(2.重庆大学城市建设与环境工程学院, 重庆 400045)
【摘要】在三氯乙烯(TCE)胁迫条件下,从生活垃圾填埋场覆盖土中富集得到了可高效降解TCE的混合菌群SWA1。考察了铜离子浓度0-15μmol/L范围内混合菌群对TCE的降解,当铜离子浓度为0.03μmol/L时,降解速率最大为29.60 nmol/min,降解率达95.75%。此条件下的pmo A和mmo X表达量均达最大值,pmo A的相对表达量(4.22 E-03)比mmo X(9.30 E-06)和Lmp H(0)高3个数量级。在0-0.75μmol/L和1-15μmol/L两个铜离子浓度区间,分别出现了TCE降解峰值,高通量测序结果表明,甲基孢囊菌科Methylocystaceae的甲烷氧化菌为优势微生物。随着铜离子浓度提高,混合菌群SWA1生物多样性显著降低。铜离子浓度的变化影响了混合菌群的结构和活性,进而影响了TCE降解机制。当铜离子浓度为0.03μmol/L时,降解机制包括TCE直接降解和甲烷氧化菌共代谢降解。当铜离子浓度为5μmol/L时,降解率可达到84.75%。此时,降解机制包括TCE直接降解以及甲烷氧化菌和含苯酚羟化酶菌群的共代谢降解。
【基金资助】 国家自然科学基金(Nos.51378522,41502328); 重庆市基础科学与前沿技术研究项目(No.cstc2015jcyjB0015)资助;
Effects of copper on biodegradation mechanism of trichloroethylene by mixed microorganisms
(2.College of Urban Construction and Environmental Engineering, Chongqing University, Chongqing, China 400045)
【Abstract】We isolated and enriched mixed microorganisms SWA1 from landfill cover soils supplemented with trichloroethylene (TCE). The microbial mixture could degrade TCE effectively under aerobic conditions. Then, we investigated the effect of copper ion (0 to 15 μmol/L) on TCE biodegradation. Results showed that the maximum TCE degradation speed was 29.60 nmol/min with 95.75% degradation when copper ion was at 0.03 μmol/L. In addition, genes encoding key enzymes during biodegradation were analyzed by Real-time quantitative reverse transcription PCR (RT-qPCR). The relative expression abundance of pmoA gene (4.22E-03) and mmoX gene (9.30E-06) was the highest when copper ion was at 0.03 μmol/L. Finally, we also used MiSeq pyrosequencing to investigate the diversity of microbial community. Methylocystaceae that can co-metabolic degrade TCE were the dominant microorganisms; other microorganisms with the function of direct oxidation of TCE were also included in SWA1 and the microbial diversity decreased significantly along with increasing of copper ion concentration. Based on the above results, variation of copper ion concentration affected the composition of SWA1 and degradation mechanism of TCE. The degradation mechanism of TCE included co-metabolism degradation of methanotrophs and oxidation metabolism directly at copper ion of 0.03 μmol/L. When copper ion at 5 μmol/L (biodegradation was 84.75%), the degradation mechanism of TCE included direct-degradation and co-metabolism degradation of methanotrophs and microorganisms containing phenol hydroxylase. Therefore, biodegradation of TCE by microorganisms was a complicated process, the degradation mechanism included co-metabolism degradation of methanotrophs and bio-oxidation of non-methanotrophs.
【Keywords】 mixed microorganisms; trichloroethylene; key enzymes; community structure; degradation mechanism;
【Funds】 National Natural Science Foundation of China (Nos. 51378522, 41502328); Fundamental and Advanced Research Projects of Chongqing (No. cstc2015jcyjB0015);
 Shukla AK, Upadhyay SN, Dubey SK. Current trends in trichloroethylene biodegradation: a review. Crit Rev Biotechnol, 2014, 34(2): 101–114.
 Chee GJ. Biodegradation analyses of trichloroethylene (TCE) by bacteria and its use for biosensing of TCE. Talanta, 2011, 85(4): 1778–1782.
 Liu JB, Amemiya T, Chang Q, et al. Toluene dioxygenase expression correlates with trichloroethylene degradation capacity in Pseudomonas putida F1 cultures. Biodegradation, 2012, 23(5): 683–691.
 Guo Y, Cui KP. Effect of sulfate reduction on biodegradation of trichloroethylene. Chin J Environ Engin, 2014, 8(10): 4159–4162 (in Chinese).
 Schmidt KR, Gaza S, Voropaev A, et al. Aerobic biodegradation of trichloroethene without auxiliary substrates. Water Res, 2014, 59: 112–118.
 Li Y, Pan T, Liu F, et al. Co-metabolism biodegradation of tetrachloroethylene under different groundwater conditions. Rock Min Anal, 2012, 31(4): 682–688 (in Chinese).
 Paszczynski AJ, Paidisetti R, Johnson AK, et al. Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase. Biodegradation, 2011, 22(6): 1045–1059.
 Xing ZL, Zhang LJ, Zhao TT. Advances in degradation of chlorinated hydrocarbons by obligate and facultative methanotrophs. Chin J Biotech, 2014, 30(4): 531–544 (in Chinese).
 Pant P, Pant S. A review: advances in microbial remediation of trichloroethylene (TCE). J Environ Sci (China), 2010, 22(1): 116–126.
 Wang JN, Shi YY, Zheng LY, et al. Isolation and identification of petroleum degradation bacteria and interspecific interactions among four Bacillus strains. Environ Sci, 2015, 36(6): 2245–2251 (in Chinese).
 Mukherjee P, Roy P. Identification and characterisation of a bacterial isolate capable of growth on trichloroethylene as the sole carbon source. Adv Microbiol, 2012, 2(3): 284–294.
 Oldenhuis R, Oedzes JY, Van der Waarde JJ, et al. Kinetics of chlorinated hydrocarbon degradation by Methylosinus trichosporium OB3b and toxicity of trichloroethylene. Appl Environ Microbiol, 1991, 57(1): 7–14.
 Dedysh SN, Liesack W, Khmelenina VN, et al. Methylocella palustris gen. nov., sp. nov., a new methane-oxidizing acidophilic bacterium from peat bogs, representing a novel subtype of serine-pathway methanotrophs. Int J Syst Evol Microbiol, 2000, 50(3): 955–969.
 Vorobev AV, Baani M, Doronina NV, et al. Methyloferula stellata gen. nov., sp. nov., an acidophilic, obligately methanotrophic bacterium that possesses only a soluble methane monooxygenase. Int J Syst Evol Microbiol, 2011, 61(10): 2456–2463.
 Semrau JD, Di Spirito AA, Yoon S. Methanotrophs and copper. FEMS Microbiol Rev, 2010, 34(4): 496–531.
 Zhang YR, Chen HQ, Gao YH, et al. Sequence analysis of 16S rDNA and pmoCAB gene cluster of trichloroethylene-degrading methanotroph. Chin J Biotech, 2014, 30(12): 1912–1923 (in Chinese).
 Choi DW, Kunz RC, Boyd ES, et al. The membrane-associated methane monooxygenase (pMMO)and pMMO-NADH: quinone oxidoreductase complex from Methylococcus capsulatus Bath. J Bacteriol, 2003, 185(19): 5755–5764.
 Gilbert B, Mc Donald IR, Finch R, et al. Molecular analysis of the pmo (particulate methane monooxygenase) operons from two type II methanotrophs. Appl Environ Microbiol, 2000, 66(3): 966–975.
 Mc Donald IR, Bodrossy L, Chen Y, et al. Molecular ecology techniques for the study of aerobic methanotrophs. Appl Environ Microbiol, 2008, 74(5): 1305–1315.
 Basu P, Katterle B, Andersson KK, et al. The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein. Biochem J, 2003, 369(2): 417–427.
 Hanson RS, Hanson TE. Methanotrophic bacteria. Microbiol Rev, 1996, 60(2): 439–471.
 Green J, Prior SD, Dalton H. Copper ions as inhibitors of protein C of soluble methane monooxygenase of Methylococcus capsulatus (Bath). Eur J Biochem, 1985, 153(1): 137–144.
 Han B, Su T, Li X, et al. Research progresses of methanotrophs and methane monooxygenases. Chin J Biotech, 2008, 24(9): 1511–1519 (in Chinese).
 Liebner S, Svenning MM. Environmental transcription of mmoX by methane-oxidizing Proteobacteria in a subarctic palsa peatland. Appl Environ Microbiol, 2013, 79(2): 701–706.
 Jagadevan S, Semrau JD. Priority pollutant degradation by the facultative methanotroph, Methylocystis strain SB2. Appl Microbiol Biotech, 2013, 97(11): 5089–5096.
 Smith SM, Rawat S, Telser J, et al. Crystal structure and characterization of particulate methane monooxygenase from Methylocystis species strain M. Biochemistry, 2011, 50(47): 10231–10240.
 Mukherjee P, Roy P. Copper enhanced monooxygenase activity and FT-IR spectroscopic characterisation of biotransformation products in trichloroethylene degrading bacterium: Stenotrophomonas maltophilia PM102. Biomed Res Int, 2013, (6): 643–653.
 Zhao T, Zhang LJ, Zhang YR, et al. Characterization of Methylocystis strain JTA1isolated from aged refuse and its tolerance to chloroform. J Environ Sci, 2013, 25(4): 770–775.
 Zhao TT, Xiang JX, Zhang LJ, et al. Screening and biological characteristics of amphitrophic methane-oxidizing bacteria from aged-refuse. Environ Sci, 2012, 33(5): 1670–1675 (in Chinese).
 Zhao TT, He CM, Zhang LJ, et al. Kinetics of affinity to methane oxidation by Chryseobacterium sp. from aged-refuse. CIESC J, 2011, 62(7): 1915–1921 (in Chinese).
 Song LY, Wang YQ, Tang W, et al. Bacterial community diversity in municipal waste landfill sites. Appl Microbiol Biotechnol, 2015, 99(18): 7745–7756.
 Heyer KU, Hupe K, Stegmann R. Methane emissions from MBT landfills. Waste Manag, 2013, 33(9): 1853–1860.
 Harborth P, FußR, Münnich K, et al. Spatial variability of nitrous oxide and methane emissions from an MBT landfill in operation: strong N2O hotspots at the working face. Waste Manag, 2013, 33(10): 2099–2107.
 Galvão TC, Mohn WW, De Lorenzo V. Exploring the microbial biodegradation and biotransformation gene pool. Trends Biotechnol, 2005, 23(10): 497–506.
 Luo MF, Wu H, Wang L, et al. Growth characteristics of a methane-utilizing mixed consortia MY9. Chin J Proc Eng, 2009, 9(1): 113–117 (in Chinese).
 Fierer N, Jackson JA, Vilgalys R, et al. Assessment of soil microbial community structure by use of taxon-specific quantitative PCR assays. Appl Environ Microbiol, 2005, 71(7): 4117–4120.
 Youssef N, Sheik CS, Krumholz LR, et al. Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rRNA gene-based environmental surveys. Appl Environ Microbiol, 2009, 75(16): 5227–5236.
 Hong C, Si Y, Xing Y, et al.Illumina Miseq sequencing investigation on the contrasting soil bacterial community structures in different iron mining areas. Environ Sci Pollut Res, 2015, 22(14): 10788–10799.
 Prior SD, Dalton H. The effect of copper ions on membrane content and methane monooxygenase activity in methanol-grown cells of Methylococcus capsulatus (Bath). J Gen Microbiol, 1985, 131(1): 155–163.
 Ho A, Lüke C, Reim A, et al. Selective stimulation in a natural community of methane oxidizing bacteria: effects of copper on pmoA transcription and activity. Soil Biolog Biochem, 2013, 65: 211–216.
 Zhang Y, Hu M, Li PF, et al. Trichloroethylene removal and bacterial variations in the up-flow anaerobic sludge blanket reactor in response to temperature shifts. Appl Microbiol Biotechnol, 2015, 99(14): 6091–6102.
 Zhang Y, Wang X, Hu M, et al.Effect of hydraulic retention time (HRT) on the biodegradation of trichloroethylene wastewater and anaerobic bacterial community in the UASB reactor. Appl Microbiol Biotechnol, 2015, 99(4): 1977–1987.
 Dey K, Roy P. Degradation of trichloroethylene by Bacillus sp.: isolation strategy, strain characteristics, and cell immobilization. Curr Microbiol, 2009, 59(3): 256–260.
 Toribio-Jiménez J, Rodríguez-Barrera MÁ, Lucena MV, et al. Production of biosurfactants by bacteria isolated from a mine tailing zone in southern Mexico and their resistance to heavy metals. African J Bacter Res, 2014, 6(4): 23–31.
 Peña-Montenegro TD, Lozano L, Dussán J. Genome sequence and description of the mosquitocidal and heavy metal tolerant strain Lysinibacillus sphaericus CBAM5. Stand Genomic Sci, 2015, 10(1): 2.
 Jenkins O, Byrom D, Jones D. Methylophilus: a new genus of methanol-utilizing bacteria. Int J Syst Evol Microbiol, 1987, 37(4): 446–448.
 Stourman NV, Rose JH, Vuilleumier S, et al. Catalytic mechanism of dichloromethane dehalogenase from Methylophilus sp. strain DM11. Biochemistry, 2003, 42(37): 11048–11056.
 Mera N, Iwasaki K. Use of plate-wash samples to monitor the fates of culturable bacteria in mercury and trichloroethylene-contaminated soils. Appl Microbiol Biotechnol, 2007, 77(2): 437–445.
 Futamata H, Harayama S, Hiraishi A, et al. Functional and structural analyses of trichloroethylene-degrading bacterial communities under different phenol-feeding conditions: laboratory experiments. Appl Microbiol Biotechnol, 2003, 60(5): 594–600.
 Movahedyan H, Khorsandi H, Salehi R, et al. Detection of phenol degrading bacteria and Pseudomonas putida in activated sludge by polymerase chain reaction. Iran J Environ Health Sci Engin, 2009, 6(2): 115–120.
 Van Der Ha D, Vanwonterghem I, Hoefman S, et al. Selection of associated heterotrophs by methane-oxidizing bacteria at different copper concentrations. Antonie Van Leeuwenhoek, 2013, 103(3): 527–537.
 Agarry SE, Durojaiye AO, Solomon BO. Microbial degradation of phenols: a review. Int J Environ Pollut, 2008, 32(1): 12–28.
 Shukla AK, Vishwakarma P, Singh RS, et al. Bio-filtration of trichloroethylene using diazotrophic bacterial community. Bioresour Technol, 2010, 101(7): 2126–2133.