固氮微生物修复石油污染土壤研究进展

杜显元; 张晗昱; 李丹丹; 李兴春; 樊金娟; 陈宏坤; 王清威; 艾绍磊; 魏炜; 王磊

doi:10.13198/j.issn.1001-6929.2023.08.07

固氮微生物修复石油污染土壤研究进展

doi: 10.13198/j.issn.1001-6929.2023.08.07

杜显元^1,,
张晗昱^{1, 2},
李丹丹^1, ,,
李兴春¹,
樊金娟²,
陈宏坤¹,
王清威¹,
艾绍磊^{1, 3},
魏炜^{1, 4},
王磊⁵

1.
中国石油集团安全环保技术研究院有限公司，石油石化污染物控制与处理国家重点实验室，北京　102206
2.
沈阳农业大学生物科学技术学院，辽宁沈阳　110866
3.
中国石油集团渤海石油装备制造有限公司，天津　300450
4.
中国石油集团渤海装备辽河钻采有限公司，辽宁盘锦　124000
5.
中国农业大学生物学院微生物学与免疫学系，农业生物技术国家重点实验室，北京　100193

基金项目: 中国博士后科学基金资助项目(No.2022M713468)；中国石油集团安全环保技术研究院有限公司科学研究与技术开发项目(No.RISE2022KY08，2021DJ0806)

详细信息

作者简介:
杜显元（1983-），男，山东烟台人，高级工程师，博士，主要从事石油石化场地调查评估与修复技术研究，duxianyuan@cnpc.com.cn

通讯作者:
李丹丹(1988-)，女，黑龙江哈尔滨人，工程师，博士，主要从事微生物修复技术开发，1293162207@qq.com

中图分类号: X703.5
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出版历程
- 收稿日期: 2023-05-05
- 修回日期: 2023-08-18
- 网络出版日期: 2023-08-18

Research Progress of Remediation of Petroleum-Contaminated Soil by Nitrogen-Fixing Microorganisms

DU Xianyuan^1
,,
ZHANG Hanyu^{1, 2},
LI Dandan^{1
, ,},
LI Xingchun¹,
FAN Jinjuan²,
CHEN Hongkun¹,
WANG Qingwei¹,
AI Shaolei^{1, 3},
WEI Wei^{1, 4},
WANG Lei⁵

1.
State Key Laboratory of Petroleum Pollutants Control, China National Petroleum Corporation Research Institute of Safety and Environment Technology, Beijing 102206, China
2.
College of Biological Science and Technology, Shenyang Agricultural University, Shenyang 110866, China
3.
China Petroleum Bohai Petroleum Equipment Manufacturing Co., Ltd., Tianjin 300450, China
4.
CNPC Bohai Equipment Liaohe Drilling and Production Co., Ltd., Panjin 124000, China
5.
State Key Laboratory of Agricultural Biotechnology, Department of Microbiology and Immunology, School of Biology, China Agricultural University, Beijing 100193, China

Funds: China Postdoctoral Science Foundation (No.2022M713468); Scientific Research and Technology Development Program of Petro China Company Limited (No.RISE2022KY08, 2021DJ0806)

摘要

摘要: 微生物修复技术因低成本和环境友好的优势已成为当前石油污染土壤修复技术领域的研究热点. 然而，石油污染土壤中氮元素限制将会直接影响微生物的修复效果. 固氮微生物可以将大气中的氮气(N₂)转化成生物可利用性氮素，有利于提升环境氮素水平，对生物修复石油污染场地具有重要作用. 本文汇总了石油污染土壤固氮微生物主要类群、固氮微生物修复石油污染土壤的现状以及影响固氮微生物活性的主要因素，以期为石油污染土壤的高效生物修复提供参考. 文献资料表明，不同地理区域的固氮微生物种类存在差异，而同一地理区域固氮微生物的群落组成上也存在差异；通过改变碳源可以从混合菌群中分离出兼具固氮作用和石油污染物降解功能的固氮菌株，利用这些土著固氮菌提高了石油污染物的降解率，且复配的菌群在提升石油烃降解效率的同时也往往具有协同作用；温度、氧气和酸碱度是影响固氮微生物功能活性的关键因素. 在未来，需要利用先进的技术手段加强固氮微生物资源深度挖掘，为石油污染场地的生态修复提供更多的菌株资源；深入开展固氮微生物及固氮微生物与植物之间的互作机制研究，系统性分析多因素交互作用对固氮微生物的影响，是实现石油污染土壤微生物高效修复的重要基础.
- 石油污染 /
- 固氮菌 /
- 微生物修复 /
- 微生物活性
Abstract: Bioremediation has become a hotspot in the study of the remediation of petroleum-contaminated soil because it is both economic and environmentally friendly. However, nitrogen limitation in petroleum-contaminated soil can directly affect the microbial remediation. Nitrogen-fixing microorganisms convert atmospheric nitrogen into bioavailable nitrogen, which can significantly contribute to the microbial degradation of petroleum. In this paper, we summarized the main groups of nitrogen-fixing microorganisms in petroleum-contaminated soils and the research progress of the remediation of petroleum-contamination soils by nitrogen-fixing microorganisms, and discussed the key factors affecting the activity of nitrogen-fixing microorganisms. We hope this study can provide useful references for the microbial remediation of petroleum-contaminated soils. We found that the groups of nitrogen-fixing microorganisms were distinguishable geographically, but also in the same geographic region due to the variety of microenvironments. Strains with functions of both nitrogen fixation and petroleum-pollutant degradation could be isolated from a mixed flora by changing carbon source. Indigenous nitrogen-fixing bacteria can improve the degradation rate of petroleum pollutants, while compounded strains normally have synergistic effects in enhancing the degradation rate of petroleum hydrocarbons. We also found that environmental factors, such as temperature, oxygen concentration and pH were important in determining the functional activities of nitrogen-fixing microorganisms. Therefore, we recommend more advanced technologies to further use microbial resources and provide more nitrogen-fixing strains for further ecological restoration of petroleum-contaminated soils. In addition, more work needs to be done on the interaction mechanisms among nitrogen-fixing microorganisms and plants, and the systematic analysis of multi-factor interactions of nitrogen-fixing microorganisms. We believe such studies will provide valuable information for the improving the microbial remediation of petroleum-contaminated soils.
- petroleum pollution /
- nitrogen-fixing microorganism /
- microbial remediation /
- microbial activity

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图 1 石油烃降解菌与固氮菌协同代谢修复石油污染土壤

Figure 1. Synergism between the petroleum-degrading microbes and nitrogen-fixing bacteria for the remediation of petroleum-contaminated soil

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表 1 常见的自生、联合和共生固氮微生物

Table 1. The most frequently studied free-living, associative and symbiotic nitrogen-fixing microorganisms

固氮类型	主要菌属	数据来源
自生固氮菌	固氮菌属(Azotobacter)、假单胞菌属(Pseudomonas)、固氮单胞菌属(Azomonas)、绿硫菌属(Clathrochloris)、拜氏固氮菌属(Azotobacterbeijerinckii)、迪茨氏菌属(Dietzia)、水生螺菌属(Aquaspirillum)、鱼腥藻属(Anabaena)、梭菌属(Clostridium)、克雷伯氏杆菌属(Klebsiella)、黄杆菌属(Flavobacterium)、脱硫肠状杆菌属(Desulfotomaculum)、脱硫弧菌属(Desulfovibrio)、红螺菌属(Rhodospirillum)、红硫菌属(Chromatium)、拜耶林克氏菌属(Beijerinckia)、氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)等	文献[12-13]
共生固氮菌	根瘤菌属(Rhizobium)、弗兰克氏菌属(Frankia)等	文献[14]
联合固氮微生物	固氮螺菌属(Azospirillum)、假单胞菌属(Pseudomonas)、产碱菌属(Alcaligenes)、克雷伯氏菌属(Klebsiella)、草螺菌属(Herbaspirillum seropedicae)、芽孢杆菌属(Bacillus)、肠杆菌属(Enterobacter)、固氮根瘤菌属(Azorhizobium)等	文献[15-16]

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表 2 石油污染环境中常见的固氮微生物

Table 2. The most frequently studied nitrogen-fixing microorganisms in petroleum-contaminated sites

样品来源	主要菌属	数据来源
油藏水	短芽孢杆菌属(Brevibacillus)、布鲁氏菌属(Brucella)、农杆菌属(Agrobacterium)、固氮菌属(Azotobacter)、假单胞菌属(Pseudomonas)、固氮螺菌属(Azospirillum)、水生螺菌属(Aquaspirillum)和迪茨氏菌属(Dietzia)等	文献[9,17-18]
油泥	芽孢杆菌属(Bacillus)、类芽孢杆菌属(Paemibacillus)、原单孢菌属(Promicromonospora)、假单胞菌属(Pseudomonas)、布鲁氏杆菌属(Brucella)、根瘤菌属(Rhizobium)、弗兰克氏菌属(Frankiadicotyledon)、红细菌属(Rhodobacter)和红螺旋菌属(Rhodospirillum)等	文献[19]
石油污染土壤	固氮菌属(Azotobacter)、假单胞菌属(Pseudomonas)、巴西固氮螺菌属(Azospirillum brasilense)、水生螺菌属(Hydrospirillum)	文献[20-21]
沉积物	芽孢杆菌属(Bacillus)、迪茨氏菌属(Dietzia)、盐单胞菌属(Halomonas)、白蚁菌属(Isoptericola)、考克氏菌属(Kocuria)、海杆菌属(Marinobacter)、分枝杆菌属(Mycobacterium)、副球菌属(Paracoccus)、假交替单胞菌属(Pseudoalteromonas)、假单胞菌属(Pseudomonas)、嗜冷杆菌属(Psychrobacter)、红球菌属(Rhodococcus)、希瓦式菌属(Shewanella)等	文献[11,22]

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表 3 固氮菌固氮能力测定方法

Table 3. Determination method for the nitrogen-fixing ability of nitrogen-fixing bacteria

方法	原理	优点	缺点	数据来源
乙炔还原法	测定固氮酶催化乙炔生成乙烯的量来计算固氮酶活	费用低、速度快、检测方法简单	精度较差	文献[28]
凯氏定氮法	利用硫酸高温消煮法测定氮含量	费用较低、检测方法简单	操作时间长、精度差	文献[29]
¹⁵N示踪法	通过比较¹⁵N₂处理与对照原子丰度的差异计算固氮速率	灵敏度和准确度高、可靠性强	¹⁵N价格昂贵，检测方法繁琐	文献[30-31]

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表 4 石油污染环境中分离的主要固氮菌

Table 4. Nitrogen-fixing bacteria isolated from petroleum-contaminated sites

样品来源	碳源	主要菌属	数据来源
阿拉伯海湾沿岸	重质原油(3%，V/V)	芽孢杆菌(Bacillus)、迪茨氏菌属(Dietzia)、盐单胞菌属(Halomonas)、考克氏菌属(Kocuria)、海杆菌属(Marinobacter)、分枝杆菌属(Mycobacterium)、副球菌属(Paracoccus)、假交替单胞菌属(Pseudoalteromonas)、假单胞菌属(Pseudomonas)、嗜冷杆菌属(Psychrobacter)、红球菌属(Rhodococcus)、希瓦氏菌属(Shewanella)	文献[9,11]
辽河油田	葡萄糖(1%，m/V)	土壤杆菌属(Agrobacterium)、新根瘤菌属(Neorhizobium)、假单胞菌属(Pseudomonas)、克雷伯菌属(Klebsiella)	文献[17]
阿拉伯海湾沿岸	轻质原油(3%，V/V)	海杆菌属(Marinobacter)	文献[18]
南极燃油污染土壤	苹果酸(5%，V/V)	固氮螺菌属(Azospirillum)	文献[20]
尼日利亚轻质原油污染土壤	葡萄糖(2%，m/V)	固氮菌属(Azotobacter)	文献[21]
辽宁盘锦油田	葡萄糖(1%，m/V)	鞘脂单胞菌属(Sphingomonas)	文献[28]
中国石油总公司高雄炼油厂	葡萄糖(5%，m/V)	固氮弓菌属(Azoarcus)	文献[32]
美国夏威夷州多环芳烃污染土壤	菲(2%，V/V)	中华根瘤菌属(Sinorhizobium)	文献[33]
泰国石油污染土壤	苊烯(6%，V/V)	根瘤菌属(Rhizobium)	文献[34]

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