Screening,Identification and Characteristics of Petroleum Degrading Bacteria from the Contaminated Soil of Yanchi Aera

doi:10.13560/j.cnki.biotech.bull.1985.2020-1273

Abstract

Abstract:

Highly efficient petroleum degrading bacteria were isolated and screened from the crude oil contaminated soil in Yanchi,Ningxia. The degradation characteristics of the strains and their remediation effect on the contaminated soil were studied,aiming to provide efficient microbial resource and theoretical basis for microbial remediation of oil contaminated soil in this area. The petroleum degrading bacteria were screened by the methods of enrichment culture and isolation and purification. The strains were identified based on physiological and biochemical characteristics and the 16S rRNA gene sequence analysis. According to the ability of producing surfactant and the degradation rate of crude oil,two efficient strains were selected to prepare single microbial agent and compound microbial agent,and their remediation effects on petroleum contaminated soil was studied. The results showed that 25 strains of petroleum degrading bacteria were isolated from petroleum contaminated soil,and most of them were gram positive. The strains belonged to 12 genera,including 6 strains each of Streptomyces and Pseudomonas as dominant genus,3 strains of Rhodococcus and 2 strains of Bacillus,and 1 strain each of the other 8 genera,with rich diversity. The strains showed different surfactant producing abilities and crude oil degradation rates. Among them,Rhodococcus strains S25 and S18 presented the highest degradation rates,50.0% and 47.2%,respectively;followed by Cytobacillus S19 and Streptomyces S10 with 44.4% degradation rate. The degradation rate of petroleum by Pseudomonas S23 was the lowest,only 25.0%. Soil remediation experiments demonstrated that the degradation rate of petroleum was 62.7% and 75.4% with the single bacterial agent prepared by S18 and S25 incubated in the contaminated soil for 90 d. The degradation effect of the composite bacterial agent prepared by the two strains was significantly better than that of the single bacterial agent,and the degradation rate reached 84.7%,indicating that there was a synergistic promotion effect between them,and they have the potential to be developed as soil remediation agents.

Key words: oil-degrading bacteria, screening and identification, degradation characteristics, soil repair

SHEN Cong, LIU Shuang, WANG Chun-xia, YAN Xue-mei, DAI Jin-xia. Screening,Identification and Characteristics of Petroleum Degrading Bacteria from the Contaminated Soil of Yanchi Aera[J]. Biotechnology Bulletin, 2021, 37(6): 127-135.

Figures/Tables 6

References 26

[1]	Sharma JK, Gautam RK, Nanekar SV, et al. Advances and perspective in bioremediation of polychlorinated biphenyls contaminated soils[J]. Environmental Science and Pollution Research International, 2018, 25(17):16355-16375. doi: 10.1007/s11356-017-8995-4 URL
[2]	Ruiqi Y, Gaosen Z, Shiweng L, et al. Degradation of crude oil by mixed cultures of bacteria isolated from the Qinghai-Tibet plateau and comparative analysis of metabolic mechanisms[J]. Environmental Science and Pollution Research, 2019, 26(2):1834-1847. doi: 10.1007/s11356-018-3718-z
[3]	Maddela NR, Masabanda M, Leiva-Mora M. Novel diesel-oil-degrading bacteria and fungi from the Ecuadorian Amazon rainforest[J]. Water Science & Technology, 2015, 71(10):1554-1561.
[4]	Ossai IC, Ahmed A, Hassan A, et al. Remediation of soil and water contaminated with petroleum hydrocarbon:A review[J]. Environmental Technology & Innovation 2020, 17
[5]	胥九兵, 迟建国, 邱维忠, 等. 微生物菌剂对石油污染土壤的修复研究[J]. 环境工程学报, 2011, 5(6):1414-1418.
	Xu JB, Chi JG, Qiu WZ, et al. Remediation of petroleum contaminated soil by bacterium microbial agent[J]. Chinese Journal of Environmental Engineering, 2011, 5(6):1414-1418.
[6]	曾玲玲, 刘德福, 张兴梅. 石油污染土壤的微生物治理研究进展[J]. 生物技术通报, 2006(6):48-51.
	Zeng LL, Liu DF, Zhang XM. Study progress on microorganic remediation of petroleum-polluted soil[J]. Biotechnology Bulletin, 2006(6):48-51.
[7]	张娟, 方祥位, 刘汉龙, 等. 石油污染土中微生物的分离鉴定及降解特性[J]. 土木与环境工程学报, 2020, 42(1):144-152.
	Zhang J, Fang XW, Liu HL, et al. Isolation, identification and degradation characteristics of microorganisms in petroleum contaminated soil[J]. Journal of Civil and Environmental Engineering, 2020, 42(1):144-152.
[8]	杨智, 陈吉祥, 秦波, 等. 3株石油降解红球菌(Rhodococcus spp. )特性及相关基因分析[J]. 应用与环境生物学报, 2015, 21(5):805-812.
	Yang Z, Chen JX, Qin B, et al. Characterization and catabolic genes detection of three oil-degrading Rhodococcus spp.[J]. Chinese Journal of Applied & Environmental Biology, 2015, 21(5):805-812.
[9]	张海荣, 唐景春, 孙克静, 等. 耐盐碱石油烃降解菌的筛选、鉴定及其耐盐碱性研究[J]. 生物技术通报, 2015, 31(1):151-159.
	Zhang HR, Tang JC, Sun KJ, et al. Isolation and identification of saline-alkaline tolerant hydrocarbon-degrading strains and study on their saline-alkaline tolerant characteristics[J]. Biotechnology Bulletin, 2015, 31(1):151-159.
[10]	陈政阳, 刘国, 唐彬彬, 等. 油基泥浆钻井岩屑中高效石油降解菌的筛选及其降解特性[J]. 环境工程, 2018, 36(2):48-53.
	Chen ZY, Liu G, Tang BB, et al. Isolation of degranding bacteria from oil base mud drilling cuttings and its degrading characteristics[J]. Environmental Engineering, 2018, 36(2):48-53.
[11]	李璐, 刘梅, 赵景联, 等. 微生物法修复陕北油田污染土壤的研究现状与展望[J]. 土壤通报, 2011, 42(4):1010-1014.
	Li L, Liu M, Zhao JL, et al. Current status and prospects of microbial remediation of soil pollution in oil fields in northern shaanxi[J]. Chinese Journal of Soil Science, 2011, 42(4):1010-1014.
[12]	Wu M, Dick WA, Li W, et al. Bioaugmentation and biostimulation of hydrocarbon degradation and the microbial community in a petroleum contaminated soil[J]. Int Biodeterior Biodegrad, 2016, 107:158-164. doi: 10.1016/j.ibiod.2015.11.019 URL
[13]	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.
	Dong XZ, Cai MY. Handbook of systematic identification of common bacteria[M]. Beijing: Science Press, 2001.
[14]	刘超. 铜绿假单胞菌NY3产表面活性剂发酵条件优化及表面活性剂性能研究[D]. 西安:西安建筑科技大学, 2011.
	Liu C. Study on medium optimization and characteristics of biosurfactant produced by Pseudomonas aeruginosa NY3[D]. Xi’an:Xi’an University of Architecture and Technology, 2011.
[15]	赵硕伟, 沈嘉澍, 沈标. 复合菌群的构建及其对石油污染土壤修复的研究[J]. 农业环境科学学报, 2011, 30(8):1567-1572.
	Zhao SW, Shen JS, Shen B. Construction of multiple bacterial consortium and its application in bioremediation of petroleum-contaminated soil[J]. Journal of Agro-Environment Science, 2011, 30(8):1567-1572.
[16]	吴涛, 谢文军, 依艳丽, 等. 耐盐石油烃降解菌的筛选鉴定及其特性研究[J]. 环境科学, 2012, 33(11):3949-3955.
	Wu T, Xie WJ, Yi YL, et al. Isolation, identification and characterization of halotolerant petroleum-degrading bacteria[J]. Environmental Science, 2012, 33(11):3949-3955. doi: 10.1021/es990200e URL
[17]	林标声, 陈雪英, 江胜滔, 等. 土壤中高效石油降解菌的筛选及其降解特征的研究[J]. 福建师大福清分校学报, 2010(2):11-16.
	Lin BS, Chen XY, Jiang ST, et al. Screening of crude oil degrading strains from the soil and research on their degration characteristics[J]. Journal of Fuqing Branch of Fujian Normal University, 2010(2):11-16.
[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 X, Wu ML, Cao BX, 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]	张楠, 陈波水, 杨致邦, 等. 润滑油降解菌的分离、鉴定及降解特性[J]. 环境科学研究, 2010, 23(6):748-753.
	Zhang N, Chen BS, Yang ZB, et al. Isolation, identification and degradation characteristics of bacterial strains for biodegradation of lubricating oil[J]. Research of Environmental Sciences, 2010, 23(6):748-753.
[20]	夏铁骑, 常慧萍, 张建清. 产生物表面活性剂石油降解菌的筛选及高效降解菌群的构建[J]. 农业灾害研究, 2013(6):49-51.
	Xia TQ, Chang HP, Zhang JQ. Screening of petroleum-degrading bacteria producing biosurfactant and community construction[J]. Journal of Agricultural Catastrophology, 2013(6):49-51.
[21]	Abalos A, Vinas M, Sabate J, et al. Enhanced biodegradation of casablanca crude oil by a microbal consortium in presence of a rhamnolipid produced by Pseudomonas aerufinosa AT10[J]. Biodegradation, 2004, 15(4):249-260. pmid: 15473554
[22]	曹文娟. 石油降解菌的筛选及其对石油污染土壤的生物修复特性研究[D]. 西安:西安建筑科技大学, 2014.
	Cao WJ. Isolation of petroleum-degrading strains and their bioremediation effects on petroleum-contaminated soil[D]. Xi’an:Xi’an University of Architecture and Technology, 2014.
[23]	孔令姣. 石油污染土壤的生物修复及细菌多样性研究[D]. 兰州:兰州理工大学, 2017.
	Kong LJ. Studies on the bioremediation of oil-contaminated soil and bacterial diversity[D]. Lanzhou:Lanzhou University of Technology, 2017.
[24]	王丽娟, 王哲, 张翼龙, 等. 土壤中石油烃降解菌群的构建及其在降解过程的特征分析[J]. 安徽农业科学, 2010, 38(20):10834-10836.
	Wang LJ, Wang Z, Zhang YL, et al. Construction of the flora degrading oil in the siol and characteristics analysisis in the degradation process[J]. Journal of Anhui Agricultural Sciences, 2010, 38(20):10834-10836.
[25]	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 degradation bacteria and interspecific interactions among four Bacillus strains[J]. Environmental Science, 2015, 36(6):2245-2251.
[26]	Gong XC, Liu ZS, Guo P, et al. Bacteria in crude oil survived autoclaving and stimulated differentially by exogenous bacteria[J]. PLoS One, 2012, 7(9):e40842. doi: 10.1371/journal.pone.0040842 URL

菌株编号 Strain No.	菌落形态 Colony morphology	革兰氏染色 Gram stain	硝酸盐还原 Nitrate reduction	淀粉水解 Starch hydrolysis	明胶实验 Gelatin experiment	V.P实验 V.P experiment	甲基红实验 Methyl red experiment	H₂O₂酶 H₂0₂ enzyme
S1	乳白色,湿润	+	+	+	-	+	+	+
S2	乳白色,湿润	-	+	+	-	+	+	-
S3	米白色,湿润	+	+	+	+	-	-	+
S4	淡黄色,干燥	+	+	+	+	-	-	+
S5	白色,干燥	+	+	+	+	+	+	+
S6	乳白色,湿润	-	+	-	-	-	-	+
S7	乳白色,湿润	+	+	+	+	-	-	+
S8	乳白色,湿润	-	+	-	-	-	-	+
S9	白色,干燥	+	-	-	+	-	+	+
S10	米色,干燥	+	-	-	-	-	-	+
S11	淡黄色,干燥	+	+	+	-	-	-	+
S12	乳白色,湿润	-	+	+	-	-	+	-
S13	米黄色,干燥	+	+	+	+	-	+	-
S14	乳白色,湿润	-	+	-	-	-	-	+
S15	乳白色,湿润	+	-	-	+	-	-	+
S16	橙色,湿润	+	+	-	+	-	-	+
S17	黄色,湿润	+	-	+	-	+	+	+
S18	淡粉色,湿润	+	+	-	-	+	-	+
S19	乳黄色,湿润	+	-	+	-	+	-	+
S20	乳黄色,湿润	+	+	+	+	+	-	+
S21	乳黄色,湿润	-	-	-	-	-	-	-
S22	黄色,湿润	+	+	+	+	-	+	+
S23	乳黄色,湿润	-	-	-	-	+	+	+
S24	乳白色,湿润	+	+	-	-	+	-	-
S25	乳黄色,湿润	+	-	-	-	-	+	-

菌株编号 Strain No.	菌落形态 Colony morphology	革兰氏染色 Gram stain	硝酸盐还原 Nitrate reduction	淀粉水解 Starch hydrolysis	明胶实验 Gelatin experiment	V.P实验 V.P experiment	甲基红实验 Methyl red experiment	H₂O₂酶 H₂0₂ enzyme
S1	乳白色,湿润	+	+	+	-	+	+	+
S2	乳白色,湿润	-	+	+	-	+	+	-
S3	米白色,湿润	+	+	+	+	-	-	+
S4	淡黄色,干燥	+	+	+	+	-	-	+
S5	白色,干燥	+	+	+	+	+	+	+
S6	乳白色,湿润	-	+	-	-	-	-	+
S7	乳白色,湿润	+	+	+	+	-	-	+
S8	乳白色,湿润	-	+	-	-	-	-	+
S9	白色,干燥	+	-	-	+	-	+	+
S10	米色,干燥	+	-	-	-	-	-	+
S11	淡黄色,干燥	+	+	+	-	-	-	+
S12	乳白色,湿润	-	+	+	-	-	+	-
S13	米黄色,干燥	+	+	+	+	-	+	-
S14	乳白色,湿润	-	+	-	-	-	-	+
S15	乳白色,湿润	+	-	-	+	-	-	+
S16	橙色,湿润	+	+	-	+	-	-	+
S17	黄色,湿润	+	-	+	-	+	+	+
S18	淡粉色,湿润	+	+	-	-	+	-	+
S19	乳黄色,湿润	+	-	+	-	+	-	+
S20	乳黄色,湿润	+	+	+	+	+	-	+
S21	乳黄色,湿润	-	-	-	-	-	-	-
S22	黄色,湿润	+	+	+	+	-	+	+
S23	乳黄色,湿润	-	-	-	-	+	+	+
S24	乳白色,湿润	+	+	-	-	+	-	-
S25	乳黄色,湿润	+	-	-	-	-	+	-

菌序号 Strain No.	参比菌株及序列号 Reference strains and serial No.	16S rDNA序列同源性 16S rDNA sequence homology/%	菌株登录号 Strain accession No.
S1	Paenibacillus tundrae A10b(EU558284)	99.6	MN859976
S2	Pseudomonas urumqiensis T3(MH355542)	97.0	MN859977
S3	Bacillus velezensis CR-502(AY603658)	99.8	MN859978
S4	Streptomyces pulveraceus LMG 20322(AJ781377)	98.9	MN859979
S5	Streptomyces rubrogriseus LMG 20318(AJ781373)	99.4	MN859980
S6	Pseudomonas brassicacearum ATCC 49054(EU391388)	99.8	MN859981
S7	Isoptericola halotolerans YIM 70177(AY789835)	99.2	MN859982
S8	Pseudomonas brassicacearum ATCC 49054(EU391388)	99.8	MN859983
S9	Streptomyces xinghaiensis S187(CP023202)	99.9	MN859984
S10	Streptomyces heliomycini NBRC 15899(AB184712)	99.3	MN859985
S11	Streptomyces lomondensis NBRC 15426(AB184673)	99.6	MN859986
S12	Pseudomonas urumqiensis T3(MH355542)	97.0	MN859987
S13	Streptomyces albidoflavus DSM 40455(Z76676)	99.6	MN859988
S14	Bordetella petrii DSM 12804(AM902716)	98.9	MN859989
S15	Brevibacillus schisleri ATCC 35690(MXAR01000082)	99.0	MN859990
S16	Rhodococcus ruber DSM 43338(LRRL01000064)	100.0	MN859991
S17	Microbacterium arabinogalactanolyticum IFO 14344(AB004715)	98.9	MN859992
S18	Rhodococcus rhodochrous NBRC 16069(BBXP01000056)	99.9	MN859993
S19	Cytobacillus oceanisediminis H2(GQ292772)	99.6	MN859994
S20	Bacillus tequilensis KCTC 13622(AYTO01000043)	99.8	MN859995
S21	Pseudomonas nitrititolerans GL14(MH917718)	100.0	MN859996
S22	Cellulosimicrobium cellulans LMG 16121(CAOI01000359)	99.8	MN859997
S23	Pseudomonas nitrititolerans GL14(MH917718)	100.0	MN859998
S24	Nocardioides aromaticivorans H-1(AB087721)	98.3	MN859999
S25	Rhodococcus rhodochrous NBRC 16069(BBXP01000056)	99.7	MW026030

菌序号 Strain No.	参比菌株及序列号 Reference strains and serial No.	16S rDNA序列同源性 16S rDNA sequence homology/%	菌株登录号 Strain accession No.
S1	Paenibacillus tundrae A10b(EU558284)	99.6	MN859976
S2	Pseudomonas urumqiensis T3(MH355542)	97.0	MN859977
S3	Bacillus velezensis CR-502(AY603658)	99.8	MN859978
S4	Streptomyces pulveraceus LMG 20322(AJ781377)	98.9	MN859979
S5	Streptomyces rubrogriseus LMG 20318(AJ781373)	99.4	MN859980
S6	Pseudomonas brassicacearum ATCC 49054(EU391388)	99.8	MN859981
S7	Isoptericola halotolerans YIM 70177(AY789835)	99.2	MN859982
S8	Pseudomonas brassicacearum ATCC 49054(EU391388)	99.8	MN859983
S9	Streptomyces xinghaiensis S187(CP023202)	99.9	MN859984
S10	Streptomyces heliomycini NBRC 15899(AB184712)	99.3	MN859985
S11	Streptomyces lomondensis NBRC 15426(AB184673)	99.6	MN859986
S12	Pseudomonas urumqiensis T3(MH355542)	97.0	MN859987
S13	Streptomyces albidoflavus DSM 40455(Z76676)	99.6	MN859988
S14	Bordetella petrii DSM 12804(AM902716)	98.9	MN859989
S15	Brevibacillus schisleri ATCC 35690(MXAR01000082)	99.0	MN859990
S16	Rhodococcus ruber DSM 43338(LRRL01000064)	100.0	MN859991
S17	Microbacterium arabinogalactanolyticum IFO 14344(AB004715)	98.9	MN859992
S18	Rhodococcus rhodochrous NBRC 16069(BBXP01000056)	99.9	MN859993
S19	Cytobacillus oceanisediminis H2(GQ292772)	99.6	MN859994
S20	Bacillus tequilensis KCTC 13622(AYTO01000043)	99.8	MN859995
S21	Pseudomonas nitrititolerans GL14(MH917718)	100.0	MN859996
S22	Cellulosimicrobium cellulans LMG 16121(CAOI01000359)	99.8	MN859997
S23	Pseudomonas nitrititolerans GL14(MH917718)	100.0	MN859998
S24	Nocardioides aromaticivorans H-1(AB087721)	98.3	MN859999
S25	Rhodococcus rhodochrous NBRC 16069(BBXP01000056)	99.7	MW026030