接种混合功能细菌降低黑麦草体内菲和芘污染的机理初探
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摘要
本研究通过批量降解试验,探讨了功能菌株Massilia sp. Pn2和Mycobacterium flavescens 033降解菲和芘的基本动力学过程和规律;重点采用温室盆栽试验,研究了接种混合菌株对黑麦草体内PAHs含量及多酚氧化酶(PPO)和过氧化物酶(POD)活性的影响.结果表明,菌株Pn2和033可以分别利用菲和芘作为碳源和能源进行生长;在30℃、pH=7.0条件下,菌株Pn2和033对100 mg·L~(-1)菲和50 mg·L~(-1)芘的降解率分别高达99.7%和98.3%,降解半衰期分别为0.34 d和0.95 d(R~2>0.98).与接种灭活混合菌株对比,接种混合菌株Pn2和033显著地降低了黑麦草体内菲和芘的含量和积累量(p<0.05),并阻控菲和芘由黑麦草根向茎叶转移.同时,接种混合菌株Pn2和033显著地提高了黑麦草根和茎叶中POD(p<0.05)活性,该酶能够促进黑麦草体内超氧自由基的清除,并保护细胞免受PAHs损伤,进而影响PAHs在黑麦草体内的代谢过程.研究结果为阐明接种混合功能菌降低植物体内PAHs污染的作用机理提供了一定的参考价值.
Polycyclic aromatic hydrocarbons(PAHs) are a category of persistent organic contaminants extensively found in soil environments. PAHs can be taken up, accumulated, and translocated by plants, which pose a significant risk worldwide to food safety and hence human health due to their high bio-accumulation, carcinogenicity, toxicity, and biodegradation-resistant. It is well documented that inoculation of PAH-degrading bacterium is a simple and effective strategy for mitigating the contamination of PAHs in plants. However, microscopic information is available on inoculation with mixed PAH-degrading bacteria to regulate plant PAH contamination and the activities of related to metabolic enzymes in plants. In this study, the biodegradation kinetics and regularity of phenanthrene and pyrene were respectively investigated using Massilia sp. Pn2 and Mycobacterium flavescens 033 in in vitro. In particular, greenhouse pot experiments of inoculated two PAH-degrading bacteria were conducted to reduce PAHs residues in ryegrass(Lolium multiflorum Lam), and the effect of strains Pn2 and 033 on the activities of polyphenol oxidase(PPO) and peroxidase(POD) was also explored in in vivo. Results indicated that both strains Pn2 and 033 could use phenanthrene and pyrene as the sources of carbon and energy for growth of tested strains. They were effective in biodegrading PAHs in in vitro, and the biodegradation rates of phenanthrene and pyrene were respectively 99.7% and 98.3%, the half-life values were respectively 0.34 d and 0.95 d(R~2>0.98). Compared with the mixed dead bacteria control(CRD), inoculation with mixed PAH-degrading strains altered the migration and transformation of PAHs in soil-ryegrass systems, and reduced the concentration and accumulation of PAHs into ryegrass roots and shoots(p<0.05). In addition, inoculation with mixed PAH-degrading bacteria also impacted the activities of PPO(p>0.05) and POD(p<0.05) in plants. Thus, strains Pn2 and 033 could be a useful bacterial resource for reducing PAHs contents via regulating the activity of POD inside plants. These findings provide a novel perspective in utilizing plant-bacteria partnerships to reduce plan PAH residues and regulate the activities of enzymes in plants, with ultimate goal of protecting agricultural products safety and human health.
Polycyclic aromatic hydrocarbons(PAHs) are a category of persistent organic contaminants extensively found in soil environments. PAHs can be taken up, accumulated, and translocated by plants, which pose a significant risk worldwide to food safety and hence human health due to their high bio-accumulation, carcinogenicity, toxicity, and biodegradation-resistant. It is well documented that inoculation of PAH-degrading bacterium is a simple and effective strategy for mitigating the contamination of PAHs in plants. However, microscopic information is available on inoculation with mixed PAH-degrading bacteria to regulate plant PAH contamination and the activities of related to metabolic enzymes in plants. In this study, the biodegradation kinetics and regularity of phenanthrene and pyrene were respectively investigated using Massilia sp. Pn2 and Mycobacterium flavescens 033 in in vitro. In particular, greenhouse pot experiments of inoculated two PAH-degrading bacteria were conducted to reduce PAHs residues in ryegrass(Lolium multiflorum Lam), and the effect of strains Pn2 and 033 on the activities of polyphenol oxidase(PPO) and peroxidase(POD) was also explored in in vivo. Results indicated that both strains Pn2 and 033 could use phenanthrene and pyrene as the sources of carbon and energy for growth of tested strains. They were effective in biodegrading PAHs in in vitro, and the biodegradation rates of phenanthrene and pyrene were respectively 99.7% and 98.3%, the half-life values were respectively 0.34 d and 0.95 d(R~2>0.98). Compared with the mixed dead bacteria control(CRD), inoculation with mixed PAH-degrading strains altered the migration and transformation of PAHs in soil-ryegrass systems, and reduced the concentration and accumulation of PAHs into ryegrass roots and shoots(p<0.05). In addition, inoculation with mixed PAH-degrading bacteria also impacted the activities of PPO(p>0.05) and POD(p<0.05) in plants. Thus, strains Pn2 and 033 could be a useful bacterial resource for reducing PAHs contents via regulating the activity of POD inside plants. These findings provide a novel perspective in utilizing plant-bacteria partnerships to reduce plan PAH residues and regulate the activities of enzymes in plants, with ultimate goal of protecting agricultural products safety and human health.
引文
Arslan M,Imran A,Khan Q M,et al.2017.Plant-bacteria partnerships for the remediation of persistent organic pollutants[J].Environmental Science and Pollution Research,24(5):4322-4336
Afzal M,Khan Q M,Sessitsch A.2014.Endophytic bacteria:Prospects and applications for the phytoremediation of organic pollutants[J].Chemosphere,117:232-242
Bacosa H P,Inoue C.2015.Polycyclic aromatic hydrocarbons (PAHs) biodegradation potential and diversity of microbial consortia enriched from tsunami sediments in Miyagi,Japan[J].Journal of Hazardous Materials,283:689-697
Barnier C,Ouvrard S,Robin C,et al.2014.Desorption kinetics of PAHs from aged industrial soils for availability assessment[J].Science of the Total Environment,470:639-645
?akmak?i R,D?nmez F,Ayd?n A,et al.2006.Growth promotion of plants by plant growth-promoting rhizobacteria under greenhouse and two different field soil conditions[J].Soil Biology & Biochemistry,38(6):1482-1487
Cai C Y,Li J Y,Wu D,et al.2017.Spatial distribution,emission source and health risk of parent PAHs and derivatives in surface soils from the Yangtze River Delta,eastern China[J].Chemosphere,178:301-308
Campos V,Merino I,Casado R,et al.2008.Phytoremediation of organic pollutants[J].Spanish Journal of Agricultural Research,6:38-47
Chen Z X,Ni H G,Jing X,et al.2015.Plant uptake,translocation,and return of polycyclic aromatic hydrocarbons via fine root branch orders in a subtropical forest ecosystem[J].Chemosphere,131:192-200
Chaudhry Q,Schr?der P,Werck-Reichhart D,et al.2002.Prospects and limitations of phytoremediation for the removal of persistent pesticides in the environment[J].Environmental Science and Pollution Research,9:4-17
陈保冬,赵方杰,张莘,等.2015.土壤生物与土壤污染研究前沿与展望[J].生态学报,35(20):6604-6613
刁硕,王红旗,许洁,等.2017.低温耐盐芘降解菌的筛选鉴定及降解特性研究[J].中国环境科学,37(2):677-685
Dean-Ross D,Cerniglia C E.1996.Degradation of pyrene by Mycobacterium flavescens[J].Applied Microbiology and Biotechnology,46:307-312
Glick B R.2014.Bacteria with ACC deaminase can promote plant growth and help to feed the world[J].Microbiological Research,169(1):30-39
Gao Y,Zhu L.2004.Plant uptake,accumulation and translocation of phenanthrene and pyrene in soils[J].Chemosphere,55(9):1169-1178
Iwabuchi T,Harayama S.1998.Biochemical and molecular characterization of 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp.KP7[J].Journal of Biological Chemistry,273(14):8332-8336
Kim K H,Jahan S A,Kabir E,et al.2013.A review of airborne polycyclic aromatic hydrocarbons (PAHs) and their human health effects[J].Environment International,60:71-80
Kvesitadze E,Sadunishvili T,Kvesitadze G.2009.Mechanisms of organic contaminants uptake and degradation in plants[J].World Academy of Science,Engineering and Technology,55(6):458-468
Liu J,Liu S,Sun K,et al.2014.Colonization on root surface by a phenanthrene-degrading endophytic bacterium and its application for reducing plant phenanthrene contamination[J].PloS One,9(9):e108249
Liu J,Xiang Y,Zhang Z,et al.2017.Inoculation of a phenanthrene-degrading endophytic bacterium reduces the phenanthrene level and alters the bacterial community structure in wheat[J].Applied Microbiology and Biotechnology,101(12):5199-5212
Liu S H,Zeng G M,Niu Q Y,et al.2016.Bioremediation mechanisms of combined pollution of PAHs and heavy metals by bacteria and fungi:A mini review[J].Bioresource Technology,224:35-33
雷东锋,冯怡,蒋大宗.2004.植物中多酚氧化酶的特征[J].自然科学进展,14(6):606-614
李全霞,范丙全,龚明波,等.2008.降解芘的分枝杆菌M11的分离鉴定和降解特性[J].环境科学,29(3):763-768
刘爽,刘娟,凌婉婷,等.2013.一株高效降解菲的植物内生细菌筛选及其生长特性[J].中国环境科学,33(1):95-102
Menzie C A,Potocki B B,Santodonato J.1992.Exposure to carcinogenic PAHs in the environment[J].Environmental Science & Technology,26:1278-1284
倪雪,刘娟,高彦征,等.2013.2株降解菲的植物内生细菌筛选及其降解特性[J].环境科学,34(2):746-752
Sprocati A,Alisi C,PintoV,et al.2014.Assessment of the applicability of a “toolbox” designed for microbially assisted phytoremediation:The case study at Ingurtosu mining site (Italy)[J].Environmental Science and Pollution Research,21:6939-6951
Sinsabaugh R L.2010.Phenol oxidase,peroxidase and organic matter dynamics of soil[J].Soil Biology and Biochemistry,42(3):391-404
Siciliano S D,Fortin N,Mihoc A,et al.2001.Selection of specific endophytic bacterial genotypes by plants in response to soil contamination[J].Applied and Environmental Microbiology,67(6):2469-2475
Shah K,Kumar R G,Verma S,et al.2001.Effect of cadmium on lipid peroxidation,superoxide anion generation and activities of antioxidant enzymes in growing rice seedlings[J].Plant Science,161(6):1135-1144
Sun K,Liu J,Gao Y,et al.2014a.Isolation,plant colonization potential,and phenanthrene degradation performance of the endophytic bacterium Pseudomonas sp.Ph6-gfp[J].Scientific Reports,4:5462
Sun K,Liu J,Jin L,et al.2014b.Utilizing pyrene-degrading endophytic bacteria to reduce the risk of plant pyrene contamination[J].Plant and Soil,374(1/2):251-262
Sams?e-Petersen L,Larsen E H,Larsen P B,et al.2002.Uptake of trace elements and PAHs by fruit and vegetables from contaminated soils[J].Environmental Science & Technology,36(14):3057-3063
盛月慧,刘娟,高彦征,等.2013.黑麦草体内POD和PPO活性及可培养内生细菌种群对不同浓度菲污染的响应[J].南京农业大学学报,36(6):51-59
孙凯,刘娟,李欣,等.2014.两株具有芘降解功能的植物内生细菌的分离筛选及其特性[J].生态学报,34(4):853-861
Taghavi S,Barac T,Greenberg B,et al.2005.Horizontal gene transfer to endogenous endophytic bacteria from poplar improves phytoremediation of toluene[J].Applied and Environmental Microbiology,71(12):8500-8505
Takaki K,Wade A J,Collins C D.2014.Assessment of plant uptake models used in exposure assessment tools for soils contaminated with organic pollutants[J].Environmental Science & Technology,48:12073-12082
Wilson M S,Herrick J B,Jeon C O,et al.2003.Horizontal transfer of phnAc dioxygenase genes within one of two phenotypically and genotypically distinctive naphthalene-degrading guilds from adjacent soil environments[J].Applied and Environmental Microbiology,69(4):2172-2181
Wang J,Liu J,Ling W,et al.2017.Composite of PAH-degrading endophytic bacteria reduces contamination and health risks caused by PAHs in vegetables[J].Science of the Total Environment,598:471-478
Waigi M G,Sun K,Gao Y.2017.Sphingomonads in microbe-assisted phytoremediation:Tackling soil pollution[J].Trends in Biotechnology,35(9):883-899
Wang Y,Xiao M,Geng X,et al.2007.Horizontal transfer of genetic determinants for degradation of phenol between the bacteria living in plant and its rhizosphere[J].Applied Microbiology and Biotechnology,77(3):733-739
Wang Y,Tian Z,Zhu H,et al.2012.Polycyclic aromatic hydrocarbons (PAHs) in soils and vegetation near an e-waste recycling site in South China:Concentration,distribution,source,and risk assessment[J].Science of the Total Environment,439:187-193
Weyens N,Van Der Lelie D,Artois T,et al.2009.Bioaugmentation with engineered endophytic bacteria improves contaminant fate in phytoremediation[J].Environmental Science & Technology,43(24):9413-9418
王涛,蓝慧,田云,等.2016.多环芳烃的微生物降解机制研究进展[J].化学与生物工程,33(2):8-14
夏红霞,朱启红.2013.Pb、Zn及其交互作用对蜈蚣草抗氧化酶及叶绿素含量的影响[J].环境化学,32(6):959-963
徐成斌,王闻烨,李鲜珠,等.2015.一株菲降解菌的鉴定及降解特性[J].环境科学学报,35(3):684-691
Zhu X,Ni X,Liu J,et al.2014.Application of endophytic bacteria to reduce persistent organic pollutants contamination in plants[J].CLEAN-Soil Air Water,42(3):306-310
Zhu X,Ni X,Waigi M G,et al.2016.Biodegradation of mixed PAHs by PAH-degrading endophytic bacteria[J].International Journal of Environmental Research and Public Health,13(8):805
Zielińska A,Oleszczuk P.2015.Evaluation of sewage sludge and slow pyrolyzed sewage sludge-derived biochar for adsorption of phenanthrene and pyrene[J].Bioresource Technology,192:618-626
Zhang W,Wang H,Zhang R,et al.2010.Bacterial communities in PAH contaminated soils at an electronic-waste processing center in China[J].Ecotoxicology,19:96-104
Afzal M,Khan Q M,Sessitsch A.2014.Endophytic bacteria:Prospects and applications for the phytoremediation of organic pollutants[J].Chemosphere,117:232-242
Bacosa H P,Inoue C.2015.Polycyclic aromatic hydrocarbons (PAHs) biodegradation potential and diversity of microbial consortia enriched from tsunami sediments in Miyagi,Japan[J].Journal of Hazardous Materials,283:689-697
Barnier C,Ouvrard S,Robin C,et al.2014.Desorption kinetics of PAHs from aged industrial soils for availability assessment[J].Science of the Total Environment,470:639-645
?akmak?i R,D?nmez F,Ayd?n A,et al.2006.Growth promotion of plants by plant growth-promoting rhizobacteria under greenhouse and two different field soil conditions[J].Soil Biology & Biochemistry,38(6):1482-1487
Cai C Y,Li J Y,Wu D,et al.2017.Spatial distribution,emission source and health risk of parent PAHs and derivatives in surface soils from the Yangtze River Delta,eastern China[J].Chemosphere,178:301-308
Campos V,Merino I,Casado R,et al.2008.Phytoremediation of organic pollutants[J].Spanish Journal of Agricultural Research,6:38-47
Chen Z X,Ni H G,Jing X,et al.2015.Plant uptake,translocation,and return of polycyclic aromatic hydrocarbons via fine root branch orders in a subtropical forest ecosystem[J].Chemosphere,131:192-200
Chaudhry Q,Schr?der P,Werck-Reichhart D,et al.2002.Prospects and limitations of phytoremediation for the removal of persistent pesticides in the environment[J].Environmental Science and Pollution Research,9:4-17
陈保冬,赵方杰,张莘,等.2015.土壤生物与土壤污染研究前沿与展望[J].生态学报,35(20):6604-6613
刁硕,王红旗,许洁,等.2017.低温耐盐芘降解菌的筛选鉴定及降解特性研究[J].中国环境科学,37(2):677-685
Dean-Ross D,Cerniglia C E.1996.Degradation of pyrene by Mycobacterium flavescens[J].Applied Microbiology and Biotechnology,46:307-312
Glick B R.2014.Bacteria with ACC deaminase can promote plant growth and help to feed the world[J].Microbiological Research,169(1):30-39
Gao Y,Zhu L.2004.Plant uptake,accumulation and translocation of phenanthrene and pyrene in soils[J].Chemosphere,55(9):1169-1178
Iwabuchi T,Harayama S.1998.Biochemical and molecular characterization of 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp.KP7[J].Journal of Biological Chemistry,273(14):8332-8336
Kim K H,Jahan S A,Kabir E,et al.2013.A review of airborne polycyclic aromatic hydrocarbons (PAHs) and their human health effects[J].Environment International,60:71-80
Kvesitadze E,Sadunishvili T,Kvesitadze G.2009.Mechanisms of organic contaminants uptake and degradation in plants[J].World Academy of Science,Engineering and Technology,55(6):458-468
Liu J,Liu S,Sun K,et al.2014.Colonization on root surface by a phenanthrene-degrading endophytic bacterium and its application for reducing plant phenanthrene contamination[J].PloS One,9(9):e108249
Liu J,Xiang Y,Zhang Z,et al.2017.Inoculation of a phenanthrene-degrading endophytic bacterium reduces the phenanthrene level and alters the bacterial community structure in wheat[J].Applied Microbiology and Biotechnology,101(12):5199-5212
Liu S H,Zeng G M,Niu Q Y,et al.2016.Bioremediation mechanisms of combined pollution of PAHs and heavy metals by bacteria and fungi:A mini review[J].Bioresource Technology,224:35-33
雷东锋,冯怡,蒋大宗.2004.植物中多酚氧化酶的特征[J].自然科学进展,14(6):606-614
李全霞,范丙全,龚明波,等.2008.降解芘的分枝杆菌M11的分离鉴定和降解特性[J].环境科学,29(3):763-768
刘爽,刘娟,凌婉婷,等.2013.一株高效降解菲的植物内生细菌筛选及其生长特性[J].中国环境科学,33(1):95-102
Menzie C A,Potocki B B,Santodonato J.1992.Exposure to carcinogenic PAHs in the environment[J].Environmental Science & Technology,26:1278-1284
倪雪,刘娟,高彦征,等.2013.2株降解菲的植物内生细菌筛选及其降解特性[J].环境科学,34(2):746-752
Sprocati A,Alisi C,PintoV,et al.2014.Assessment of the applicability of a “toolbox” designed for microbially assisted phytoremediation:The case study at Ingurtosu mining site (Italy)[J].Environmental Science and Pollution Research,21:6939-6951
Sinsabaugh R L.2010.Phenol oxidase,peroxidase and organic matter dynamics of soil[J].Soil Biology and Biochemistry,42(3):391-404
Siciliano S D,Fortin N,Mihoc A,et al.2001.Selection of specific endophytic bacterial genotypes by plants in response to soil contamination[J].Applied and Environmental Microbiology,67(6):2469-2475
Shah K,Kumar R G,Verma S,et al.2001.Effect of cadmium on lipid peroxidation,superoxide anion generation and activities of antioxidant enzymes in growing rice seedlings[J].Plant Science,161(6):1135-1144
Sun K,Liu J,Gao Y,et al.2014a.Isolation,plant colonization potential,and phenanthrene degradation performance of the endophytic bacterium Pseudomonas sp.Ph6-gfp[J].Scientific Reports,4:5462
Sun K,Liu J,Jin L,et al.2014b.Utilizing pyrene-degrading endophytic bacteria to reduce the risk of plant pyrene contamination[J].Plant and Soil,374(1/2):251-262
Sams?e-Petersen L,Larsen E H,Larsen P B,et al.2002.Uptake of trace elements and PAHs by fruit and vegetables from contaminated soils[J].Environmental Science & Technology,36(14):3057-3063
盛月慧,刘娟,高彦征,等.2013.黑麦草体内POD和PPO活性及可培养内生细菌种群对不同浓度菲污染的响应[J].南京农业大学学报,36(6):51-59
孙凯,刘娟,李欣,等.2014.两株具有芘降解功能的植物内生细菌的分离筛选及其特性[J].生态学报,34(4):853-861
Taghavi S,Barac T,Greenberg B,et al.2005.Horizontal gene transfer to endogenous endophytic bacteria from poplar improves phytoremediation of toluene[J].Applied and Environmental Microbiology,71(12):8500-8505
Takaki K,Wade A J,Collins C D.2014.Assessment of plant uptake models used in exposure assessment tools for soils contaminated with organic pollutants[J].Environmental Science & Technology,48:12073-12082
Wilson M S,Herrick J B,Jeon C O,et al.2003.Horizontal transfer of phnAc dioxygenase genes within one of two phenotypically and genotypically distinctive naphthalene-degrading guilds from adjacent soil environments[J].Applied and Environmental Microbiology,69(4):2172-2181
Wang J,Liu J,Ling W,et al.2017.Composite of PAH-degrading endophytic bacteria reduces contamination and health risks caused by PAHs in vegetables[J].Science of the Total Environment,598:471-478
Waigi M G,Sun K,Gao Y.2017.Sphingomonads in microbe-assisted phytoremediation:Tackling soil pollution[J].Trends in Biotechnology,35(9):883-899
Wang Y,Xiao M,Geng X,et al.2007.Horizontal transfer of genetic determinants for degradation of phenol between the bacteria living in plant and its rhizosphere[J].Applied Microbiology and Biotechnology,77(3):733-739
Wang Y,Tian Z,Zhu H,et al.2012.Polycyclic aromatic hydrocarbons (PAHs) in soils and vegetation near an e-waste recycling site in South China:Concentration,distribution,source,and risk assessment[J].Science of the Total Environment,439:187-193
Weyens N,Van Der Lelie D,Artois T,et al.2009.Bioaugmentation with engineered endophytic bacteria improves contaminant fate in phytoremediation[J].Environmental Science & Technology,43(24):9413-9418
王涛,蓝慧,田云,等.2016.多环芳烃的微生物降解机制研究进展[J].化学与生物工程,33(2):8-14
夏红霞,朱启红.2013.Pb、Zn及其交互作用对蜈蚣草抗氧化酶及叶绿素含量的影响[J].环境化学,32(6):959-963
徐成斌,王闻烨,李鲜珠,等.2015.一株菲降解菌的鉴定及降解特性[J].环境科学学报,35(3):684-691
Zhu X,Ni X,Liu J,et al.2014.Application of endophytic bacteria to reduce persistent organic pollutants contamination in plants[J].CLEAN-Soil Air Water,42(3):306-310
Zhu X,Ni X,Waigi M G,et al.2016.Biodegradation of mixed PAHs by PAH-degrading endophytic bacteria[J].International Journal of Environmental Research and Public Health,13(8):805
Zielińska A,Oleszczuk P.2015.Evaluation of sewage sludge and slow pyrolyzed sewage sludge-derived biochar for adsorption of phenanthrene and pyrene[J].Bioresource Technology,192:618-626
Zhang W,Wang H,Zhang R,et al.2010.Bacterial communities in PAH contaminated soils at an electronic-waste processing center in China[J].Ecotoxicology,19:96-104
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