嗜盐碱高环PAHs降解菌的分离及其降解特性研究
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摘要
为获取嗜盐碱高环多环芳烃(Polycyclic aromatic hydrocarbons,PAHs)降解菌,并研究其降解特性,利用盐度为5%、pH为8.6的无机盐培养液从延长油田石油污染土壤中富集分离出6株能以芘为唯一碳源和能源的嗜盐碱菌SYP-1、SYP-2、SYP-3、SYP-4、SYP-5和SYP-11。经形态学观察、生理生化特征分析和16S rRNA基因序列对比对菌株进行了鉴定,确定SYP-1为代尔夫特菌属(Delftia sp.),SYP-2、SYP-4和SYP-11为海杆菌属(Marinobacter sp.),SYP-3和SYP-5为芽孢杆菌属(Bacillus sp.)。初步的降解能力试验表明,6株降解菌7 d内可使初始浓度为50 mg·L~(-1)的芘降解率达42.3%～68.8%,使初始浓度为5 mg·L~(-1)的苯并[a]芘降解率达27.0%～49.4%。经耐盐碱特性分析,株菌SYP-1、SYP-2、SYP-3和SYP-11的可生长盐度范围为0～20%,SYP-4和SYP-5的可生长盐度范围为0～15%,6株菌均可在pH为5～10的环境中生长。选择了对芘和苯并[a]芘同时具有较高降解能力的4株菌SYP-2、SYP-3、SYP-4和SYP-11,分析了其在不同盐度和pH条件下对芘降解效率的影响,结果表明,无论在0～15%的盐度范围内还是在5～10的pH范围内,4株菌对芘均具有良好的降解效果。研究表明,所分离菌株具有较高的耐盐碱性,且对降解4环以上高环PAHs具有很大潜力。
To obtain the haloalkaliphilic high molecular-weight polycyclic aromatic hydrocarbon(PAH)-degrading bacteria and study their degradation characteristics, six haloalkaliphilic strains(SYP-1, SYP-2, SYP-3, SYP-4, SYP-5, and SYP-11)were isolated from oilpolluted soil in the Yanchang oilfield with pyrene as the sole carbon and energy source in a mineral salt medium with a salinity of 5% and a pH of 8.6. Based on morphological observations, physiochemical characteristics, and 16 S rRNA sequences analysis, SYP-1 was identified as Delftia sp.; SYP-2, SYP-4, and SYP-11 were identified as Marinobacter sp.; and SYP-3 and SYP-5 were identified as Bacillus sp. A preliminary degradation ability experiment showed that 42.3%～68.8% of pyrene with an initial concentration of 50 mg·L~(-1) was degraded and 27.0%～49.4% of benzo(a)pyrene with an initial concentration of 5 mg·L~(-1) was degraded by the six degrading strains in 7 days. Four strains(SYP-2, SYP-3, SYP-4, and SYP-11)with high degradation ability for pyrene and benzo[a]pyrene were selected to analyze the effects of different salinity and pH on the degradation efficiency of pyrene. In the range of 0～15% salinity or pH range of 5～10, all four strains showed efficient degradation ability of pyrene. Thus, the isolated strains have high salinity and alkali resistance, and great potential to degrade high molecular weight PAHs above four rings.
To obtain the haloalkaliphilic high molecular-weight polycyclic aromatic hydrocarbon(PAH)-degrading bacteria and study their degradation characteristics, six haloalkaliphilic strains(SYP-1, SYP-2, SYP-3, SYP-4, SYP-5, and SYP-11)were isolated from oilpolluted soil in the Yanchang oilfield with pyrene as the sole carbon and energy source in a mineral salt medium with a salinity of 5% and a pH of 8.6. Based on morphological observations, physiochemical characteristics, and 16 S rRNA sequences analysis, SYP-1 was identified as Delftia sp.; SYP-2, SYP-4, and SYP-11 were identified as Marinobacter sp.; and SYP-3 and SYP-5 were identified as Bacillus sp. A preliminary degradation ability experiment showed that 42.3%～68.8% of pyrene with an initial concentration of 50 mg·L~(-1) was degraded and 27.0%～49.4% of benzo(a)pyrene with an initial concentration of 5 mg·L~(-1) was degraded by the six degrading strains in 7 days. Four strains(SYP-2, SYP-3, SYP-4, and SYP-11)with high degradation ability for pyrene and benzo[a]pyrene were selected to analyze the effects of different salinity and pH on the degradation efficiency of pyrene. In the range of 0～15% salinity or pH range of 5～10, all four strains showed efficient degradation ability of pyrene. Thus, the isolated strains have high salinity and alkali resistance, and great potential to degrade high molecular weight PAHs above four rings.
引文
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[19]李倩,崔志松,赵爱芬,等.一株石油烃降解菌新种Marinobacter sp.PY97S的鉴定[J].微生物学报,2011,51(5):648-655.LI Qian,CUI Zhi-song,ZHAO Ai-fen,et al.Identification and characterization of a novel hydrocarbon-degrading Marinobacter sp.PY97S[J].Acta Microbiologica Sinica,2011,51(5):648-655.
[20]Zhou H,Wang H,Huang Y,et al.Characterization of pyrene degradation by halophilic Thalassospira sp.strain TSL5-1 isolated from the coastal soil of Yellow Sea,China[J].International Biodeterioration&Biodegradation,2016,107:62-69.
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[23]Minai-Tehrani D,Minoui S,Herfatmanesh A.Effect of salinity on biodegradation of polycyclic aromatic hydrocarbons(PAHs)of heavy crude oil in soil[J].Bulletin of Environmental Contamination and Toxicology,2009,82(2):179-184.
[24]Arulazhagan P,Sivaraman C,Kumar S A,et al.Co-metabolic degradation of benzo(e)pyrene by halophilic bacterial consortium at different saline conditions[J].Journal of Environmental Biology,2014,35(3):445-453.
[25]Qi Y B,Wang C Y,Lv C Y,et al.Removal capacities of polycyclic aromatic hydrocarbons(PAHs)by a newly isolated strain from oilfield produced water[J].International Journal of Environmental Research&Public Health,2017,14(2):215.
[26]宋立超,李培军,刘宛,等.盐碱土壤PAHs降解菌的筛选鉴定及其降解特性[J].微生物学通报,2011,38(2):282-287.SONG Li-chao,LI Pei-jun,LIU Wan,et al.Isolation,identification and degradation characteristics of a PAHs-degrading bacteria from salt-alkaline soil[J].Microbiology China,2011,38(2):282-287.
[27]顾平,周启星,王鑫,等.一株土著B[a]P降解菌的筛选及降解特性研究[J].农业环境科学学报,2018,37(5):926-932.GU Ping,ZHOU Qi-xing,WANG Xin,et al.The screening-out and degradation character of an indigenous benzo[a]pyrene-degrading bacteria[J].Journal of Agro-Environment Science,2018,37(5):926-932.
[28]Cui Z,Yang B,Xu G,et al.Isolation and characterization of Cycloclasticus strains from Yellow Sea sediments and biodegradation of pyrene and fluoranthene by their syntrophic association with Marinobacter strains[J].International Biodeterioration&Biodegradation,2014,91:45-51.
[29]Deng M C,Li J,Liang F R,et al.Isolation and characterization of a novel hydrocarbon-degrading bacterium Achromobacter sp.HZ01from the crude oil-contaminated seawater at the Daya Bay,southern China[J].Marine Pollution Bulletin,2014,83(1):79-86.
[30]Liu T,Zhang Z,Dong W,et al.Bioremediation of PAHs contaminated river sediment by an integrated approach with sequential injection of co-substrate and electron acceptor:Lab-scale study[J].Environmental Pollution,2017,230:413-421.
[31]Lu X Y,Li B,Zhang T,et al.Enhanced anoxic bioremediation of PAHs-contaminated sediment[J].Bioresource Technology,2012,104(1):51-58.
[2]Wise S A,Sander L C,Schantz M M.Analytical methods for determination of polycyclic aromatic hydrocarbons(PAHs)-A historical perspective on the 16 U.S.EPA priority pollutant PAHs[J].Polycyclic Aromatic Compounds,2015,35(2/3/4):187-247.
[3]Means J C.Influence of salinity upon sediment-water partitioning of aromatic hydrocarbons[J].Marine Chemistry,1995,51(1):3-16.
[4]Brinda Lakshmi M,Anandaraj V P,Velan M.Bioremediation of phenanthrene by Mycoplana sp.MVMB2 isolated from contaminated soil[J].Clean-Soil,Air,Water,2013,41(1):86-93.
[5]范瑞娟,郭书海,李凤梅.石油降解菌群的构建及其对混合烃的降解特性[J].农业环境科学学报,2017,36(3):522-530.FAN Rui-juan,GUO Shu-hai,LI Feng-mei.Construction of petroleum degrading bacteria consortium and its degradation properties of mixed hydrocarbons[J].Journal of Agro-Environment Science,2017,36(3):522-530.
[6]李凤梅,郭书海,张灿灿,等.多环芳烃降解菌的筛选及其在焦化场地污染土壤修复中的应用[J].环境污染与防治,2016,38(4):1-5.LI Feng-mei,GUO Shu-hai,ZHANG Can-can,et al.Isolation of PAHs degrading bacteria and its application to mediation of pulluted soil in coking site[J].Environmental Pollution&Control,2016,38(4):1-5.
[7]姜岩,杨颖,张贤明.典型多环芳烃生物降解及转化机制的研究进展[J].石油学报(石油加工),2014,30(6):1137-1150.JIANG Yan,YANG Ying,ZHANG Xian-ming.Review on the biodegradation and conversion mechanism of typical polycyclic aromatic hydrocarbons[J].Acta Petrolei Sinica(Petroleum Processing Section),2014,30(6):1137-1150.
[8]Woo N Y S,Chung K C.Tolerance of pomacanthus imperator to hypoosmotic salinities:Changes in body composition and hepatic enzyme activities[J].Journal of Fish Biology,1995,47(1):70-81.
[9]Onishi H,Fuchi H,Konomi K,et al.Isolation and distribution of a variety of halophilic bacteria and their classification by salt-response[J].Journal of the Agricultural Chemical Society of Japan,1980,44(6):1253-1258.
[10]Hedlund B P,Geiselbrecht A D,Bair T J,et al.Polycyclic aromatic hydrocarbon degradation by a new marine bacterium,Neptunomonas naphthovorans gen.nov.,sp.nov[J].Applied and Environmental Microbiology,1999,65(1):251-259.
[11]Cui C Z,Feng T C,Yu Y Q,et al.Isolation,charcaterization of an anthracene degrading bacterium Martelella sp.AD-3 and cloning of dioxygenase gene[J].Environmental Science,2012,33(11):4062-4068.
[12]厉阗,钱坤,肖伟,等.嗜盐拟香味菌Y6降解硝基苯的特性研究[J].环境科学,2013,34(2):753-759.LI Tian,QIAN Kun,XIAO Wei,et al.Biodegradation of nitrobenzene by a Halophilic Myroides odoratimimus strain Y6[J].Environmental Science,2013,34(2):753-759.
[13]Wang C,Guo G,Huang Y,et al.Salt adaptation and evolutionary implication of a Nah-related PAHs dioxygenase cloned from a halophilic phenanthrene degrading consortium[J].Scientific Reports,2017,7(1):1-13.
[14]Dastgheib S M,Amoozegar M A,Khajeh K,et al.Biodegradation of polycyclic aromatic hydrocarbons by a halophilic microbial consortium[J].Applied Microbiology and Biotechnology,2012,95(3):789-798.
[15]王慧,周海燕,黄勇,等.一株高环多环芳烃降解嗜盐菌Thalassospira sp.的分离及降解特性[J].清华大学学报(自然科学版),2015,55(1):87-92.WANG Hui,ZHOU Hai-yan,HUANG Yong,et al.Isolation and degradation characteristics of a HMW-PAHs degrading halophilic strain Thalassospira sp.[J].Journal of Tsinghua University(Science and Technology),2015,55(1):87-92.
[16]李康,范文玉,郭光,等.盐环境下降解菌群对芘的降解特性研究[J].环境污染与防治,2018,40(7):755-759.LI Kang,FAN Wen-yu,GUO Guang,et al.Degradation characteristics of pyrene by bacteria consortium in saline environment[J].Environmental Pollution&Control,2018,40(7):755-759.
[17]蔡妙英,东秀珠.常见细菌系统鉴定手册[M].北京:科学出版社,2001.CAI Miao-ying,DONG Xiu-zhu.Common bacterial system identification manual[M].Beijing:Science Press,2001.
[18]Wu W Y,Huang H Y,Ling Z M,et al.Genome sequencing reveals mechanisms for heavy metal resistance and polycyclic aromatic hydrocarbon degradation in Delftia lacustris strain LZ-C[J].Ecotoxicology,2016,25(1):234-247.
[19]李倩,崔志松,赵爱芬,等.一株石油烃降解菌新种Marinobacter sp.PY97S的鉴定[J].微生物学报,2011,51(5):648-655.LI Qian,CUI Zhi-song,ZHAO Ai-fen,et al.Identification and characterization of a novel hydrocarbon-degrading Marinobacter sp.PY97S[J].Acta Microbiologica Sinica,2011,51(5):648-655.
[20]Zhou H,Wang H,Huang Y,et al.Characterization of pyrene degradation by halophilic Thalassospira sp.strain TSL5-1 isolated from the coastal soil of Yellow Sea,China[J].International Biodeterioration&Biodegradation,2016,107:62-69.
[21]Boopathy R.Factors limiting bioremediation technologies[J].Bioresource Technology,2000,74(1):63-67.
[22]赵百锁,王慧,毛心慰.嗜盐微生物在环境修复中的研究进展[J].微生物学通报,2007,34(6):1209-1212.ZHAO Bai-suo,WANG Hui,MAO Xin-wei.The recent advances in halophilic microorganisms for environmental bioremediation[J].Microbiology China,2007,34(6):1209-1212.
[23]Minai-Tehrani D,Minoui S,Herfatmanesh A.Effect of salinity on biodegradation of polycyclic aromatic hydrocarbons(PAHs)of heavy crude oil in soil[J].Bulletin of Environmental Contamination and Toxicology,2009,82(2):179-184.
[24]Arulazhagan P,Sivaraman C,Kumar S A,et al.Co-metabolic degradation of benzo(e)pyrene by halophilic bacterial consortium at different saline conditions[J].Journal of Environmental Biology,2014,35(3):445-453.
[25]Qi Y B,Wang C Y,Lv C Y,et al.Removal capacities of polycyclic aromatic hydrocarbons(PAHs)by a newly isolated strain from oilfield produced water[J].International Journal of Environmental Research&Public Health,2017,14(2):215.
[26]宋立超,李培军,刘宛,等.盐碱土壤PAHs降解菌的筛选鉴定及其降解特性[J].微生物学通报,2011,38(2):282-287.SONG Li-chao,LI Pei-jun,LIU Wan,et al.Isolation,identification and degradation characteristics of a PAHs-degrading bacteria from salt-alkaline soil[J].Microbiology China,2011,38(2):282-287.
[27]顾平,周启星,王鑫,等.一株土著B[a]P降解菌的筛选及降解特性研究[J].农业环境科学学报,2018,37(5):926-932.GU Ping,ZHOU Qi-xing,WANG Xin,et al.The screening-out and degradation character of an indigenous benzo[a]pyrene-degrading bacteria[J].Journal of Agro-Environment Science,2018,37(5):926-932.
[28]Cui Z,Yang B,Xu G,et al.Isolation and characterization of Cycloclasticus strains from Yellow Sea sediments and biodegradation of pyrene and fluoranthene by their syntrophic association with Marinobacter strains[J].International Biodeterioration&Biodegradation,2014,91:45-51.
[29]Deng M C,Li J,Liang F R,et al.Isolation and characterization of a novel hydrocarbon-degrading bacterium Achromobacter sp.HZ01from the crude oil-contaminated seawater at the Daya Bay,southern China[J].Marine Pollution Bulletin,2014,83(1):79-86.
[30]Liu T,Zhang Z,Dong W,et al.Bioremediation of PAHs contaminated river sediment by an integrated approach with sequential injection of co-substrate and electron acceptor:Lab-scale study[J].Environmental Pollution,2017,230:413-421.
[31]Lu X Y,Li B,Zhang T,et al.Enhanced anoxic bioremediation of PAHs-contaminated sediment[J].Bioresource Technology,2012,104(1):51-58.