苯并[a]芘对毛霉EPS特征的影响
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摘要
为了明确毛霉胞外聚合物(Extracellular polymertic substances,EPS)在多环芳烃修复中的作用机理,研究了不同浓度苯并[a]芘(BaP)诱导下毛霉EPS的提取量(TOC)和主要化学成分含量(糖类、蛋白质、腐植酸和DNA)的变化规律,采用三维荧光光谱(3D-EEM)、傅里叶红外光谱(FTIR)和扫描电镜(SEM)等方法分析了不同条件下EPS蛋白含量和自身结构的变化。结果表明,当BaP浓度为40 mg·L~(-1)时,单位生物量所产生的毛霉EPS的提取量、糖类含量、蛋白质含量和腐植酸含量均达到最大值,分别为1 407.22 mg·g-1、700 g·g-1、564 g·g-1和174 g·g-1;3D-EEM结果显示,当BaP浓度为40 mg·L~(-1)时,荧光类蛋白质(峰A和峰B)强度分别达到最大值68.91 a.u和245.6 a.u;FTIR分析结果表明,在BaP的诱导下,与空白相比,EPS的细胞质蛋白质酰胺Ⅰ带和酰胺Ⅱ带向长波方向发生了红移现象;SEM分析结果显示,随BaP浓度的增大,毛霉EPS表面逐渐呈现板结现象,当BaP浓度为40 mg·L~(-1)时,毛霉EPS呈毛绒状,孔隙达到最大。以上分析结果表明,BaP破坏毛霉EPS原有结构,低浓度BaP促进真菌EPS的分泌,EPS中的蛋白质和多糖在毛霉代谢降解BaP过程中起到了主要作用。
To elucidate the role of Mucor mucedo EPS in the remediation of polycyclic aromatic hydrocarbon(PAH)pollution, the extracted concentration of EPS(TOC)and the content of proteins(PN), polysaccharides(PS), humic acid(HC), and DNA in EPS were determined after the exposure of M. mucedo to different concentrations of benzo[a]pyrene(BaP). Three-dimensional fluorescence spectroscopy(3 DEEM)and flourier transform infrared spectroscopy(FTIR)were used to verify the changes in the concentration of proteins when different concentrations of BaP were used, and scanning electron microscopy(SEM)was employed to study the changes in the structure of EPS. The results showed that the changes in the concentration of TOC, PN, PS, and HC showed the same trend as the exposed BaP concentrations increased, and their content increased initially, and then decreased. When the concentration of BaP was 40 mg·L~(-1), the concentration of TOC,PN, PS, and HC produced by unit biomass reached the maximum values of 1 407.2 mg·g-1, 700 g·g-1, 564 g·g-1, and 174 g·g-1, respectively. According to the results of 3 D-EEM, when the concentration of BaP was 40 mg·L~(-1), the intensity of fluorescence protein(peak A and peak B)reached the maximum value of 68.91 a.u and 245.6 a.u. The analysis of FTIR showed that compared with that of the blank control,the cytoplasmic protein amideⅠand amide Ⅱ bands of the EPS shifted to the long wave direction under the induction of BaP. The results of SEM indicated that the increase in BaP concentration gradually hardened the EPS surface, the EPS was wool-like, and the voids reached the maximum number when the concentration of BaP was 40 mg·L~(-1). These findings show that BaP can break the original structure of M.mucedo EPS, low concentrations of BaP can promote the production of fungus EPS, and PN and PS might play a major role during the metabolic degradation of BaP by M. mucedo.
To elucidate the role of Mucor mucedo EPS in the remediation of polycyclic aromatic hydrocarbon(PAH)pollution, the extracted concentration of EPS(TOC)and the content of proteins(PN), polysaccharides(PS), humic acid(HC), and DNA in EPS were determined after the exposure of M. mucedo to different concentrations of benzo[a]pyrene(BaP). Three-dimensional fluorescence spectroscopy(3 DEEM)and flourier transform infrared spectroscopy(FTIR)were used to verify the changes in the concentration of proteins when different concentrations of BaP were used, and scanning electron microscopy(SEM)was employed to study the changes in the structure of EPS. The results showed that the changes in the concentration of TOC, PN, PS, and HC showed the same trend as the exposed BaP concentrations increased, and their content increased initially, and then decreased. When the concentration of BaP was 40 mg·L~(-1), the concentration of TOC,PN, PS, and HC produced by unit biomass reached the maximum values of 1 407.2 mg·g-1, 700 g·g-1, 564 g·g-1, and 174 g·g-1, respectively. According to the results of 3 D-EEM, when the concentration of BaP was 40 mg·L~(-1), the intensity of fluorescence protein(peak A and peak B)reached the maximum value of 68.91 a.u and 245.6 a.u. The analysis of FTIR showed that compared with that of the blank control,the cytoplasmic protein amideⅠand amide Ⅱ bands of the EPS shifted to the long wave direction under the induction of BaP. The results of SEM indicated that the increase in BaP concentration gradually hardened the EPS surface, the EPS was wool-like, and the voids reached the maximum number when the concentration of BaP was 40 mg·L~(-1). These findings show that BaP can break the original structure of M.mucedo EPS, low concentrations of BaP can promote the production of fungus EPS, and PN and PS might play a major role during the metabolic degradation of BaP by M. mucedo.
引文
[1] Wang C, Sun H, Li J, et al. Enzyme activities during degradation of polycyclic aromatic hydrocarbons by white rot fungus Phanerochaete chrysosporium in soils[J]. Chemosphere, 2009, 77(6):733-738.
[2] Juhasz A L, Naidu R. Bioremediation of high molecular weight polycyclic aromatic hydrocarbons:A review of the microbial degradation of benzo[a]pyrene[J]. International Biodeterioration&Biodegradation,2000, 45(1):57-88.
[3] Sun R, Belcher R W, Liang J, et al. Effects of cowpea(Vigna unguiculata)root mucilage on microbial community response and capacity for phenanthrene remediation[J]. Journal of Environmental Sciences, 2015,33(7):45-59.
[4] Li X, Li P, Lin X, et al. Biodegradation of aged polycyclic aromatic hydrocarbons(PAHs)by microbial consortia in soil and slurry phases[J].Journal of Hazardous Materials, 2008, 150(1):21-26.
[5] Seo J S, Keum Y S, Li Q X. Bacterial degradation of aromatic compounds[J]. International Journal of Environmental Research&Public Health, 2009, 6(1):278.
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[8] Zhang Z Q, Xia S Q, Wang X J, et al. A novel biosorbent for dye removal:Extracellular polymeric substance(EPS)of Proteus mirabilis TJ-1.[J]. Journal of Hazardous Materials, 2009, 163(1):279-284.
[9] Morgan J W, Forster C F, Evison L. A comparative study of the nature of biopolymers extracted from anaerobic and activated sludges[J]. Water Research, 1990, 24(6):743-750.
[10] Jia C, Li X, Zhang L, et al. Extracellular polymeric substances from a fungus are more effective than those from a bacterium in polycyclic aromatic hydrocarbon biodegradation[J]. Water Air&Soil Pollution,2017, 228(6):195.
[11] Jia C, Li X, Allinson G, et al. Composition and morphology characterization of exopolymeric substances produced by the PAH-degrading fungus of Mucor mucedo[J]. Environ Sci Pollut Res Int, 2016, 23(9):8421-8430.
[12]陶玉贵,倪正,王文强,等.活性污泥胞外聚合物(EPS)对Pb2+吸附性能的研究[J].农业环境科学学报, 2012, 31(5):1021-1027.TAO Yu-gui, NI Zheng, WANG Wen-qiang, et al. The characteristics study on adsorption of Pb2+by extracellular polymeric substances from activated sludge[J]. Journal of Agro-Environment Science, 2012,31(5):1021-1027.
[13]Antonio D B. Composition, fate and transformation of extracellular polymers in wastewater and sludge treatment processes[D]. New York,USA:Cornell University, 2000:684.
[14] Hudson N, Baker A, Ward D, et al. Can fluorescence spectrometry be used as a surrogate for the biochemical oxygen demand(BOD)test in water quality assessment? An example from South West England[J].Science of the Total Environment, 2008, 391(1):149-158.
[15] Karunakaran E, Biggs C A. Mechanisms of Bacillus cereus biofilm formation:An investigation of the physicochemical characteristics of cell surfaces and extracellular proteins[J]. Applied Microbiology&Biotechnology, 2011, 89(4):1161-1175.
[16] Davis M W, Glaser J A, Evans J W, et al. Field evaluation of the lignin-degrading fungus Phanerochaete sordida to treat creosote-contaminated soil[J]. Journal of Environmental Science&Technology,1993, 27(12):2572-2576.
[17] Dohse D M, Lion L W. Effect of microbial polymers on the sorption and transport of phenanthrene in a low-carbon sand[J]. Environmental Science&Technology, 1994, 28(4):541-548.
[18]张宇,梁彦秋,贾春云,等.不同方法提取PAHs高效降解菌EPS的特性[J].生态学杂志, 2014, 33(4):1027-1033.ZHANG Yu, LIANG Yan-qiu, JIA Chun-yun, et al. Characteristics of EPS extracted from a high efficient PAHs-degrading bacterium by different methods[J]. Chinese Journal of Ecology, 2014, 33(4):1027-1033.
[19] Henriques I D S, Love N G. The role of extracellular polymeric substances in the toxicity response of activated sludge bacteria to chemical toxins[J]. Water Research, 2007, 41(18):4177-4185.
[20] Chen J, Gu B, Leboeuf E J, et al. Spectroscopic characterization of the structural and functional properties of natural organic matter fractions[J]. Chemosphere, 2002, 48(1):59-68.
[21] Swietlik J, Dabrowska A, Raczykstanis?awiak U, et al. Reactivity of natural organic matter fractions with chlorine dioxide and ozone[J].Water Research, 2004, 38(3):547-558.
[22] Chen W, Westerhoff P, Leenheer J A, et al. Fluorescence excitationemission matrix regional integration to quantify spectra for dissolved organic matter[J]. Environmental Science&Technology, 2003, 37(24):5701-5710.
[23]牛秋雅.基于堆肥化和高效降解菌的多环芳烃降解研究[D].长沙:湖南大学, 2013.NIU Qiu-ya. Researches on the biodegradation of PAHs based on composting and high efficient PAHs-degrading bacteria[D]. Changsha:Hunan University, 2013.
[24]潘进权.毛霉AS3. 2778胞外蛋白酶组分的纯化、鉴定及性质研究[D].广州:华南理工大学, 2008.PAN Jin-quan. Purification, identification and characterization of extracellular protease fraction from Mucor AS3. 2778[D]. Guangzhou:South China University of Technology, 2008.
[25] Zhu L, Zhou J, Lv M, et al. Specific component comparison of extracellular polymeric substances(EPS)in flocs and granular sludge using EEM and SDS-PAGE[J]. Chemosphere, 2015, 121(5):26-32.
[26] Busi S, Karuganti S, Rajkumari J, et al. Sludge settling and algal flocculating activity of extracellular polymeric substance(EPS)derived from Bacillus cereus SK[J]. Water and Environment Journal, 2017, 31(1):97-104.
[27] Nielsen P H, Jahn A, Palmgren R. Conceptual model for production and composition of exopolymers in biofilms[J]. Water Science&Technology, 1997, 36(1):11-19.
[2] Juhasz A L, Naidu R. Bioremediation of high molecular weight polycyclic aromatic hydrocarbons:A review of the microbial degradation of benzo[a]pyrene[J]. International Biodeterioration&Biodegradation,2000, 45(1):57-88.
[3] Sun R, Belcher R W, Liang J, et al. Effects of cowpea(Vigna unguiculata)root mucilage on microbial community response and capacity for phenanthrene remediation[J]. Journal of Environmental Sciences, 2015,33(7):45-59.
[4] Li X, Li P, Lin X, et al. Biodegradation of aged polycyclic aromatic hydrocarbons(PAHs)by microbial consortia in soil and slurry phases[J].Journal of Hazardous Materials, 2008, 150(1):21-26.
[5] Seo J S, Keum Y S, Li Q X. Bacterial degradation of aromatic compounds[J]. International Journal of Environmental Research&Public Health, 2009, 6(1):278.
[6] Jia C, Li P, Li X, et al. Degradation of pyrene in soils by extracellular polymeric substances(EPS)extracted from liquid cultures[J]. Process Biochemistry, 2011, 46(8):1627-1631.
[7] Zhang Y, Wang F, Zhu X, et al. Extracellular polymeric substances govern the development of biofilm and mass transfer of polycyclic aromatic hydrocarbons for improved biodegradation[J]. Bioresour Technol,2015, 193(1):274-280.
[8] Zhang Z Q, Xia S Q, Wang X J, et al. A novel biosorbent for dye removal:Extracellular polymeric substance(EPS)of Proteus mirabilis TJ-1.[J]. Journal of Hazardous Materials, 2009, 163(1):279-284.
[9] Morgan J W, Forster C F, Evison L. A comparative study of the nature of biopolymers extracted from anaerobic and activated sludges[J]. Water Research, 1990, 24(6):743-750.
[10] Jia C, Li X, Zhang L, et al. Extracellular polymeric substances from a fungus are more effective than those from a bacterium in polycyclic aromatic hydrocarbon biodegradation[J]. Water Air&Soil Pollution,2017, 228(6):195.
[11] Jia C, Li X, Allinson G, et al. Composition and morphology characterization of exopolymeric substances produced by the PAH-degrading fungus of Mucor mucedo[J]. Environ Sci Pollut Res Int, 2016, 23(9):8421-8430.
[12]陶玉贵,倪正,王文强,等.活性污泥胞外聚合物(EPS)对Pb2+吸附性能的研究[J].农业环境科学学报, 2012, 31(5):1021-1027.TAO Yu-gui, NI Zheng, WANG Wen-qiang, et al. The characteristics study on adsorption of Pb2+by extracellular polymeric substances from activated sludge[J]. Journal of Agro-Environment Science, 2012,31(5):1021-1027.
[13]Antonio D B. Composition, fate and transformation of extracellular polymers in wastewater and sludge treatment processes[D]. New York,USA:Cornell University, 2000:684.
[14] Hudson N, Baker A, Ward D, et al. Can fluorescence spectrometry be used as a surrogate for the biochemical oxygen demand(BOD)test in water quality assessment? An example from South West England[J].Science of the Total Environment, 2008, 391(1):149-158.
[15] Karunakaran E, Biggs C A. Mechanisms of Bacillus cereus biofilm formation:An investigation of the physicochemical characteristics of cell surfaces and extracellular proteins[J]. Applied Microbiology&Biotechnology, 2011, 89(4):1161-1175.
[16] Davis M W, Glaser J A, Evans J W, et al. Field evaluation of the lignin-degrading fungus Phanerochaete sordida to treat creosote-contaminated soil[J]. Journal of Environmental Science&Technology,1993, 27(12):2572-2576.
[17] Dohse D M, Lion L W. Effect of microbial polymers on the sorption and transport of phenanthrene in a low-carbon sand[J]. Environmental Science&Technology, 1994, 28(4):541-548.
[18]张宇,梁彦秋,贾春云,等.不同方法提取PAHs高效降解菌EPS的特性[J].生态学杂志, 2014, 33(4):1027-1033.ZHANG Yu, LIANG Yan-qiu, JIA Chun-yun, et al. Characteristics of EPS extracted from a high efficient PAHs-degrading bacterium by different methods[J]. Chinese Journal of Ecology, 2014, 33(4):1027-1033.
[19] Henriques I D S, Love N G. The role of extracellular polymeric substances in the toxicity response of activated sludge bacteria to chemical toxins[J]. Water Research, 2007, 41(18):4177-4185.
[20] Chen J, Gu B, Leboeuf E J, et al. Spectroscopic characterization of the structural and functional properties of natural organic matter fractions[J]. Chemosphere, 2002, 48(1):59-68.
[21] Swietlik J, Dabrowska A, Raczykstanis?awiak U, et al. Reactivity of natural organic matter fractions with chlorine dioxide and ozone[J].Water Research, 2004, 38(3):547-558.
[22] Chen W, Westerhoff P, Leenheer J A, et al. Fluorescence excitationemission matrix regional integration to quantify spectra for dissolved organic matter[J]. Environmental Science&Technology, 2003, 37(24):5701-5710.
[23]牛秋雅.基于堆肥化和高效降解菌的多环芳烃降解研究[D].长沙:湖南大学, 2013.NIU Qiu-ya. Researches on the biodegradation of PAHs based on composting and high efficient PAHs-degrading bacteria[D]. Changsha:Hunan University, 2013.
[24]潘进权.毛霉AS3. 2778胞外蛋白酶组分的纯化、鉴定及性质研究[D].广州:华南理工大学, 2008.PAN Jin-quan. Purification, identification and characterization of extracellular protease fraction from Mucor AS3. 2778[D]. Guangzhou:South China University of Technology, 2008.
[25] Zhu L, Zhou J, Lv M, et al. Specific component comparison of extracellular polymeric substances(EPS)in flocs and granular sludge using EEM and SDS-PAGE[J]. Chemosphere, 2015, 121(5):26-32.
[26] Busi S, Karuganti S, Rajkumari J, et al. Sludge settling and algal flocculating activity of extracellular polymeric substance(EPS)derived from Bacillus cereus SK[J]. Water and Environment Journal, 2017, 31(1):97-104.
[27] Nielsen P H, Jahn A, Palmgren R. Conceptual model for production and composition of exopolymers in biofilms[J]. Water Science&Technology, 1997, 36(1):11-19.