一株中度嗜盐菌Salinicola sp.在高盐环境中的烷烃降解特性
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摘要
从青海油田附近被石油污染的土壤中分离得到一株可利用原油为唯一碳源的菌株,将其命名为X4菌株。经16SrDNA分析鉴定,该菌株与中度嗜盐菌Salinicola zeshunii strain N4~T(GenBank序列号为EU056581)同源性高达99%。X4菌株的最适温度为30℃,最适盐度为8%,最适pH为6.5,最佳碳源为甘油,最佳氮源为氯化铵。该菌可产生生物乳化剂,具有较强的细胞疏水性,对正辛烷、十六烷、二甲苯等典型烃类物质具有良好的乳化能力,细胞CSH值达到60%以上。在含5%盐度的无机盐培养基中,以3g/L的柴油为唯一碳源,采用GC-MS定量分析X4菌株的烃降解特征,结果表明菌株X4培养5天后柴油的总降解率达56%,菌株X4优先降解中长链烃类;C_7～C_(13)烃类的平均降解率为64.1%,C_(14)～C_(20)烃类的平均降解率为52.3%,C_(21)～C_(31)烃类的平均降解率约26.8%。离子型表面活性剂TTAB和SDS对X4菌株生长具有较强的毒性:在浓度达到100mg/L和400mg/L时能完全抑制菌体生长;在40mg/L的浓度下,使得菌株对柴油的降解率降低到20%。而X4菌株对非离子型表活剂——吐温80和生物表面活性剂——鼠李糖脂的耐受浓度均可达400mg/L。鼠李糖脂是嗜盐菌X4菌株的合适复配表活剂。
A crude oil resistant strain X4 was isolated from oil contaminated soil near Qinghai oilfield. It was identified as Salinicola sp. and named as Salinicola sp. X4, since the 16 Sr DNA sequencing homologie to Salinicola zeshunii strain N4~T(GenBank serial number EU056581) was up to 99%. The optimal temperature, NaCl concentration, pH, carbon source and nitrogen source of Salinicola sp. X4 was 30℃, 8%, 6.5, glycerol and NH_4Cl, respectively. The bacterium could produce bioemulsifier, and it had good emulsification ability toward some typical hydrocarbons such as octane, cetane, xylene. The CSH values of Salinicola sp. X4 could reach 60%. Using diesel oil(3 g/L) as the sole carbon source in the inorganic salt medium containing 5% NaCl, the Salinicola sp. X4 could degrade 55% of the total diesel for five days. GC-MS analysis showed that the strain preferred to degrade medium-chain length hydrocarbons firstly; the average degradation rate of C_7―C_(13), C_(14)―C_(20), C_(21)―C_(31) was 64.1%, 52.3% and 26.8%, respectively. The ionic surfactant SDS and TTAB had strong toxicity to the cells: strain X4 wasn't able to survive when the concentration reached 100 mg/L and 400 mg/L, respectively; under the concentration of 40 mg/L, the degradation rate of diesel oil was reduced to 20%. While the tolerance concentration of the strain to the non-ionic surfactants――Twain80 and bio-surfactant――rhamnolipid reached 400 mg/L;and rhamnolipid is a suitable compound surfactant for strain X4.
A crude oil resistant strain X4 was isolated from oil contaminated soil near Qinghai oilfield. It was identified as Salinicola sp. and named as Salinicola sp. X4, since the 16 Sr DNA sequencing homologie to Salinicola zeshunii strain N4~T(GenBank serial number EU056581) was up to 99%. The optimal temperature, NaCl concentration, pH, carbon source and nitrogen source of Salinicola sp. X4 was 30℃, 8%, 6.5, glycerol and NH_4Cl, respectively. The bacterium could produce bioemulsifier, and it had good emulsification ability toward some typical hydrocarbons such as octane, cetane, xylene. The CSH values of Salinicola sp. X4 could reach 60%. Using diesel oil(3 g/L) as the sole carbon source in the inorganic salt medium containing 5% NaCl, the Salinicola sp. X4 could degrade 55% of the total diesel for five days. GC-MS analysis showed that the strain preferred to degrade medium-chain length hydrocarbons firstly; the average degradation rate of C_7―C_(13), C_(14)―C_(20), C_(21)―C_(31) was 64.1%, 52.3% and 26.8%, respectively. The ionic surfactant SDS and TTAB had strong toxicity to the cells: strain X4 wasn't able to survive when the concentration reached 100 mg/L and 400 mg/L, respectively; under the concentration of 40 mg/L, the degradation rate of diesel oil was reduced to 20%. While the tolerance concentration of the strain to the non-ionic surfactants――Twain80 and bio-surfactant――rhamnolipid reached 400 mg/L;and rhamnolipid is a suitable compound surfactant for strain X4.
引文
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[2]郑刚,杨志坚,董博林,等.高盐含油废水中微生物筛选及系统发育研究[J].微生物学杂志, 2016, 36(6):40-47.ZHENG G, YANG Z J, DONG B L, et al. Screening and phylogenetic studies of halophiles in high salinity wastewater[J]. Journal of Microbiology, 2016, 36(6):40-47.
[3]刘铁汉,周培瑾.嗜盐微生物[J].微生物学通报, 1999, 26(3):232.LIU T H, ZHOU P J. Halophilic microorganism[J]. Microbiology China, 1999, 26(3):232.
[4] KUSHNER D J. Life in high salt and solute concentrations:halophilic bacteria[J]. Microbial Life in Extreme Environments, 1978:317-368.
[5] GAO P, TIAN H, WANG Y, et al. Spatial isolation and environmental factors drive distinct bacterial and archaeal communities in different types of petroleum reservoirs in China[J]. Scientific Reports, 2016, 6:20174.
[6] SAITOU N, NEI M. The neighbor-joining method—A new method for reconstructing phylogenetic trees.[J]. Mol. Biol. E, 1987, 4(4):406-425.
[7] KUMAR S, TAMURA K, NEI M. MEGA3:integrated software for molecular evolutionary genetics analysis and sequence alignment[J].Briefings in Bioinformatics, 2004, 5(2):150-163.
[8] COOPER D G, GOLDENBERG B G. Surface-active agents from two bacillus species[J]. Applied&Environmental Microbiology, 1987, 53(2):224-229.
[9] JONSSON P, WADSTR?M T. Cell surface hydrophobicity of Staphylococcus aureus measured by the salt aggregation test(SAT)[J].Current Microbiology, 1984, 10(4):203-209.
[10]胡春辉,于浩,赵阳国,等.高效耐盐柴油降解菌的筛选、鉴定及降解基因[J].中国环境科学, 2017, 37(11):4251-4258.HU C H,YU H, ZHAO Y G,et al. Isolation and identification of a highefficient diesel degrading bacterial strain acinetobacter sp. L7[J]. China Environmental Science, 2017, 37(11):4251-4258.
[11] WANG G, CHANG X, WANG T G, et al. Pregnanes as molecular indicators for depositional environments of sediments and petroleum source rocks[J]. Organic Geochemistry, 2015, 78:110-120.
[12] CAO L, YAN Q, NI H, et al. Salinicola zeshunii sp. nov. a moderately halophilic bacterium isolated from soil of a chicken farm[J]. Current Microbiology, 2013, 66(2):192-196.
[13]许爱清,周士镭,冯杏杏,等.一株墨鱼干源中度嗜盐菌的系统发育分析与生长特性[J].食品工业科技, 2013, 34(21):175-179.XU A Q, ZHOU S L, FENG X X, et al. Phylogenetic analysis and growth characteristics of a moderate halophilic bacterium isolated from dried cuttlefish[J]. Science and Technology of Food Industry, 2013, 34(21):175-179.
[14] OLSSON B E, KORSAKOVA E S, ANAN’INA L N, et al. Draft genome sequences of strains Salinicola socius SMB35T, Salinicola sp.MH3R3-1 and Chromohalobacter sp. SMB17 from the Verkhnekamsk potash mining region of Russia[J]. Standards in Genomic Sciences,2017, 12(1):39.
[15]汪淼,于琪,苟敏.一株栖盐田菌Salinicola sp. W1的柴油降解特性研究[J].湖北农业科学, 2016, 55(12):3074-3078.WANG M, YU Q, GOU M. Characterization of diesel degradation by Salinicola sp. W1[J]. Hubei Agricultural Sciences, 2016, 55(12):3074-3078.
[16] CHEN G Q, IVAN H. The‘PHAome’[J]. Trends in Biotechnology,2015, 33(10):559-64.
[17] YE J, HU D, CHE X, et al. Engineering of Halomonas bluephagenesis for low cost production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)from glucose[J]. Metabolic Engineering, 2018, 47:143-152.
[18]杨波,郝建安,张晓青,等.复合嗜盐微生物处理高含盐废水的研究[J].盐业与化工, 2015, 44(11):33-39.YANG B, HAO J A, ZHANG X Q, et al. Research on hypersaline wastewater treatment by composite halophilic microorganism[J].Journal of Salt and Chemical industry, 2015, 44(11):33-39.
[19] WANG C, HUANG Y, ZHANG Z, et al. Salinity effect on the metabolic pathway and microbial function in phenanthrene degradation by a halophilic consortium[J]. AMB Express, 2018, 8(1):67.
[20]刘金峰,牟伯中.油藏极端环境中的微生物[J].微生物学杂志,2004, 24(4):31-34.LIU J F, MOU B Z. Microorganisms in extreme environments of reservoirs[J]. Journal of Microbiology, 2004, 24(4):31-34.
[21] KIM K K, JIN L, YANG H C, et al. Halomonas gomseomensis sp. nov.,Halomonas janggokensis sp. nov., Halomonas salaria sp. nov. and Halomonas denitrificans sp. nov. moderately halophilic bacteria isolated from saline water[J]. International Journal of Systematic&Evolutionary Microbiology, 2007, 57(4):675-81.
[22] ANAN'INA L N, PLOTNIKOVA E G, EIU G, et al. Salinicola sociusgen. nov. sp. nov., a moderately halophilic bacterium from a naphthalene-utilizing microbial association[J]. Microbiologiia, 2007,76(3):369.
[23] ARAHAL D R, GARCíA M T, VARGAS C, et al. Chromohalobacter salexigens sp. nov., a moderately halophilic species that includes halomonas elongata DSM 3043 and ATCC 33174[J]. International Journal of Systematic&Evolutionary Microbiology, 2001, 51(4):1457-1462.
[24] TAN D, XUE Y S, AIBAIDULA G, et al. Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01[J]. Bioresource Technology, 2011, 102(17):8130-8136.
[25] FRANZETTI A, GANDOLFI I, RAIMONDI C, et al. Environmental fate, toxicity, characteristics and potential applications of novel bioemulsifiers produced by variovorax paradoxus 7bCT5[J].Bioresource Technology, 2012, 108(3):245-251.
[26]李国强,纪凯华,李佳斌,等.嗜热解烃菌DM-2产生的生物乳化剂[J].微生物学通报, 2014, 41(4):585-591.LI G Q, JI K H,LI J B, et al. Bioemulsifier produced by a thermophilic hydrocarbon-degrading strain DM-2[J]. Microbiology China, 2014, 41(4):585-591.
[27]刘洋,李蔚,单厚菲,等.细胞疏水性对采油微生物运移吸附作用的影响[J].西南石油大学学报(自然科学版), 2009, 31(5):117-120.LIU Y, LI W, SHAN H F, et al. Effect of cell hydrophobicity on microorganism migration and adsorption of oil recovery[J]. Journal of Southweat Petroleum University(Science&Technology Edition),2009, 31(5):117-120.
[28]谢鲲鹏,周集体,曲媛媛,等.一株耐盐原油降解菌的分离鉴定及其降解特性研究[J].海洋环境科学, 2009, 28(6):680-683.XIE K P, ZHOU J T,QU Y Y, et al. Studies on isolation,identification of halophil curde oildegrading bacteria and their degradation capability[J]. Marine Environmental Science, 2009, 28(6):680-683.
[29]章慧,郭楚玲,卢桂宁,等.具有产表面活性剂功能石油降解菌的筛选及其发酵条件优化[J].农业环境科学学报, 2013, 32(11):2185-2191.ZHANG H, GUO C L, LU G N, et al. Screening and fermentation optimization of a biosurfactant-producing oil-degrading bacterium[J].Journal of Agro—Environment Science, 2013, 32(11):2185-2191.
[30]杨蕊琪,薛林贵,常思静,等.一株耐低温原油降解菌的分离鉴定及降解特性[J].浙江农业学报, 2016, 28(10):1781-1789.YANG X Q, XUE L G, CHANG S J, et al. Studies on isolation,identification of cold-resistant petroleum-degrading strain and its degradation capability[J]. Acta Agriculturae Zhejiangensis, 2016, 28(10):1781-1789.
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