牛粪堆肥系统环境因子对抗性基因的影响
|
摘要
本研究利用实时定量PCR技术检测牛粪60 d好氧堆肥过程中典型四环素类抗性基因(tet Q、tet W、tet M、tet G、tet A和tet X)和大环内酯类抗性基因(erm35、erm36、erm B、erm F、erm T和erm X)的数量变化规律,系统研究了温度、含水率、挥发性有机质(VS)含量、碳氮比(C/N)、pH值、氧化还原电位(ORP)等堆肥系统环境因子和细菌数量(TCB)对这两类典型抗生素抗性基因的影响.结果表明,堆肥升温阶段牛粪中tet Q、tet W、tet G和tet X数量分别增加1. 7、0. 3、84. 8和4. 5倍,而tet M丰度降低了83. 1%;堆肥腐熟阶段牛粪中tet G和tet X数量分别增加23. 8和11. 5倍,而tet W、tet Q和tet M数量均降低90%.整个牛粪堆肥过程中erm35、erm36、erm B、erm F、erm T和erm X丰度分别增加2. 1、430. 4、0. 6、11. 5、2. 1和49. 1倍. RDA分析表明,牛粪堆肥过程中对四环素与大环内酯类抗性基因数量影响较大的3个环境因子分别为:ORP(54. 8%)、温度(34%)和VS(11. 3%),其中ORP与erm X和erm B、温度与tet Q和erm F以及VS与tet W数量均呈明显地正相关.
The effects of environmental factors and bacterial numbers on typical tetracycline resistance genes( tet Q,tet W,tet M,tet G,tet A,and tet X) and macrolide resistance genes( erm35,erm36,erm B,erm F,erm T,and erm X) during cow manure composting were investigated and quantified via real-time quantitative PCR. The results suggested that the abundance of tet Q,tet W,tet G,and tet X increased by 1. 7,0. 3,84. 8,and 4. 5 times respectively,whereas tet M decreased by 83. 1% during the heating stages of cow manure composting. The abundance of tet G and tet X increased by 23. 8 and 11. 5 times respectively,whereas tet W,tet Q,and tet M decreased by 90% during the maturity stage. However,the macrolide resistance genes( erm35,erm36,erm B,erm F,erm T,and erm X)increased by 2. 1,430. 4,0. 6,11. 5,2. 1,and 49. 1 times,respectively. Redundancy analysis showed that ORP,temperature,and VS were crucial factors that could explain the variations of 54. 8%,34%,and 11. 3%,respectively,in ARGs. ORP,temperature,and VS also had the greatest positive correlation with erm X,erm B,tet Q,erm F,and tet W,in that order.
The effects of environmental factors and bacterial numbers on typical tetracycline resistance genes( tet Q,tet W,tet M,tet G,tet A,and tet X) and macrolide resistance genes( erm35,erm36,erm B,erm F,erm T,and erm X) during cow manure composting were investigated and quantified via real-time quantitative PCR. The results suggested that the abundance of tet Q,tet W,tet G,and tet X increased by 1. 7,0. 3,84. 8,and 4. 5 times respectively,whereas tet M decreased by 83. 1% during the heating stages of cow manure composting. The abundance of tet G and tet X increased by 23. 8 and 11. 5 times respectively,whereas tet W,tet Q,and tet M decreased by 90% during the maturity stage. However,the macrolide resistance genes( erm35,erm36,erm B,erm F,erm T,and erm X)increased by 2. 1,430. 4,0. 6,11. 5,2. 1,and 49. 1 times,respectively. Redundancy analysis showed that ORP,temperature,and VS were crucial factors that could explain the variations of 54. 8%,34%,and 11. 3%,respectively,in ARGs. ORP,temperature,and VS also had the greatest positive correlation with erm X,erm B,tet Q,erm F,and tet W,in that order.
引文
[1]苏建强,黄福义,朱永官.环境抗生素抗性基因研究进展[J].生物多样性,2013,21(4):481-487.Su J Q,Huang F Y,Zhu Y G.Antibiotic resistance genes in the environment[J].Biodiversity Science,2013,21(4):481-487.
[2]Wu D,Huang X H,Sun J Z,et al.Antibiotic resistance genes and associated microbial community conditions in aging landfill systems[J].Environmental Science&Technology,2017,51(21):12859-12867.
[3]Jechalke S,Heuer H,Siemens J,et al.Fate and effects of veterinary antibiotics in soil[J].Trends in Microbiology,2014,22(9):536-545.
[4]Kong W D,Zhu Y G,Fu B J,et al.The veterinary antibiotic oxytetracycline and Cu influence functional diversity of the soil microbial community[J].Environmental Pollution,2006,143(1):129-137.
[5]Lin H,Zhang J,Chen H J,et al.Effect of temperature on sulfonamide antibiotics degradation,and on antibiotic resistance determinants and hosts in animal manures[J].Science of the Total Environment,2017,607-608:725-732.
[6]Cheng W X,Li J N,Wu Y,et al.Behavior of antibiotics and antibiotic resistance genes in eco-agricultural system:a case study[J].Journal of Hazardous Materials,2016,304:18-25.
[7]Liu M M,Zhang Y,Yang M,et al.Abundance and distribution of tetracycline resistance genes and mobile elements in an oxytetracycline production wastewater treatment system[J].Environmental Science&Technology,2012,46(14):7551-7557.
[8]Marti E,Jofre J,Balcazar J L.Prevalence of antibiotic resistance genes and bacterial community composition in a river influenced by a wastewater treatment plant[J].PLo S One,2013,8(10):e78906.
[9]何芳,罗阳,浣成,等.高温堆肥技术在我国畜禽粪便污染治理中的应用[J].安徽农业科学,2018,46(17):41-43.He F,Luo Y,Huan C,et al.Application of high-temperature composting technology in the pollution treatment of livestock and poultry manure in China[J].Journal of Anhui Agricultural Sciences,2018,46(17):41-43.
[10]Ho Y B,Zakaria M P,Latif P A,et al.Degradation of veterinary antibiotics and hormone during broiler manure composting[J].Bioresource Technology,2013,131:476-484.
[11]Wang J,Ben W W,Zhang Y,et al.Effects of thermophilic composting on oxytetracycline,sulfamethazine,and their corresponding resistance genes in swine manure[J].Environmental Science:Processes&Impacts,2015,17(9):1654-1660.
[12]Wang K,Chu C,Li X K,et al.Succession of bacterial community function in cow manure composing[J].Bioresource Technology,2018,267:63-70.
[13]Wang K,Mao H L,Li X K.Functional characteristics and influence factors of microbial community in sewage sludge composting with inorganic bulking agent[J].Bioresource Technology,2018,249:527-535.
[14]王伟东,王小芬,朴哲,等.堆肥化过程中微生物群落的动态[J].环境科学,2007,28(11):2591-2597.Wang W D,Wang X F,Piao Z,et al.Microbial dynamics during the composting process[J].Environmental Science,2007,28(11):2591-2597.
[15]勾长龙,王雨琼,张喜庆,等.高温堆肥对猪粪中四环素类抗生素及抗性基因的影响[J].环境科学学报,2017,37(4):1454-1460.Gou C L,Wang Y Q,Zhang X Q,et al.Effects of hightemperature composting on tetracyclines and tetracycline resistance genes in swine manure[J].Acta Scientiae Circumstantiae,2017,37(4):1454-1460.
[16]Li H C,Duan M L,Gu J,et al.Effects of bamboo charcoal on antibiotic resistance genes during chicken manure composting[J].Ecotoxicology and Environmental Safety,2017,140:1-6.
[17]Chen J,Yu Z T,Michel F C Jr,et al.Development and application of real-time PCR assays for quantification of erm genes conferring resistance to macrolides-lincosamides-streptogramin b in livestock manure and manure management systems[J].Applied and Environmental Microbiology,2007,73(14):4407-4416.
[18]钱勋.好氧堆肥对畜禽粪便中抗生素抗性基因的削减条件探索及影响机理研究[D].杨凌:西北农林科技大学,2016.1-140.Qian X.Mechanism and conditions for reducing antibiotic resistance genes during aerobic composting of livestock manure[D].Yangling:Northwest A&F University,2016.1-140.
[19]康军.杨凌城市污泥高效好氧堆肥研究[D].杨凌:西北农林科技大学,2012.Kang J.High-efficiency aerobic composting of municipal sewage sludge in Yangling[D].Yangling:Northwest A&F University,2012.
[20]郑宁国.猪粪堆肥对抗生素抗性基因的影响及微生物群落变化的初步探究[D].西安:西北大学,2016.Zheng N G.Effects of swine manure composting on ARGs and the initial research of changes of microorganism community[D].Xi’an:Northwest University,2016.
[21]Gao M C,Liang F Y,Yu A,et al.Evaluation of stability and maturity during forced-aeration composting of chicken manure and sawdust at different C/N ratios[J].Chemosphere,2010,78(5):614-619.
[22]Liu C G,Xue C,Lin Y H,et al.Redox potential control and applications in microaerobic and anaerobic fermentations[J].Biotechnology Advances,2013,31(2):257-265.
[23]Su J Q,Wei B,Ou-Yang W Y,et al.Antibiotic resistome and its association with bacterial communities during sewage sludge composting[J].Environmental Science&Technology,2015,49(12):7356-7363.
[24]Guo A Y,Gu J,Wang X J,et al.Effects of superabsorbent polymers on the abundances of antibiotic resistance genes,mobile genetic elements,and the bacterial community during swine manure composting[J].Bioresource Technology,2017,244:658-663.
[25]Wang R,Zhang J Y,Sui Q W,et al.Effect of red mud addition on tetracycline and copper resistance genes and microbial community during the full scale swine manure composting[J].Bioresource Technology,2016,216:1049-1057.
[26]Yu Z T,Michel F C Jr,Hansen G,et al.Development and application of real-time PCR assays for quantification of genes encoding tetracycline resistance[J].Applied and Environmental Microbiology,2005,71(11):6926-6933.
[27]Zhang J Y,Sui Q W,Tong J,et al.Soil types influence the fate of antibiotic-resistant bacteria and antibiotic resistance genes following the land application of sludge composts[J].Environment International,2018,118:34-43.
[28]郑宁国,黄南,王卫卫,等.高温堆肥过程对猪粪来源抗生素抗性基因的影响[J].环境科学,2016,37(5):1986-1992.Zheng N G,Huang N,Wang W W,et al.Effects of thermophilic composting on antibiotic resistance genes(ARGs)of swine manure source[J].Environmental Science,2016,37(5):1986-1992.
[29]韦蓓,黄福义,李虎,等.污泥堆肥过程中磺胺类和大环内酯类抗性基因的残留[J].应用与环境生物学报,2014,20(3):395-400.Wei P,Huang F Y,Li H,et al.Persistence of sulfonamide and macrolide resistance genes during sewage sludge composting[J].Chinese Journal of Applied and Environmental Biology,2014,20(3):395-400.
[30]Zhang J Y,Chen M X,Sui Q W,et al.Impacts of addition of natural zeolite or a nitrification inhibitor on antibiotic resistance genes during sludge composting[J].Water Research,2016,91:339-349.
[31]Liao H P,Lu X M,Rensing C,et al.Hyperthermophilic composting accelerates the removal of antibiotic resistance genes and mobile genetic elements in sewage sludge[J].Environmental Science&Technology,2018,52(1):266-276.
[32]Wu D,Huang Z T,Yang K,et al.Relationships between antibiotics and antibiotic resistance gene levels in municipal solid waste leachates in shanghai,China[J].Environmental Science&Technology,2015,49(7):4122-4128.
[33]Qian X,Sun W,Gu J,et al.Variable effects of oxytetracycline on antibiotic resistance gene abundance and the bacterial community during aerobic composting of cow manure[J].Journal of Hazardous Materials,2016,315:61-69.
[34]Peng S,Li H J,Song D,et al.Influence of zeolite and superphosphate as additives on antibiotic resistance genes and bacterial communities during factory-scale chicken manure composting[J].Bioresource Technology,2018,263:393-401.
[35]Chopra I,Roberts M.Tetracycline antibiotics:mode of action,applications,molecular biology,and epidemiology of bacterial resistance[J].Microbiology and Molecular Biology Reviews,2001,65(2):232-260.
[36]Zhang Y J,Li H C,Gu J,et al.Effects of adding different surfactants on antibiotic resistance genes and intI1 during chicken manure composting[J].Bioresource Technology,2016,219:545-551.
[37]Zhang R R,Gu J,Wang X J,et al.Contributions of the microbial community and environmental variables to antibiotic resistance genes during co-composting with swine manure and cotton stalks[J].Journal of Hazardous Materials,2018,358:82-91.
[38]Dobal-Amador V,Nieto-Cid M,Guerrero-Feijóo E,et al.Vertical stratification of bacterial communities driven by multiple environmental factors in the waters(0-5000 m)off the Galician coast(NW Iberian margin)[J].Deep Sea Research Part I:Oceanographic Research Papers,2016,114:1-11.
[2]Wu D,Huang X H,Sun J Z,et al.Antibiotic resistance genes and associated microbial community conditions in aging landfill systems[J].Environmental Science&Technology,2017,51(21):12859-12867.
[3]Jechalke S,Heuer H,Siemens J,et al.Fate and effects of veterinary antibiotics in soil[J].Trends in Microbiology,2014,22(9):536-545.
[4]Kong W D,Zhu Y G,Fu B J,et al.The veterinary antibiotic oxytetracycline and Cu influence functional diversity of the soil microbial community[J].Environmental Pollution,2006,143(1):129-137.
[5]Lin H,Zhang J,Chen H J,et al.Effect of temperature on sulfonamide antibiotics degradation,and on antibiotic resistance determinants and hosts in animal manures[J].Science of the Total Environment,2017,607-608:725-732.
[6]Cheng W X,Li J N,Wu Y,et al.Behavior of antibiotics and antibiotic resistance genes in eco-agricultural system:a case study[J].Journal of Hazardous Materials,2016,304:18-25.
[7]Liu M M,Zhang Y,Yang M,et al.Abundance and distribution of tetracycline resistance genes and mobile elements in an oxytetracycline production wastewater treatment system[J].Environmental Science&Technology,2012,46(14):7551-7557.
[8]Marti E,Jofre J,Balcazar J L.Prevalence of antibiotic resistance genes and bacterial community composition in a river influenced by a wastewater treatment plant[J].PLo S One,2013,8(10):e78906.
[9]何芳,罗阳,浣成,等.高温堆肥技术在我国畜禽粪便污染治理中的应用[J].安徽农业科学,2018,46(17):41-43.He F,Luo Y,Huan C,et al.Application of high-temperature composting technology in the pollution treatment of livestock and poultry manure in China[J].Journal of Anhui Agricultural Sciences,2018,46(17):41-43.
[10]Ho Y B,Zakaria M P,Latif P A,et al.Degradation of veterinary antibiotics and hormone during broiler manure composting[J].Bioresource Technology,2013,131:476-484.
[11]Wang J,Ben W W,Zhang Y,et al.Effects of thermophilic composting on oxytetracycline,sulfamethazine,and their corresponding resistance genes in swine manure[J].Environmental Science:Processes&Impacts,2015,17(9):1654-1660.
[12]Wang K,Chu C,Li X K,et al.Succession of bacterial community function in cow manure composing[J].Bioresource Technology,2018,267:63-70.
[13]Wang K,Mao H L,Li X K.Functional characteristics and influence factors of microbial community in sewage sludge composting with inorganic bulking agent[J].Bioresource Technology,2018,249:527-535.
[14]王伟东,王小芬,朴哲,等.堆肥化过程中微生物群落的动态[J].环境科学,2007,28(11):2591-2597.Wang W D,Wang X F,Piao Z,et al.Microbial dynamics during the composting process[J].Environmental Science,2007,28(11):2591-2597.
[15]勾长龙,王雨琼,张喜庆,等.高温堆肥对猪粪中四环素类抗生素及抗性基因的影响[J].环境科学学报,2017,37(4):1454-1460.Gou C L,Wang Y Q,Zhang X Q,et al.Effects of hightemperature composting on tetracyclines and tetracycline resistance genes in swine manure[J].Acta Scientiae Circumstantiae,2017,37(4):1454-1460.
[16]Li H C,Duan M L,Gu J,et al.Effects of bamboo charcoal on antibiotic resistance genes during chicken manure composting[J].Ecotoxicology and Environmental Safety,2017,140:1-6.
[17]Chen J,Yu Z T,Michel F C Jr,et al.Development and application of real-time PCR assays for quantification of erm genes conferring resistance to macrolides-lincosamides-streptogramin b in livestock manure and manure management systems[J].Applied and Environmental Microbiology,2007,73(14):4407-4416.
[18]钱勋.好氧堆肥对畜禽粪便中抗生素抗性基因的削减条件探索及影响机理研究[D].杨凌:西北农林科技大学,2016.1-140.Qian X.Mechanism and conditions for reducing antibiotic resistance genes during aerobic composting of livestock manure[D].Yangling:Northwest A&F University,2016.1-140.
[19]康军.杨凌城市污泥高效好氧堆肥研究[D].杨凌:西北农林科技大学,2012.Kang J.High-efficiency aerobic composting of municipal sewage sludge in Yangling[D].Yangling:Northwest A&F University,2012.
[20]郑宁国.猪粪堆肥对抗生素抗性基因的影响及微生物群落变化的初步探究[D].西安:西北大学,2016.Zheng N G.Effects of swine manure composting on ARGs and the initial research of changes of microorganism community[D].Xi’an:Northwest University,2016.
[21]Gao M C,Liang F Y,Yu A,et al.Evaluation of stability and maturity during forced-aeration composting of chicken manure and sawdust at different C/N ratios[J].Chemosphere,2010,78(5):614-619.
[22]Liu C G,Xue C,Lin Y H,et al.Redox potential control and applications in microaerobic and anaerobic fermentations[J].Biotechnology Advances,2013,31(2):257-265.
[23]Su J Q,Wei B,Ou-Yang W Y,et al.Antibiotic resistome and its association with bacterial communities during sewage sludge composting[J].Environmental Science&Technology,2015,49(12):7356-7363.
[24]Guo A Y,Gu J,Wang X J,et al.Effects of superabsorbent polymers on the abundances of antibiotic resistance genes,mobile genetic elements,and the bacterial community during swine manure composting[J].Bioresource Technology,2017,244:658-663.
[25]Wang R,Zhang J Y,Sui Q W,et al.Effect of red mud addition on tetracycline and copper resistance genes and microbial community during the full scale swine manure composting[J].Bioresource Technology,2016,216:1049-1057.
[26]Yu Z T,Michel F C Jr,Hansen G,et al.Development and application of real-time PCR assays for quantification of genes encoding tetracycline resistance[J].Applied and Environmental Microbiology,2005,71(11):6926-6933.
[27]Zhang J Y,Sui Q W,Tong J,et al.Soil types influence the fate of antibiotic-resistant bacteria and antibiotic resistance genes following the land application of sludge composts[J].Environment International,2018,118:34-43.
[28]郑宁国,黄南,王卫卫,等.高温堆肥过程对猪粪来源抗生素抗性基因的影响[J].环境科学,2016,37(5):1986-1992.Zheng N G,Huang N,Wang W W,et al.Effects of thermophilic composting on antibiotic resistance genes(ARGs)of swine manure source[J].Environmental Science,2016,37(5):1986-1992.
[29]韦蓓,黄福义,李虎,等.污泥堆肥过程中磺胺类和大环内酯类抗性基因的残留[J].应用与环境生物学报,2014,20(3):395-400.Wei P,Huang F Y,Li H,et al.Persistence of sulfonamide and macrolide resistance genes during sewage sludge composting[J].Chinese Journal of Applied and Environmental Biology,2014,20(3):395-400.
[30]Zhang J Y,Chen M X,Sui Q W,et al.Impacts of addition of natural zeolite or a nitrification inhibitor on antibiotic resistance genes during sludge composting[J].Water Research,2016,91:339-349.
[31]Liao H P,Lu X M,Rensing C,et al.Hyperthermophilic composting accelerates the removal of antibiotic resistance genes and mobile genetic elements in sewage sludge[J].Environmental Science&Technology,2018,52(1):266-276.
[32]Wu D,Huang Z T,Yang K,et al.Relationships between antibiotics and antibiotic resistance gene levels in municipal solid waste leachates in shanghai,China[J].Environmental Science&Technology,2015,49(7):4122-4128.
[33]Qian X,Sun W,Gu J,et al.Variable effects of oxytetracycline on antibiotic resistance gene abundance and the bacterial community during aerobic composting of cow manure[J].Journal of Hazardous Materials,2016,315:61-69.
[34]Peng S,Li H J,Song D,et al.Influence of zeolite and superphosphate as additives on antibiotic resistance genes and bacterial communities during factory-scale chicken manure composting[J].Bioresource Technology,2018,263:393-401.
[35]Chopra I,Roberts M.Tetracycline antibiotics:mode of action,applications,molecular biology,and epidemiology of bacterial resistance[J].Microbiology and Molecular Biology Reviews,2001,65(2):232-260.
[36]Zhang Y J,Li H C,Gu J,et al.Effects of adding different surfactants on antibiotic resistance genes and intI1 during chicken manure composting[J].Bioresource Technology,2016,219:545-551.
[37]Zhang R R,Gu J,Wang X J,et al.Contributions of the microbial community and environmental variables to antibiotic resistance genes during co-composting with swine manure and cotton stalks[J].Journal of Hazardous Materials,2018,358:82-91.
[38]Dobal-Amador V,Nieto-Cid M,Guerrero-Feijóo E,et al.Vertical stratification of bacterial communities driven by multiple environmental factors in the waters(0-5000 m)off the Galician coast(NW Iberian margin)[J].Deep Sea Research Part I:Oceanographic Research Papers,2016,114:1-11.