高效原油污染降解菌的筛选、鉴定及菌群的构建

doi:10.13560/j.cnki.biotech.bull.1985.2020-1094

摘要/Abstract

摘要：

为获得高效原油降解菌株,从山东某油田的含油污泥中和实验室自有菌剂以及原油污染的土壤中分离筛选得到9株微生物,分别编号SF1、SF2、SF3、SF4、SF5、SF6、SF7、SF8和SF9。研究表明,9株菌对原油均有一定的降解能力。通过16S rRNA鉴定表明：SF1为中间苍白杆菌属（Ochrobactrum intermedium）,SF2、SF8属于肠杆菌属（Enterobacter sp.）,SF3、SF7为不动杆菌属（Acinetobacter sp.）,SF4、SF5、SF6、SF9属于假单胞菌菌属（Pseudomonas sp.）。其中,菌株SF1降解能力最强,降解率达48.73%,菌株SF2、SF7、SF9的降解能力较强,降解率分别为41.90%、45.35%、32.25%,其他菌株的降解率也高于20%。通过正交实验得出,由菌株SF1、SF2、SF3、SF4、SF5、SF6、SF7和SF9且添加量为2∶1∶1∶1∶1∶1∶2∶2组成的菌群对原油的降解效果优于单个菌株,该菌群为最佳高效菌群,降解率达57.88%。

关键词: 原油污染, 高效降解菌, 筛选, 鉴定, 菌群构建

Abstract:

In order to obtain efficient crude oil-degrading strains,9 strains of microorganisms were isolated and screened from the oily sludge of an oil field in Shandong,laboratory’s own bacterial agents and crude oil contaminated soil,which were numbered SF1,SF2,SF3,SF4,SF5,SF6,SF7,SF8 and SF9. Studies have shown that 9 strains presented certain abilities of degrading crude oil. Via 16S rRNA identification,SF1 belonged to Ochrobactrum intermedium,SF2 and SF8 belonged to Enterobacter sp,SF3 and SF7 belonged to Acinetobacter sp,SF4,SF5,SF6 and SF9 belonged to Pseudomonas sp. Among them,strain SF1 has the strongest degradability,with a degradation rate of 48.73%,strains SF2,SF7,and SF9 showed strong degradability,with degradation rates of 41.90%,45.35%,and 32.25%,respectively. The degradation rates of other strains were higher than 20%. Through orthogonal experiments,it is concluded that the bacterial flora composed of strains SF1,SF2,SF3,SF4,SF5,SF6,SF7and SF9 with the addition amount of 2∶1∶1∶1∶1∶1∶2∶2 has an effect on crude oil,and the degradation effect is better than that by a single strain,and this flora is the best efficient one,with a degradation rate of 57.88%.

Key words: crude oil pollution, highly efficient degrading bacteria, screening, identification, flora construction

朱淑芳, EHENEDEN Iyobosa, 宁海军, 尚洁昊, 李武阳, 孟宪刚. 高效原油污染降解菌的筛选、鉴定及菌群的构建[J]. 生物技术通报, 2021, 37(4): 107-115.

ZHU Shu-fang, EHENEDEN Iyobosa, NING Hai-jun, SHANG Jie-hao, LI Wu-yang, MENG Xian-gang. Screening and Identification of Highly Efficient Crude Oil-degrading Bacteria and Construction of Bacterial Consortium[J]. Biotechnology Bulletin, 2021, 37(4): 107-115.

图/表 6

参考文献 33

[1]	一苇. 能源与环境科普知识系列石油是怎么生成的?[J]. 中国石化, 2008(9):41-42.
	Yi W. Popular science knowledge series on energy and environment how is petroleum produced?[J]. Sinopec, 2008(9):41-42.
[2]	唐未庆. 适用于炼化一体化企业原油分类方法的探讨[J]. 石油炼制与化工, 2011,42(8):12-15.
	Tang WQ. Discussion on crude oil classification method suitable for integrated refining and chemical enterprises[J]. Petroleum Refining and Chemical Industry, 2011,42(8):12-15.
[3]	李鹏举, 张澍, 何耀庭. 原油分类问题[J]. 油气储运, 1990,9(1):74-77.
	Li PJ, Zhang S, He YT. The problem of crude oil classification[J]. Oil and Gas Storage and Transportation, 1990,9(1):74-77.
[4]	郑瑾, 陈宏坤, 杜显元, 等. 微生物降解苯系污染物的机理及研究进展[J]. 中国资源综合利用, 2020,38(1):118-120.
	Zheng J, Chen HK, Du XY, et al. The mechanism and research progress of microbial degradation of benzene series pollutants[J]. China Resources Comprehensive Utilization, 2020,38(1):118-120.
[5]	罗资琴. 石油及其产品中硫危害的分析[J]. 宁夏石油化工, 2003,22(3):8-10.
	Luo ZQ. Analysis of sulfur hazards in petroleum and its products[J]. Ningxia Petrochemical Industry, 2003,22(3):8-10.
[6]	Ayotamuno JM, Okparanma RN, Amadi F, et al. Enhanced remediation of an oily sludge with saline water[J]. African Journal of Environmental Science and Technology, 2011,5(4):262-267.
[7]	Bhattacharyya JK, Shekdar AV . Treatment and disposal of refinery sludges:Indian s cenario[J]. Waste Manag Res, 2003,21(3):249-261. doi: 10.1177/0734242X0302100309 URL
[8]	Meckenstock RU, Boll M, Mouttaki H, et al. Anaerobic degradation of benzene and polycyclic aromatic hydrocarbons[J]. Mol Microbiol Biotechnol, 2016,26(1-3):92-118.
[9]	Amel Bouderhem, Aminata Ould El Hadj Khelil. Isolation and characterization of crude oil degrading bacteria from soil of Ouargla(Algeria)[J]. International Journal of Biosciences, 2017,10(6):13-19.
[10]	杨丽芹, 蒋继辉. 微生物对石油烃类的降解机理[J]. 油气田环境保护, 2011,21(2):28-30, 65.
	Yang LQ, Jiang JH. Mechanism of microbial degradation of petroleum hydrocarbons[J]. Environmental Protection of Oil and Gas Fields, 2011,21(2):28-30, 65.
[11]	宋永亭, 宋欣, 高光军, 等. 一株沥青质降解菌的筛选及其对稠油作用评价[J]. 湖南大学学报:自然科学版, 2013,40(1):82-86.
	Song YT, Song X, Gao GJ, et al. Screening of an asphaltene-degrading bacteria and evaluation of its effect on heavy oil[J]. Journal of Hunan University:Natural Science Edition, 2013,40(1):82-86.
[12]	贾群超, 郭楚玲, 卢桂宁, 等. 两株稠油高效降解菌的筛选鉴定及其降解性能研究[J]. 环境工程学报, 2011,5(5):1181-1186.
	Jia QC, Guo CL, Lu GN, et al. Screening and identification of two high-efficiency heavy oil degrading bacteria and their degradation performance[J]. Journal of Environmental Engineering, 2011,5(5):1181-1186.
[13]	任妍君, 陈梅梅, 岳勇, 等. 高效稠油降解菌DL1-G的筛选及降解特性[J]. 中国环境科学, 2012,32(6):1080-1086.
	Ren YJ, Chen MM, Yue Y, et al. Screening and degradation characteristics of high-efficiency heavy oil degradation bacteria DL1-G[J]. China Environmental Science, 2012,32(6):1080-1086.
[14]	沈萍, 陈向东. 微生物学实验[M]. 北京: 高等教育出版社, 2011: 241.
	Shen P, Chen XD. Microbiology experiment[M]. Beijing: Higher Education Press, 2011: 241.
[15]	田永娥. 微生物对储油罐底油泥无公害处理初步研究[D]. 西安:西北大学, 2009.
	Tian YE. Preliminary study on the pollution-free treatment of oil sludge at the bottom of oil tank by microorganisms[D]. Xi’an:Northwest University, 2009.
[16]	Varjani Sunita J, Rana Dolly P, Bateja S, Upasani Vivek N. Isolation and screening for hydrocarbon utilizing bacteria(HUB)from petroleum samples[J]. International Journal Current Microbiology and Applied Sciences, 2013,2(4):48-60.
[17]	章慧, 郭楚玲, 卢桂宁, 等. 具有产表面活性剂功能石油降解菌的筛选及其发酵条件优化[J]. 农业环境科学学报, 2013,32(11):2185-2191.
	Zhang H, Guo CL, Lu GN, et al. Screening of petroleum-degrading bacteria with surfactant-producing function and optimization of fermentation conditions[J]. Acta Agro-Environmental Sciences, 2013,32(11):2185-2191.
[18]	沈德新, 封志纯, 杜江. 细菌DNA提取方法比较[J]. 中原医刊, 2004,31(10):20-22.
	Shen DX, Feng ZC, Du J. Comparison of bacterial DNA extraction methods[J]. Zhongyuan Medical Journal, 2004,31(10):20-22.
[19]	冯广达, 陈美标, 羊宋贞, 等. 用于PCR扩增的细菌DNA提取方法比较[J]. 华南农业大学学报, 2013,34(3):439-442.
	Feng GD, Chen MB, Yang SZ, et al. Comparison of bacterial DNA extraction methods for PCR amplification[J]. Journal of South China Agricultural University, 2013,34(3):439-442.
[20]	Gao W, Gao XX, Mi TZ, et al. Degradation potential and diversity of oil-degrading bacteria isolated from the sediments of the Jiaozhou Bay, China[J]. Acta Oceanologica Sinica, 2019,38(6):54-64. doi: 10.1007/s13131-019-1353-2 URL
[21]	王佳楠, 石妍云, 郑力燕, 等. 石油降解菌的分离鉴定及4株芽胞杆菌种间效应[J]. 环境科学, 2015,36(6):2245-2251.
	Wang JN, Shi YY, Zheng LY, et al. Isolation and identification of petroleum-degrading bacteria and the interspecific effects of 4 strains of Bacillus[J]. Environmental Science, 2015,36(6):2245-2251.
[22]	袁林杰, 彭芃, 申泰铭, 等. 石油烃降解菌的研究进展[J]. 生命科学, 2019,31(10):1088-1097.
	Yuan LJ, Peng P, Shen TM, et al. Research progress of petroleum hydrocarbon degrading bacteria[J]. Life Science, 2019,31(10):1088-1097.
[23]	田秀梅, 王晓丽, 彭士涛, 等. 一株高效原油降解不动杆菌的筛选及降解特性分析[J]. 环境工程, 2019,37(6):165-169, 95.
	Tian XM, Wang XL, Peng ST, et al. Screening of a highly efficient crude oil degrading Acinetobacter and analysis of its degradation characteristics[J]. Environmental Engineering, 2019,37(6):165-169, 95.
[24]	张攀. 高效石油烃降解菌的诱变选育及其特性研究[D]. 南京:南京工业大学, 2016.
	Zhang P. Mutation breeding and characterization of high efficient petroleum hydrocarbon degrading bacteria[D]. Nanjing:Nanjing University of Technology, 2016.
[25]	田燕, 万云洋, 孙午阳, 等. 高效石油降解菌的筛选及稳定性[J]. 中国石油大学学报:自然科学版, 2018,42(5):131-139.
	Tian Y, Wan YY, Sun WY, et al. Screening and stability of high-efficiency petroleum degradation bacteria[J]. Journal of China University of Petroleum:Edition of Natural Science, 2018,42(5):131-139.
[26]	刘玉华, 王慧, 胡晓珂. 不动杆菌属(Acinetobacter)细菌降解石油烃的研究进展[J]. 微生物学通报, 2016,43(7):1579-1589.
	Liu YH, Wang H, Hu XK. Research progress of petroleum hydrocarbon degradation by Acinetobacter bacteria[J]. Bulletin of Microbiology, 2016,43(7):1579-1589.
[27]	Nwinyi OC, Ajayi OO, Amund OO. Degradation of polynuclear aromatic hydrocarbons by two strains of Pseudomonas[J]. Brazilian Journal of Microbiology, 2016,47(3):551-562. doi: 10.1016/j.bjm.2016.04.026 URL
[28]	蓝慧, 王翀, 杨辉, 等. 原油降解细菌ODB01的筛选、鉴定及降解特性[J]. 生态与农村环境学报, 2017,33(5):466-473.
	Lan H, Wang C, Yang H, et al. Screening, identification and degradation characteristics of crude oil degrading bacteria ODB01[J]. Journal of Ecology and Rural Environment, 2017,33(5):466-473.
[29]	周婷, 陈吉祥, 杨智, 等. 一株嗜油不动杆菌(Acinetobacter oleivorans)的分离鉴定及石油降解特性[J]. 环境工程学报, 2015,9(11):5626-5632.
	Zhou T, Chen JX, Yang Z, et al. Isolation and identification of a strain of Acinetobacter oleivorans and its petroleum degradation characteristics[J]. Environmental Engineering Journal, 2015,9(11):5626-5632.
[30]	高鹏飞, 刘虹, 刘翠珠, 等. 炼油废水中筛选的一株高效降解石油烃菌株的鉴定及降解特性[J]. 环境污染与防治, 2018,40(12):51-55.
	Gao PF, Liu H, Liu CZ, et al. Identification and degradation characteristics of a petroleum hydrocarbon degrading strain screened from refinery wastewater[J]. Environmental Pollution and Prevention, 2018,40(12):51-55.
[31]	崔志松, 李倩, 高伟, 等. 复合菌液在模拟溢油岸滩修复中的应用[J]. 应用与环境生物学报, 2013,19(2):324-329.
	Cui ZS, Li Q, Gao Wei, et al. Application of composite bacterial liquid in the restoration of simulated oil spill bank[J]. Chinese Journal of Applied and Environmental Biology, 2013,19(2):324-329.
[32]	Owsianiak M, Chrzanowski L, Szulc A, et al. Biodegradation of diesel/biodiesel blends by a consortium of hydrocarbon degraders:Effect of the type of blend and the addition of biosurfactants[J]. Bioresour Technol, 2009,99(3):1497-1500. doi: 10.1016/j.biortech.2007.01.050 URL
[33]	王海峰, 李阳, 赵丽萍, 等. 稠油降解菌的筛选、鉴定与菌群构建[J]. 环境工程学报, 2011,5(4):925-929.
	Wang HF, Li Y, Zhao LP, et al. Screening, identification and flora construction of heavy oil degrading bacteria[J]. Techniques and Equipment for Environmental Pollution Control, 2011,5(4):925-929.

实验组Experimental group	SF1	SF2	SF3	SF4	SF5	SF6	SF7	SF8	SF9	降解率Degradation rate/%
1	0	0	1	0.5	0.5	0	0.5	0	0	30.20
2	1	0.5	1	0	0	0.5	0.5	0	0	38.00
3	0.5	0.5	1	0.5	0	0	1	0.5	0.5	41.25
4	0.5	0.5	0.5	1	0.5	0	0	0.5	0	43.60
5	1	0	0.5	0	1	1	0	0.5	1	37.10
6	0.5	1	0.5	0	0	1	1	0	0	31.1
7	0	1	0.5	0.5	0	0.5	0	0.5	0.5	34.45
8	0.5	0	0.5	0.5	1	0.5	0.5	1	0	28.60
9	1	0.5	0.5	0.5	0.5	0.5	1	0	1	57.88
10	1	1	1	0.5	1	0	0	1	0	24.90
11	0.5	0	0	1	0	0.5	1	1	1	28.55
12	0	0.5	0	0.5	1	1	1	1	0	40.90
13	0	1	0	1	0.5	0.5	0.5	0.5	0	19.85
14	1	1	0	0	0.5	0	1	1	0.5	28.75
15	0	0	0.5	1	1	0	1	0	0.5	30.05
16	0.5	0	1	0	0.5	0.5	0	1	0.5	19.30
17	0	0.5	1	1	0	1	0	1	1	25.80
18	0	0	0	0	0	0	0	0	0	2.50
19	1	0	0	0.5	0	1	0.5	0.5	0.5	31.60
20	0	1	1	0	1	0.5	1	0.5	1	27.30
21	1	0.5	0	1	1	0.5	0	0	0.5	11.10
22	0.5	1	0	0.5	0.5	1	0	0	1	34.05
23	0	0.5	0.5	0	0.5	1	0.5	1	0.5	17.35
24	1	1	0.5	1	0	0	0.5	1	1	28.65
25	0.5	1	1	1	1	1	0.5	0	0.5	14.35
26	1	0	1	1	0.5	1	1	0.5	0	7.60
27	0.5	0.5	0	0	1	0	0.5	0.5	1	23.80

实验组Experimental group	SF1	SF2	SF3	SF4	SF5	SF6	SF7	SF8	SF9	降解率Degradation rate/%
1	0	0	1	0.5	0.5	0	0.5	0	0	30.20
2	1	0.5	1	0	0	0.5	0.5	0	0	38.00
3	0.5	0.5	1	0.5	0	0	1	0.5	0.5	41.25
4	0.5	0.5	0.5	1	0.5	0	0	0.5	0	43.60
5	1	0	0.5	0	1	1	0	0.5	1	37.10
6	0.5	1	0.5	0	0	1	1	0	0	31.1
7	0	1	0.5	0.5	0	0.5	0	0.5	0.5	34.45
8	0.5	0	0.5	0.5	1	0.5	0.5	1	0	28.60
9	1	0.5	0.5	0.5	0.5	0.5	1	0	1	57.88
10	1	1	1	0.5	1	0	0	1	0	24.90
11	0.5	0	0	1	0	0.5	1	1	1	28.55
12	0	0.5	0	0.5	1	1	1	1	0	40.90
13	0	1	0	1	0.5	0.5	0.5	0.5	0	19.85
14	1	1	0	0	0.5	0	1	1	0.5	28.75
15	0	0	0.5	1	1	0	1	0	0.5	30.05
16	0.5	0	1	0	0.5	0.5	0	1	0.5	19.30
17	0	0.5	1	1	0	1	0	1	1	25.80
18	0	0	0	0	0	0	0	0	0	2.50
19	1	0	0	0.5	0	1	0.5	0.5	0.5	31.60
20	0	1	1	0	1	0.5	1	0.5	1	27.30
21	1	0.5	0	1	1	0.5	0	0	0.5	11.10
22	0.5	1	0	0.5	0.5	1	0	0	1	34.05
23	0	0.5	0.5	0	0.5	1	0.5	1	0.5	17.35
24	1	1	0.5	1	0	0	0.5	1	1	28.65
25	0.5	1	1	1	1	1	0.5	0	0.5	14.35
26	1	0	1	1	0.5	1	1	0.5	0	7.60
27	0.5	0.5	0	0	1	0	0.5	0.5	1	23.80