低温萘降解菌的筛选、鉴定及降解条件优化
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摘要
研究采用富集培养法从黑龙江省大庆油田地区污染土壤中筛选能以萘为唯一碳源和能源的低温菌株,采用气相色谱-质谱法(GC-MS)研究降解菌在萘-无机盐培养基中对萘的降解情况,通过单因素试验与正交试验测定降解菌的培养条件并进行优化,同时分析降解阶段其主控因素。结果表明:筛选出2株在低温条件下高效降解萘的菌株,编号为GN1和GN2。在低温条件下GN1和GN2可以快速降解萘,在对照组非生物因素影响基础上,萘(300 mg/L)的降解率在4 d内达到94.43%和95.47%,在耐受能力和降解速度方面具明显优势;经形态观察、生理生化特性和16S rDNA基因序列鉴定两株降解菌皆属于假单胞菌属(Pseudomonas);均在萘-无机盐培养基(萘浓度300 mg/L),培养温度15℃,初始pH 6.0,培养转数180 r/min,培养时间7 d的条件下生长最佳。2株降解菌的生长与5种环境因素均有显著关系。
The enrichment culture method was used to screen low temperature strains using naphthalene as the sole carbon source and energy source from the contaminated soil in Daqing Oilfield,Heilongjiang Province. The gas chromatography-mass spectrometry(GC-MS)was employed to study the degradation of naphthalene by degrading bacteria in naphthalene-inorganic salt medium. The single-factor test and orthogonal test were applied to determine and optimize the culture conditions of the degrading bacteria,and the main controlling factors in the degradation stage were analyzed. The results showed that 2 strains efficiently degrading naphthalene under low temperature condition were screened and named as GN1 and GN2. GN1 and GN2 rapidly degraded naphthalene under low temperature conditions. On the basis of the nonbiological factors of the control group,the degradation rate of naphthalene(300 mg/L)reached 94.43% and 95.47% in 4 days,indicating that they had obvious advantages in tolerance and degradation rate. The 2 strains were identified as Pseudomonas by morphological observation,physiological and biochemical characteristics and 16 S r DNA gene. The strains GN1 and GN2 grew best in naphthalene-inorganic salt medium(Naphthalene concentration 300 mg/L),culture temperature 15℃,initial pH 6.0,culture rotation 180 r/min,and culture time 7 d. The growth of the 2 bacteria was significantly related to 5 environmental factors.
The enrichment culture method was used to screen low temperature strains using naphthalene as the sole carbon source and energy source from the contaminated soil in Daqing Oilfield,Heilongjiang Province. The gas chromatography-mass spectrometry(GC-MS)was employed to study the degradation of naphthalene by degrading bacteria in naphthalene-inorganic salt medium. The single-factor test and orthogonal test were applied to determine and optimize the culture conditions of the degrading bacteria,and the main controlling factors in the degradation stage were analyzed. The results showed that 2 strains efficiently degrading naphthalene under low temperature condition were screened and named as GN1 and GN2. GN1 and GN2 rapidly degraded naphthalene under low temperature conditions. On the basis of the nonbiological factors of the control group,the degradation rate of naphthalene(300 mg/L)reached 94.43% and 95.47% in 4 days,indicating that they had obvious advantages in tolerance and degradation rate. The 2 strains were identified as Pseudomonas by morphological observation,physiological and biochemical characteristics and 16 S r DNA gene. The strains GN1 and GN2 grew best in naphthalene-inorganic salt medium(Naphthalene concentration 300 mg/L),culture temperature 15℃,initial pH 6.0,culture rotation 180 r/min,and culture time 7 d. The growth of the 2 bacteria was significantly related to 5 environmental factors.
引文
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[15]Jesús L, Francisco MM, JoséAR, et al. Bacterial chemotaxis towardsaromatichydrocarbonsinPseudomonas[J].Environmental Microbiology, 2011, 13(7):1733-1744.
[16]Mangwani N, Kumari S, Das S. Effect of synthetic N-acylhomoserine lactonesoncell-cellinteractionsinmarinePseudomonas andbiofilmmediateddegradationofpolycyclicaromatic hydrocarbons[J]. Chemical Engineering Journal, 2016, 302:172-186.
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[20]Rockne KJ, Strand SE. Anaerobic biodegradation of naphthalene,phenanthrene,andbiphenylbyadenitrifyingenrichment culture[J]. Water Research, 2001, 35(1):291-299.
[21]孟建宇,张阳,李蘅,等.萘降解菌的分离、鉴定及降解特性的研究[J].中国酿造, 2017, 36(2):58-63.
[22]Bertilsson S, Widenfalk A. Photochemical degradation of PAHs in freshwaters and their impact on bacterial growth-Influence of water chemistry[J]. Hydrobiologia, 2002, 469. 1:23-32.
[23]Wu YC, Zeng J, Zhu QH, et al. pH is the primary determinant of the bacterial community structure in agricultural soils impacted by polycyclic aromatic hydrocarbon pollution[J]. Scientific Reports,2017, 7:40093.
[24]Guo JH, Liu XJ, Zhang Y, et al. Significant acidification in major Chinese croplands[J]. Science, 2010, 327:1008-1010.
[25]Ping LF, Luo YM, Zhang HB, et al. Distribution of polycyclic aromatic hydrocarbons in thirty typical soil profiles in the Yangtze River Delta region, east China[J]. Environmental Pollution,2006, 147:358-365.
[26]周晓阳,周世伟,徐明岗,等.中国南方水稻土酸化演变特征及影响因素[J].中国农业科学, 2015, 48(23):4811-4817.
[27]Yu QM, Wang P, Liu DB, et al. Degradation characteristics and metabolic pathway of 17β-estradiol(E2)by Rhodococcus sp.DS201[J]. Biotechnology and Bioprocess Engineering, 2016, 21(6):804-813.
[2]Chibwe L, Davie-Martin CL, Michael DA, et al. Identification of polar transformation products and high molecular weight polycyclic aromatic hydrocarbons(PAHs)in contaminated soil following bioremediation[J]. Science of The Total Environment, 2017,599:1099-1107.
[3]Amanda JW, Nina AD, Marie EH, et al. Urinary and breast milk biomarkers to assess exposure to naphthalene in pregnant women:an investigation of personal and indoor air sources[J].Environmental Health, 2014, 13(1):30.
[4]Weiming L, Dongsheng W, Feng H, et al. Exogenous IAA treatment enhances phytoremediation of soil contaminated with phenanthrene by promoting soil enzyme activity and increasing microbial biomass[J]. Environmental Science And Pollution Research,2016, 23(11):10656-10664.
[5]刘增俊,滕应,黄标,等.长江三角洲典型地区农田土壤多环芳烃分布特征与源解析[J].土壤学报, 2010, 47(6):1110-1111.
[6]于洋洋,王广智,王卓然,等.萘好氧降解污泥强化培养特性与降解效能研究[J].环境科学与技术, 2017, 40(8):128-133.
[7]Monica RC, Lucia G, Simone B, et al. Polycyclicaromatic hydrocarbon-contaminated soils:bioaugmentation of autochthonous bacteria and toxicological assessment of the bioremediation process by means of Vicia faba L.[J]. Environmental Science and Pollution Research, 2016, 23(8):7930-7941.
[8]Denk P, Velasco SC, Buettner A. Resolving the chemical structures of off-odorants and potentially harmful substances in toys-example of children’s swords[J]. Anal Bioanal Chem, 2017, 409(22):5249-5258.
[9]夏文迪.多环芳烃(PAHs)人体内暴露剂量与致癌风险研究[D].长沙:中南大学, 2014.
[10]Liu J, Xiang Y, Zhang Z, et al. Inoculation of a phenanthrenedegrading endophytic bacterium reduces the phenanthrene level and alters the bacterial community structure in wheat[J].Applied Microbiology and Biotechnology, 2017, 101(12):5199-5212.
[11]田兆雪,刘雪华.环境中多环芳烃污染对生物体的影响及其修复[J].环境科学与技术, 2018, 41(12):79-89.
[12]Couling NR, Towell MG, Semple KT. Biodegradation of PAHs in soil:influence of chemical structure, concentration and multiple amendment[J]. Environmental Pollution, 2010, 158:3411-3420.
[13]Hesham A, Mawad AM, Mostafa YM, et al. Study of enhancement and inhibition phenomena and genes relating to degradation of petroleum polycyclic aromatic hydrocarbons in isolated bacteria[J]. Microbiology, 2014, 83:599-607.
[14]Saranya K, Palanisami T, Surender S, et al. Polycyclic aromatic hydrocarbons(PAHs)degradationpotential,surfactant production, metal resistance and enzymatic activity of two novel cellulose-degrading bacteria isolated from koala faeces[J].Environmental Earth Sciences, 2017, 76:14.
[15]Jesús L, Francisco MM, JoséAR, et al. Bacterial chemotaxis towardsaromatichydrocarbonsinPseudomonas[J].Environmental Microbiology, 2011, 13(7):1733-1744.
[16]Mangwani N, Kumari S, Das S. Effect of synthetic N-acylhomoserine lactonesoncell-cellinteractionsinmarinePseudomonas andbiofilmmediateddegradationofpolycyclicaromatic hydrocarbons[J]. Chemical Engineering Journal, 2016, 302:172-186.
[17]Zhang YX, Tao S. Seasonal variation of polycyclic aromatic hydrocarbons(PAHs)emissions in China[J]. Environmental Pollution, 2008, 156(3):657-663.
[18]马万里.我国土壤和大气中多环芳烃分布特征和大尺度数值模拟[D].哈尔滨:哈尔滨工业大学, 2010.
[19]Buchanan RE, Gibbens NE, Murray RG, et al.伯杰细菌鉴定手册[M].北京:科学出版社, 1984.
[20]Rockne KJ, Strand SE. Anaerobic biodegradation of naphthalene,phenanthrene,andbiphenylbyadenitrifyingenrichment culture[J]. Water Research, 2001, 35(1):291-299.
[21]孟建宇,张阳,李蘅,等.萘降解菌的分离、鉴定及降解特性的研究[J].中国酿造, 2017, 36(2):58-63.
[22]Bertilsson S, Widenfalk A. Photochemical degradation of PAHs in freshwaters and their impact on bacterial growth-Influence of water chemistry[J]. Hydrobiologia, 2002, 469. 1:23-32.
[23]Wu YC, Zeng J, Zhu QH, et al. pH is the primary determinant of the bacterial community structure in agricultural soils impacted by polycyclic aromatic hydrocarbon pollution[J]. Scientific Reports,2017, 7:40093.
[24]Guo JH, Liu XJ, Zhang Y, et al. Significant acidification in major Chinese croplands[J]. Science, 2010, 327:1008-1010.
[25]Ping LF, Luo YM, Zhang HB, et al. Distribution of polycyclic aromatic hydrocarbons in thirty typical soil profiles in the Yangtze River Delta region, east China[J]. Environmental Pollution,2006, 147:358-365.
[26]周晓阳,周世伟,徐明岗,等.中国南方水稻土酸化演变特征及影响因素[J].中国农业科学, 2015, 48(23):4811-4817.
[27]Yu QM, Wang P, Liu DB, et al. Degradation characteristics and metabolic pathway of 17β-estradiol(E2)by Rhodococcus sp.DS201[J]. Biotechnology and Bioprocess Engineering, 2016, 21(6):804-813.