石油烃降解菌的筛选及其降解特性研究

冒学宇; 李勇; 吴丛杨慧; 蔡凯; 林雪峰

doi:10.12153/j.issn.1674-991X.20210203

石油烃降解菌的筛选及其降解特性研究

doi: 10.12153/j.issn.1674-991X.20210203

冒学宇^{1, 2,},
李勇^1, ,,
吴丛杨慧²,
蔡凯²,
林雪峰^{1, 2}

1.
苏州科技大学环境科学与工程学院
2.
苏州市宏宇环境科技股份有限公司

基金项目: 国家水体污染控制与治理科技重大专项（2017ZX07205-002）

详细信息

作者简介:
冒学宇（1996—），女，硕士研究生，主要研究方向为水污染控制理论与技术研究，mxy15501691531@163.com

通讯作者:
李勇（1969—），男，教授，主要从事废水生物处理理论与技术研究，yongli69@163.com

中图分类号: X172，X53
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- 被引次数: 0
出版历程
- 收稿日期: 2021-05-26
- 网络出版日期: 2022-06-07

Research on the screening of petroleum hydrocarbon degrading bacteria and their degradation characteristics

MAO Xueyu^{1, 2
,},
LI Yong^{1
, ,},
WU Congyanghui²,
CAI Kai²,
LIN Xuefeng^{1, 2}

1.
School of Environmental Science and Engineering, Suzhou University of Science and Technology
2.
Suzhou Hongyu Environmental Technology Co., Ltd.

摘要

摘要:
从连云港某废弃化工厂污染土壤中分离筛选高效石油烃降解菌株，研究菌株的生理生化特征并对其进行测序和种属鉴定，采用单因素试验对菌株降解柴油的环境因子进行分析。结果表明：从污染土壤中共分离出柴油降解菌株4株，经过测序及同源比对，与该4株菌株同源性最高的分别为阴沟肠杆菌（Enterobacter_cloacae，HY1），肺无色杆菌（Achromobacter_pulmonis，HY2），台湾假单胞菌（Pseudomonas_taiwanensis，HY3），铜绿假单胞菌（Pseudomonas_aeruginosa，HY4），同源性均达98%以上；4株菌株具备不同的产表面活性剂能力和柴油降解能力，其中菌株HY1和HY2对柴油的降解率最高，当柴油浓度为0.5%，处理时间为20 d时，二者对柴油的降解率均达37%以上；通过单因素试验对降解条件进行优化后，发现在柴油浓度为0.5%，降解时间为8 d时，菌株HY2和HY1最佳降解条件是初始pH为7，摇床转速为180 r/min，接种量为3%~4%，此时，二者对柴油的降解率分别为40.15%和43.87%。本研究可以丰富石油烃降解菌的菌种信息，为石油烃污染土壤修复提供菌种资源及数据支持。
- 微生物降解 /
- 柴油降解菌 /
- 降解特性 /
- 筛选分离 /
- 菌株鉴定
Abstract:
The efficient petroleum hydrocarbon degrading bacteria were isolated and screened from the polluted soil of an abandoned chemical plant in Lianyungang City. The physiological and biochemical characteristics of the strains were studied, sequenced and identified. The related environmental factors of diesel fuel degradation were analyzed by single factor test. The results showed that four strains of diesel degrading bacteria were isolated from polluted soil. After sequencing and homology comparison, the highest homology with the four strains were Enterobacter_cloacae (HY1), Achromobacter_pulmonis (HY2), Pseudomonas_taiwanensis (HY3), Pseudomonas_aeruginosa (HY4), and the homology was more than 98%. The four strains had different abilities of producing surfactant and degrading diesel oil. HY1 and HY2 had the highest degradation rate of diesel oil. When the diesel oil concentration was 0.5% and the treatment time was 20 days, the degradation rate of diesel oil by the two strains was more than 37%. After optimizing the degradation conditions by single factor experiment, when the diesel oil concentration was 0.5% and the degradation time was 8 days, the optimal degradation conditions of HY2 and HY1 were initial pH 7, shaking table speed 180 r/min and inoculation amount 3%-4%. At this time, the degradation rates of diesel oil by the two strains were 40.15% and 43.87%, respectively. This study can enrich the strain information of petroleum hydrocarbon degrading bacteria and provide strain resources and data support for the remediation of petroleum hydrocarbon contaminated soil.
- microbial degradation /
- diesel oil degrading bacteria /
- degradation characteristics /
- screening and separation /
- strain identification

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图 1 4株菌的系统发育树

Figure 1. Phylogenetic tree of 4 strains

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图 2 产表面活性剂试验结果

Figure 2. Surfactant production results

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图 3 各菌株的柴油降解率

Figure 3. Diesel degradation rate of each strain

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图 4 菌株HY1、HY2和HY4在不同柴油浓度平板的生长状态

注：数字为柴油浓度。

Figure 4. Growth state of plate with different diesel oil concentration of strains HY1, HY2 and HY4

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图 5 菌株HY1、HY2、HY4的生长曲线

Figure 5. Growth curve of strains HY1, HY2, HY4

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图 6 菌株HY1、HY2在不同pH时对柴油的降解率

Figure 6. Degradation rate of diesel oil by strains HY1 and HY2 at different pH

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图 7 菌株HY1、HY2在不同转速时对柴油的降解率

Figure 7. Degradation rate of diesel oil by strains HY1 and HY2 at different speeds

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图 8 菌株HY1、HY2在不同接种量时对柴油的降解率

Figure 8. Degradation rate of diesel oil by strains HY1 and HY2 under different inoculation amounts

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表 1 4株菌株理化性质

Table 1. Physicochemical properties of 4 strains

鉴定项目	HY1	HY2	HY3	HY4
菌体形态	杆状	杆状	杆状	杆状
菌落形态	白色、黏液状	圆形、淡黄色、湿润	扁平、白色、湿润	湿润、微绿
革兰氏染色	−	−	−	−
吲哚试验	+	+	+	+
柠檬酸钠	+	++	−	++
甲基红	−	−	−	−
VP试验	++	++	++	++
明胶液化	+	+	+	+
接触酶试验	+	+	+	+
注：+表示阳性，++表示强阳性，−表示阴性。

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表 2 4株菌的序列比对信息

Table 2. Sequence alignment information of 4 strains

菌株	同源微生物名称	同源性/%
HY1	阴沟肠杆菌（Enterobacter cloacae）	98
HY2	肺无色杆菌（Achromobacter pulmonis）	99
HY3	台湾假单胞菌（Pseudomonas taiwanensis）	99
HY4	铜绿假单胞菌（Pseudomonas aeruginosa）	99

下载: 导出CSV

参考文献(30)

[1]	朱立强.石油化工对环境的污染及应对措施[J]. 化工管理,2020(10):44-45. doi: 10.3969/j.issn.1008-4800.2020.10.032
[2]	代小丽, 王硕, 李佳斌, 等.石油污染土壤原位生物修复强化技术研究进展[J]. 环境工程技术学报,2020,10(3):456-466. doi: 10.12153/j.issn.1674-991X.20190141 DAI X L, WANG S, LI J B, et al. Research progress on in situ bioremediation enhancement technology of oil contaminated soil[J]. Journal of Environmental Engineering Technology,2020,10(3):456-466. doi: 10.12153/j.issn.1674-991X.20190141
[3]	吴蔓莉, 李可欣, 侯爽爽, 等.贫养分低有机质黄绵土中石油烃的生物去除特性及菌群结构变化[J]. 环境科学研究,2021,34(8):1961-1970. WU M L, LI K X, HOU S S, et al. Petroleum hydrocarbon degradation characteristics and microbial community shift by bioremediation in oligotrophic and low organic matter soil[J]. Research of Environmental Sciences,2021,34(8):1961-1970.
[4]	黄廷林, 徐金兰, 唐智新, 等.生物菌剂对石油污染土壤生物修复作用的研究[J]. 环境科学,2009,30(6):1838-1843. doi: 10.3321/j.issn:0250-3301.2009.06.047 HUANG T L, XU J L, TANG Z X, et al. Bioremediation of petroleum hydrocarbon contaminated soil by bioaugmentation products[J]. Environmental Science,2009,30(6):1838-1843. doi: 10.3321/j.issn:0250-3301.2009.06.047
[5]	M'RASSI A G, BENSALAH F, GURY J, et al. Isolation and characterization of different bacterial strains for bioremediation of n-alkanes and polycyclic aromatic hydrocarbons[J]. Environmental Science and Pollution Research International,2015,22(20):15332-15346. doi: 10.1007/s11356-015-4343-8
[6]	安玉姿.土壤石油污染的危害与修复[J]. 中国资源综合利用,2017,35(5):72-73. doi: 10.3969/j.issn.1008-9500.2017.05.031 AN Y Z. Harm and remediation of soil oil pollution[J]. China Resources Comprehensive Utilization,2017,35(5):72-73. doi: 10.3969/j.issn.1008-9500.2017.05.031
[7]	曲丽娜, 贾洪柏, 王秋玉.油污土壤降解细菌的分离鉴定及其生物强化研究[J]. 安徽农学通报,2015,21(11):17-20. doi: 10.3969/j.issn.1007-7731.2015.11.007 QU L N, JIA H B, WANG Q Y. Isolation, identification and bioaugmentation of oil-degradation bacteria from the petroleum-contaminated soil[J]. Anhui Agricultural Science Bulletin,2015,21(11):17-20. doi: 10.3969/j.issn.1007-7731.2015.11.007
[8]	赵瑞雪, 刘淑梅, 郑笑秋.石油烃类的微生物降解[J]. 长春理工大学学报,2006,29(4):100-102.
[9]	杨雪莲, 李凤梅, 刘婉婷, 等.高效石油降解菌的筛选及其降解特性[J]. 农业环境科学学报,2008,27(1):230-233. doi: 10.3321/j.issn:1672-2043.2008.01.042 YANG X L, LI F M, LIU W T, et al. Isolation of petroleum-degrading strains and their degrading characteristics[J]. Journal of Agro-Environment Science,2008,27(1):230-233. doi: 10.3321/j.issn:1672-2043.2008.01.042
[10]	齐永强, 王红旗, 刘敬奇.土壤中石油污染物微生物降解及其降解去向[J]. 中国工程科学,2003,5(8):70-75. doi: 10.3969/j.issn.1009-1742.2003.08.011 QI Y Q, WANG H Q, LIU J Q. A research on the bioremediation and the fate of oil pollutant in soil[J]. Engineering Science,2003,5(8):70-75. doi: 10.3969/j.issn.1009-1742.2003.08.011
[11]	马健波. 深层石油污染土壤微生物修复技术研究[D]. 西安: 西安石油大学, 2017.
[12]	马强. 高效石油烃降解菌的分离、鉴定及降解能力研究[D]. 北京: 北京化工大学, 2008.
[13]	杨民和. 微生物学实验[M]. 北京: 科学出版社, 2012.
[14]	卢迪. 加油站石油烃污染黄土原位修复微生物桩技术研究[D]. 兰州: 兰州理工大学, 2019.
[15]	张凡, 佘跃惠.排油圈法对生物表面活性剂的定性与定量[J]. 化学工程师,2005,19(1):14-15. doi: 10.3969/j.issn.1002-1124.2005.01.006 ZHANG F, SHE Y H. Quantitative and qualitative analysis of biosurfactant by oil spreading method[J]. Chemical Engineer,2005,19(1):14-15. doi: 10.3969/j.issn.1002-1124.2005.01.006
[16]	DAS N, CHANDRAN P. Microbial degradation of petroleum hydrocarbon contaminants: an overview[J]. Biotechnology Research International,2011,2011:941810.
[17]	任春艳. 产微生物表面活性剂菌株的筛选、性能及其应用研究[D]. 西安: 西安建筑科技大学, 2009.
[18]	杨茜, 吴蔓莉, 曹碧霄, 等.石油降解菌的筛选、降解特性及其与基因的相关性研究[J]. 安全与环境学报,2014,14(1):187-192. YANG Q, WU M L, CAO B X, et al. Isolation of petroleum degrading strains and determination their degrading and gene characteristics[J]. Journal of Safety and Environment,2014,14(1):187-192.
[19]	徐圆圆, 刘永民.阴沟肠杆菌对石油烃污染物的降解特性[J]. 化工文摘,2008(3):40-42. XU Y Y, LIU Y M. Characteristics of petroleum degradation with the strain of enterobacte cloacae[J]. China Chemicals,2008(3):40-42.
[20]	徐苗, 晁群芳.一株产胞外邻苯二酚-2, 3-双加氧酶菌种的筛选及鉴定[J]. 中国农业科技导报,2017,19(12):34-42. XU M, CHAO Q F. Screening and identification of a strain producing extracellular catechol-2,3-dioxygenase[J]. Journal of Agricultural Science and Technology,2017,19(12):34-42.
[21]	肖连冬, 臧晋, 姬鄂豫.石油降解菌的分离、表征及其降油最适状态研究[J]. 科技创新导报,2008,5(1):46. doi: 10.3969/j.issn.1674-098X.2008.01.038
[22]	RAMASAMY S, ARUMUGAM A, CHANDRAN P. Optimization of Enterobacter cloacae (KU923381) for diesel oil degradation using response surface methodology (RSM)[J]. Journal of Microbiology,2017,55(2):104-111. doi: 10.1007/s12275-017-6265-2
[23]	OSSAI I C, AHMED A, HASSAN A, et al. Remediation of soil and water contaminated with petroleum hydrocarbon: a review[J]. Environmental Technology & Innovation,2020,17:100526.
[24]	东秀珠, 蔡妙英. 常见细菌系统鉴定手册[M]. 北京: 科学出版社, 2001.
[25]	王迎, 何闪英, 吕黎, 等.柴油污染土壤的微生物修复研究进展[J]. 能源工程,2019(3):53-57. WANG Y, HE S Y, LÜ L, et al. Research advances in microbial remediation of diesel-contaminated soils[J]. Energy Engineering,2019(3):53-57.
[26]	谢云. 高效石油烷烃降解菌及原油降解基因工程菌构建研究[D]. 西安: 西北大学, 2014.
[27]	顾锡慧, 李立峰, 滕厚开, 等.耐高温石油微生物的筛选与降解能力初探[J]. 工业水处理,2010,30(11):20-24. doi: 10.11894/1005-829x.2010.30(11).20 GU X H, LI L F, TENG H K, et al. Screening and degradation capacity of heat resisting petroleum microorganism[J]. Industrial Water Treatment,2010,30(11):20-24. doi: 10.11894/1005-829x.2010.30(11).20
[28]	张小梅, 孔萌, 邢献杰, 等.双效工程菌BBb对炼化油泥中总石油烃的降解特性[J]. 环境化学,2021,40(7):2255-2264. doi: 10.7524/j.issn.0254-6108.2020031503 ZHANG X M, KONG M, XING X J, et al. Degradation characteristics of total petroleum hydrocarbons in refining sludge by dual-function bacteria BBb[J]. Environmental Chemistry,2021,40(7):2255-2264. doi: 10.7524/j.issn.0254-6108.2020031503
[29]	李燕妮. 细菌菌群降解石油的影响因素及应用研究[D]. 天津: 天津理工大学, 2013.
[30]	付保荣, 嫣雨朦, 杨丰硕, 等. 一种降解石油污染的阴沟肠杆菌及其应用: CN111154702A[P]. 2020-05-15.