盐池采油区污染土壤石油降解菌的筛选鉴定及其降解特性

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

摘要/Abstract

摘要：

从宁夏盐池原油污染土壤中分离筛选高效石油降解菌,研究菌株的降解特性及其对污染土壤的修复效果,以期为该地区石油污染土壤的微生物修复提供高效菌源和理论依据。通过富集培养和分离纯化的方法筛选石油降解菌,结合菌株生理生化特征和16S rRNA基因序列对其进行鉴定;根据菌株产表面活性剂的能力和原油降解率,筛选2株高效菌株制备单一菌剂和复合菌剂,研究其对石油污染土壤的修复作用。结果表明,共分离出石油降解菌25株,多为革兰氏阳性细菌,隶属于12个属,其中链霉菌属和假单胞菌属各6株,为优势菌,红球菌属3株,芽孢杆菌属2株,其他8属各有1株,多样性丰富。菌株具备不同的产表面活性剂能力和原油降解率,其中红球菌属菌株S25和S18原油降解率最高,分别为50.0%和47.2%,Cytobacillus属S19和链霉菌属S10次之,降解率均为44.4%,假单胞菌S23最低,仅为25.0%;土壤修复实验表明,以S18和S25制备的单一菌剂孵育污染土壤90 d后,石油降解率分别为62.7%和75.4%,而二者制备的复合菌剂降解效果要显著优于单一菌剂,降解率达到84.7%,表明菌株之间存在协同促进作用,具有开发为土壤修复剂的潜能。

关键词: 石油降解菌, 筛选与鉴定, 降解特性, 土壤修复

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

沈聪, 刘爽, 王春霞, 严雪梅, 代金霞. 盐池采油区污染土壤石油降解菌的筛选鉴定及其降解特性[J]. 生物技术通报, 2021, 37(6): 127-135.

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.

图/表 6

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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