与条件优化

邱思元(), 徐晶雪(), 张奕婷, 孙盛阳, 盛冬雪, 高佳欣, 曲丽娜

大庆师范学院生物工程学院，黑龙江大庆 163712

收稿日期:2024-09-02 接受日期:2024-12-13 出版日期:2025-03-25 发布日期:2025-04-29
通讯作者: 徐晶雪
作者简介:邱思元 E-mail: 1178735617@qq.com；
基金资助:
黑龙江省自然科学基金联合引导项目(LH2021C002);2023年国家级大学生创新创业训练计划项目(202310235B003)

Screening and Condition Optimization of Endogenous Compound Microflora in Shale Oil Driven by Biological Fracturing Fluid

Siyuan QIU(), Jingxue XU(), Yiting ZHANG, Shengyang SUN, Dongxue SHENG, Jiaxin GAO, Lina QU

School of Biological Engineering，Daqing Normal University，Heilongjiang Daqing 163712，China

Received:2024-09-02 Accepted:2024-12-13 Online:2025-03-25 Published:2025-04-29
Contact: Jingxue XU

摘要/Abstract

摘要：

水力压裂技术在高效开采页岩油中具有独特优势，但其中所含的化学物质对地下水有较高的污染风险。利用页岩油内源功能菌系所产发酵液作为生物压裂液，参与页岩油的绿色开采，能够显著提高页岩油采收效率并减少对环境的负面影响。利用血平板初筛功能菌株，再以表面活性剂产量等作为评判标准，确定3株功能菌株并进行16S rDNA测序鉴定种属信息。按1、2、3 μL的不同接种量比例，对3株菌株复配成等体积的不同菌系，获得最优菌系配比。通过单因素实验筛选出最适因素，再采用正交法及响应面法进一步优化，获得高产培养条件。结果发现，3株高效功能菌株分别为假单胞菌属、芽孢杆菌属和陶厄氏菌属，复配比例为2∶2∶1。最适培养配方为乳糖浓度13.87 g·L^-1、过硫酸铵2.13 g·L^-1、亚硫酸铁1.75 g·L^-1、pH 6，该条件下菌系的表面活性剂产量为315.51 mg·L^-1，相比初始产量（187.30 mg·L^-1），增长了59.37%。研究结果为页岩油生物压裂液的开发提供了参考。

关键词: 页岩油开采, 水力压裂技术, 生物压裂液, 生物表面活性剂, 正交及响应面优化

Abstract:

Hydraulic fracturing technology has unique advantages in the efficient extraction of shale oil， but the chemicals contained therein pose a high risk of polluting groundwater. By using the fermentation broth produced by shale oil endogenous functional bacteria as biological fracturing fluid， participating in the green exploitation of shale oil can significantly improve the recovery rate of shale oil and reduce the negative impact on the environment. The functional strains were screened by blood plates， and the yield of surfactant was used as the evaluation criterion， and the three functional strains were determined and the species information was identified by 16S rDNA sequencing. According to the different inoculation ratios of 1， 2 and 3 μL， the three strains were compounded into different strains of the same volume to obtain the optimal strain ratio， and the most suitable factors were screened out by single factor experiments， and then the orthogonal and response surfaces were further optimized to obtain high-yield culture conditions. The results showed that the three strains of high-efficiency functional bacteria were Pseudomonas， Bacillus and Taureella， with a combination ratio of 2∶2∶1. The optimal cultivation formula was lactose concentration 13.87 g·L^-1， ammonium persulfate 2.13 g·L^-1， iron sulfite 1.75 g·L^-1 and pH 6. Under these conditions， the surfactant yield of the strain was 315.51 mg·L^-1， an increase of 59.37% compared to the initial yield of 187.30 mg·L^-1. The results of this study can provide a reference for the development of biological fracturing fluid for shale oil.

Key words: shale oil extraction, hydraulic fracturing technology, biological fracturing fluid, biosurfactant, orthogonal and response surface optimization

中图分类号:

Q819

邱思元, 徐晶雪, 张奕婷, 孙盛阳, 盛冬雪, 高佳欣, 曲丽娜. 生物压裂液驱提页岩油内源复合菌群的筛选与条件优化[J]. 生物技术进展, 2025, 15(2): 305-313.

Siyuan QIU, Jingxue XU, Yiting ZHANG, Shengyang SUN, Dongxue SHENG, Jiaxin GAO, Lina QU. Screening and Condition Optimization of Endogenous Compound Microflora in Shale Oil Driven by Biological Fracturing Fluid[J]. Current Biotechnology, 2025, 15(2): 305-313.

图/表 13

图1 菌株分离纯化及筛选和性能测定结果注：数值是3次测定的平均值±标准误，下同。

Fig. 1 Strain isolation and purification and performance determination

图2 菌株Q4、Q5、Q7系统发育树注：Stutzerimonas stutzeri—施氏假单胞菌；Pseudomonas otitidis—耳炎假单胞菌；Pseudomonas guezennei—古泽内氏假单胞菌；Pseudomonas resinovorans—树脂假单胞菌；Pseudomonas alcaligenes—产碱假单胞菌；Pseudomonas indoloxydans—氧化吲哚假单胞菌；Pseudomonas oleovorans—食油假单胞菌；Pseudomonas citronellolis—香茅醇假单胞菌；Pseudomonas aeruginosa—铜绿假单胞菌；Bacillus australimaris—中国南海芽胞杆菌；Bacillus zhangzhouensis—漳州芽孢杆菌；Bacillus safensis—沙福芽孢杆菌；Bacillus pumilus—短小芽孢杆菌；Bacillus aerophilus—嗜气芽孢杆菌；Bacillus stratosphericus—平流层芽孢杆菌；Bacillus xiamenensis—厦门芽孢杆菌；Bacillus altitudinis—高地芽孢杆菌； Bacillus aerius—气芽孢杆菌；Bacillus atrophaeus—萎缩芽孢杆菌；Bacillus velezensis—贝莱斯芽孢杆菌；Bacillus vallismortis—死亡谷芽孢杆菌；Bacillus halotolerans—耐盐芽孢杆菌；Bacillus mojavensis—摩加夫芽胞杆菌；Bacillus subtilis—枯草芽孢杆菌；Bacillus inaquosorum—栖旱芽孢杆菌；Thauera propionica—丙酸陶厄氏菌；Thauera terpenica—萜烯陶厄氏菌；Thauera humireducens—腐殖质还原陶厄氏菌；Thauera phenylacetica—苯乙酸盐陶厄氏菌；Thauera selenatis—硒酸盐陶厄氏菌；Thauera aminoaromatica—湖泊陶厄氏菌；Thauera mechernichensis—梅谢尼希陶厄氏菌；Thauera chlorobenzoica—氯苯甲酸陶厄氏菌；Thauera sinica—中国陶厄氏菌；Thauera butanivorans—食丁烷陶厄氏菌；Thauera linaloolentis—芳樟醇陶厄氏菌；Thauera aromatica—芳香陶厄氏菌；Aromatoleum evansii—埃文斯芳香降解菌；Aromatoleum petrolei—石油芳香降解菌；Aromatoleum toluclasticum—解甲苯芳香降解菌；Aromatoleum buckelii—巴克尔芳香降解菌；Aromatoleum anaerobium—厌氧芳香降解菌；Aromatoleum bremense—不来梅芳香降解菌；Aromatoleum aromaticum—芳香族降解菌。

Fig. 2 Phylogenetic tree of strains Q4、Q5 and Q7

图3 复合菌系表面活性剂产量

Fig. 3 Surfactant production of compound strains

表1 菌系能力测定

Table 1 Determination of lineage ability

Q4:Q5:Q7	乳化能力/%	明胶液化能力	原油降粘能力/cP	表面张力/(mN·m^-1)
1:1:1	79±0.03	+	47.40±0.50	22.60±0.20
2:2:1	86±0.02	+	37.50±0.20	20.17±0.12
3:2:3	81±0.02	+	45.10±0.40	21.98±0.15

图4 碳源、微量元素、氮源、pH单因素筛选的实验结果

Fig. 4 Experimental results of univariate screening of carbon source, trace element, nitrogen source, and pH

表2 菌系正交试验因素水平

Table 2 Factor levels of orthogonal test for bacterial lines

水平	A：pH	B：乳糖浓度/(g·L^-1)	C：过硫酸铵浓度/(g·L^-1)	D：硫酸亚铁浓度/(g·L^-1)
1	6	10.00	2.00	1.50
2	7	12.50	2.50	1.75
3	8	15.00	3.00	2.00

表3 菌系正交试验结果直观分析

Table 3 Visual analysis of the results of bacterial lineage orthogonal tests

试验	因素水平				表面活性剂产量/(mg·L^-1)
试验	A：pH	B：乳糖浓度/(g·L^-1)	C：过硫酸铵浓度/(g·L^-1)	D：硫酸亚铁浓度/(g·L^-1)	表面活性剂产量/(mg·L^-1)
1	1	1	1	1	260.56
2	1	2	2	2	284.46
3	1	3	3	3	270.61
4	2	1	2	3	244.16
5	2	2	3	1	279.28
6	2	3	1	2	276.91
7	3	1	3	2	285.60
8	3	2	1	3	338.34
9	3	3	2	1	305.68
K1	271.877	263.440	291.937	281.840
K2	266.783	300.693	278.100	282.323
K3	309.873	284.400	278.497	284.370
R	43.090	37.253	13.837	2.530

表4 菌系正交试验结果方差分析

Table 4 ANOVA with results of orthogonal tests

因素	偏差平方和	自由度	F值	F临界值	显著性
A	3 326.48	2	307.35	19.00	显著
B	2 092.61	2	193.35	19.00	显著
C	372.24	2	34.39	19.00	显著
D	10.82	2	1.00	19.00
误差	10.82	2

表5 试验设计及结果

Table 5 Test design and results

实验号	X1	X2	X3	Y/(g·L^-1)
1	6.00	15.00	3.00	323.81
2	7.00	16.70	2.50	321.62
3	7.00	12.50	2.50	322.51
4	7.00	12.50	2.50	326.81
5	7.00	12.50	2.50	324.61
6	7.00	12.50	2.50	327.52
7	8.00	10.50	3.00	317.23
8	7.00	12.50	3.34	321.11
9	6.00	10.00	2.00	320.63
10	8.00	15.00	2.00	320.32
11	6.00	15.00	2.00	326.61
12	7.00	12.50	2.50	323.81
13	7.00	12.50	2.50	325.12
14	7.00	8.30	2.50	318.53
15	6.00	10.00	3.00	318.80
16	5.32	12.50	2.50	324.24
17	8.00	10.00	2.00	321.62
18	8.00	15.00	3.00	319.21
19	8.68	12.50	2.50	325.01
20	7.00	12.50	1.66	326.59

表6 响应面回归方程的显著性及方差分析

Table 6 Significance and variance analysis of the response surface regression equation

方差来源	平方和	自由度	均方	F值	P值	显著性
模型	140.33	9	15.59	3.97	0.021 3	显著
A	7.58	1	7.58	1.93	0.195 0
B	20.83	1	20.83	5.30	0.044 1
C	27.41	1	27.41	6.97	0.024 7
AB	13.29	1	13.29	3.38	0.095 8
AC	0.095	1	0.095	0.024	0.879 8
BC	0.67	1	0.67	0.17	0.689 0
A2	3.76	1	3.76	0.96	0.350 9
B2	64.75	1	64.75	16.48	0.002 3
C2	8.88	1	8.88	2.26	0.163 7
残差	39.30	10	3.93
失拟项	21.91	5	4.38	1.26	0.402 9	不显著
纯误差	17.39	5	3.48
总离差	179.62	19

图5 乳糖浓度和pH大小对表面活性剂产量影响的响应曲面

Fig. 5 Response surfaces for the effect of lactose concentration and pH size on surfactant production

图6 过硫酸铵浓度和pH大小对表面活性剂产量影响的响应曲面

Fig. 6 Response surfaces for the effect of ammonium persulfate concentration and pH size on surfactant production

图7 过硫酸铵浓度和乳糖浓度对表面活性剂产量影响的响应曲面

Fig. 7 Response surfaces for the effect of ammonium persulfate concentration and lactose concentration on surfactant production

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生物压裂液驱提页岩油内源复合菌群的筛选与条件优化

Screening and Condition Optimization of Endogenous Compound Microflora in Shale Oil Driven by Biological Fracturing Fluid

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