土霉素、铜对牛粪厌氧消化过程的影响机理
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摘要
随着畜禽粪便污染加剧,厌氧消化技术是使畜禽粪便有效减量化和资源化的技术之一,但是畜禽粪便中含有高浓度的抗生素和重金属会影响厌氧消化工况的稳定运行及沼气产量。四环素类抗生素作为饲料添加剂最为普遍,其用量已经占饲料添加抗生素总量的57%,四环素类抗生素中以金霉素、土霉素、四环素应用最广,铜、锌、铬、铁等重金属被广泛添加至饲料和药物中,在饲料中添加10倍于畜禽正常需求量的铜,可明显提高畜禽生产性能。为了探究抗生素和重金属对牛粪厌氧消化过程的影响,选择将土霉素和铜添加到厌氧消化过程中,使其浓度呈梯度。土霉素添加至液体物料中,2L容积内使其为三个浓度梯度:20mg/l、50mg/l、80mg/l;重金属铜添加至牛粪固体中,使牛粪固体内含铜浓度为100μg/g、200μg/g、300μg/g、400μg/g、500μg/g、600μg/g,再填装入厌氧消化罐内,含固率约为8%;土霉素和铜复合添加实验将土霉素浓度为20μg/g、50μg/g、100μg/g与铜浓度100μg/g、200μg/g、300μg/g两两组合,搭配成9种方式进行厌氧消化。联合分子生物学手段探究厌氧发酵微生物的多样性和动态变化,研究土霉素和铜单独添加及联合添加对牛粪厌氧消化过程的影响。
(1)土霉素单独添加对牛粪厌氧消化工艺运行及产气会产生抑制作用,厌氧发酵液内土霉素浓度为20mg/l已可以造成抑制作用,含有20mg/l、50mg/l、80mg/l浓度土霉素的牛粪厌氧消化产甲烷抑制率分别为70.92%、82.78%、91.44%;
(2)铜单独添加牛粪在厌氧消化过程中在100μg/g~300μg/g浓度范围内对厌氧消化有促进作用,在400μg/g~600μg/g浓度范围内则会产生抑制作用;
(3)土霉素和铜复合添加对牛粪厌氧消化过程造成的影响并不显著,并未发生抑制作用,当牛粪中Cu~(2+)浓度为200μg/g和300μg/g时,对牛粪厌氧消化有促进作用,
(4)通过分子生物学手段探究了土霉素、重金属铜单独污染和两者复合污染牛粪在厌氧消化过程中微生物多样性及动态变化,结果表明,土霉素可抑制微生物生长,在厌氧发酵运行末期微生物指数明显降低;重金属铜在Cu~(2+)浓度为400~600μg/g明显对微生物群落优势菌群造成影响;而土霉素和铜联合毒性作用于牛粪时并未对微生物群落多样性造成太大影响。
With the pollution of livestock and poultry droppings getting worse, anaerobic digestion technology is effective on the reduction and recycling of the technology, but livestock and poultry droppings containing high concentrations of antibiotics and heavy metal will affect anaerobic digestion conditions and the stable operation of the biogas yield. In order to explore the influence of antibiotics and heavy metal to cow dung anaerobic digestion process, we added oxytetracycline and heavy metal copper respectively to the fermentation liquor with different concentrations, Using molecular techniques to find the law of the inhibitory effect of anaerobic fermentation microorganisms and dynamic change, research the rules of individual inhibition and joint inhibition of oxytetracycline and copper on anaerobic digestion treatment of cow dung.
(1)Study on the individual inhibition of oxytetracycline added to the cow dung anaerobic fermentation process operation, the concentrations of oxytetracycline is 20mg/l anaerobic fermented liquid that can cause inhibition action;
(2)Study on the individual inhibitory effect of heavy metal copper. when the cooper is in certain concentration range anaerobic fermentation have promote functions, while more than a specified concentration will produce the inhibition.
(3) Study on the joint inhibitory effect of oxytetracycline and copper. when the concentration of the Cu~(2+) in the cow dung is in the range of 200~300μg/g, the compound toxicity of oxytetracycline and heavy metal copper was antagonistic.
(4) By means of molecular biology study on the diversity of anaerobic fermentation microorganisms and dynamic change, The experimental results showed the oxytetracycline can cause inhibitory on the anaerobic fermentation microorganisms, in the end of anaerobic fermentation the diversity index reduced significantly; when concentration of the Cu~(2+) in the cow dung is in the range of 400~600μg/g, the dominant microflora was influenced observably; however, the joint toxicity of oxytetracycline and cooper have not cause effects to the microbial community diversity.
(1)土霉素单独添加对牛粪厌氧消化工艺运行及产气会产生抑制作用,厌氧发酵液内土霉素浓度为20mg/l已可以造成抑制作用,含有20mg/l、50mg/l、80mg/l浓度土霉素的牛粪厌氧消化产甲烷抑制率分别为70.92%、82.78%、91.44%;
(2)铜单独添加牛粪在厌氧消化过程中在100μg/g~300μg/g浓度范围内对厌氧消化有促进作用,在400μg/g~600μg/g浓度范围内则会产生抑制作用;
(3)土霉素和铜复合添加对牛粪厌氧消化过程造成的影响并不显著,并未发生抑制作用,当牛粪中Cu~(2+)浓度为200μg/g和300μg/g时,对牛粪厌氧消化有促进作用,
(4)通过分子生物学手段探究了土霉素、重金属铜单独污染和两者复合污染牛粪在厌氧消化过程中微生物多样性及动态变化,结果表明,土霉素可抑制微生物生长,在厌氧发酵运行末期微生物指数明显降低;重金属铜在Cu~(2+)浓度为400~600μg/g明显对微生物群落优势菌群造成影响;而土霉素和铜联合毒性作用于牛粪时并未对微生物群落多样性造成太大影响。
With the pollution of livestock and poultry droppings getting worse, anaerobic digestion technology is effective on the reduction and recycling of the technology, but livestock and poultry droppings containing high concentrations of antibiotics and heavy metal will affect anaerobic digestion conditions and the stable operation of the biogas yield. In order to explore the influence of antibiotics and heavy metal to cow dung anaerobic digestion process, we added oxytetracycline and heavy metal copper respectively to the fermentation liquor with different concentrations, Using molecular techniques to find the law of the inhibitory effect of anaerobic fermentation microorganisms and dynamic change, research the rules of individual inhibition and joint inhibition of oxytetracycline and copper on anaerobic digestion treatment of cow dung.
(1)Study on the individual inhibition of oxytetracycline added to the cow dung anaerobic fermentation process operation, the concentrations of oxytetracycline is 20mg/l anaerobic fermented liquid that can cause inhibition action;
(2)Study on the individual inhibitory effect of heavy metal copper. when the cooper is in certain concentration range anaerobic fermentation have promote functions, while more than a specified concentration will produce the inhibition.
(3) Study on the joint inhibitory effect of oxytetracycline and copper. when the concentration of the Cu~(2+) in the cow dung is in the range of 200~300μg/g, the compound toxicity of oxytetracycline and heavy metal copper was antagonistic.
(4) By means of molecular biology study on the diversity of anaerobic fermentation microorganisms and dynamic change, The experimental results showed the oxytetracycline can cause inhibitory on the anaerobic fermentation microorganisms, in the end of anaerobic fermentation the diversity index reduced significantly; when concentration of the Cu~(2+) in the cow dung is in the range of 400~600μg/g, the dominant microflora was influenced observably; however, the joint toxicity of oxytetracycline and cooper have not cause effects to the microbial community diversity.
引文
[1] Ye Chen, Jay J.Cheng, Kurt S.Creamer. Inhibition of anaerobic digestion process: A review[J]. Bioresource Technology, 2008, 99: 4045-4046.
[2]孙建平.抗生素与重金属对猪场废水厌氧消化的抑制效应及其调控对策[D]. 2009:3-4.
[3]苗蕾,寇渊博,李有,李爱伟,沈连峰.河南省畜禽养殖污染现状分析及对策[J].河南科学,2010, 28(1): 94-95.
[4]周传社,谭支良.集约化畜禽养殖环境风险与技术对策[J].今日畜牧兽医,2009(12).
[5] Poels J, Van Assche P, Verstraete W. Effect of antibiotics and chemotherapeutics on biogas production from piggery waste. Bioenergy, 1984, 84: 422-426.
[6] Masse D I, Lu D, Masse L, et al. Effect of antibiotics on psychrophilic anaerobic digestion of swine manure slurry in sequencing batch reactors. Bioresource Technology, 2000, 75: 205-211.
[7] Van Nevel C J and Demeyer D I. Effect of Monensin on rumen metabolism In vitro.Appl.Environ. Microbiol, 1997, 34: 251-257
[8]郝俊虎,王俊东,张建海.铬在畜禽中的应用研究进展.兽药与饲料添加剂,2003,8(3): 17-19
[9] LIN C Y. Effect of heavy metals on acidogenesis in anaerobic digestion[J]. Water Res.,1993, 27(1): 147-152.
[10] LA WRENCE A W, MCCARTY P L. The role of sulfide in preventing heavy metal toxicity on anaerobic treatment[J]. J.Water Pollut.Control Fed., 1965, 37(3): 392-405.
[11] SHEN C F, KOSARIC N,BLASZCZYK R. The effect of selected heavy metals (Ni,Co,and Fe) on anaerobic granules and their extracellular polymeric substances (EPS) [J]. Water Res., 1993, 27 (1): 25-33.
[12] CALLANDER I J, BARFORD J P. Precipitation, chelation , and the availability of metals as nut rients in anaerobic digestion.I. Applications [J]. Biotechnol.Bioeng., 1983, 25(8): 1947-1957.
[13] Strritt R M, Lester J N. Interaction of heavy metals with bacteria.Sci. Total Environ, 1980, 14(1): 5-17.
[14] Hickey R F, Vanderwielen J,Switzenbaum Ms. The effect of heavy metals on methane production and hydrogen and carbon monoxide level during batch anaerobic sludge digestion.Water Res, 1989, 23, 207-218.
[15] Zayed G and Winter J. Inhibition of methane production from whey by heavy metals-protective effect of sulfide. Appl Microbiol Biotechnol, 2000, 53: 726-731.
[16] Ye Chen, Jay J, Cheng, Kurt S. Creamer. Inhibition of anaerobic digestion process: A review [J]. Bioresource Technology ,2008:4044-4064.
[17]岑承志,陈砺,严宗诚,王红林,邓涛.沼气发酵技术发展及应用现状[J]. 2009, 6(36): 78-79.
[18] Amann RI, Ludwig W Schleifer KH. Phylogenetic identification and in situ detection of individual microbial cells without cultivation[J]. Microbiol.Rev, 1995, 59(1): 143-169.
[19] Fischer SG, Lerman LS. Length-independent separation of DNA reatriction fragments in two-dimensional gel electrophoresis[J]. Cell, 1979(16): 191-200.
[20] Muyzer G, De Waal EC, Uitterlinden AG. Appl Environ Microbio, 1993, 59: 695-700.
[21] Murrell J C, McDonald I R, Bourne D G. Molecular metheds for the study of methanothoph ecology. FEMS Microbiology Ecology, 1998, 27: 103-114.
[22] Dearman B P,Marschner R H,Bentham.Methane production and microbial community structure in single-stage batch and sequential batch systems anaerobically co-digesting food waste and biosolids. Applied Microbiology and Biotechnology, 2006, 69: 589-596.
[23] Keyser M,Witthuhn R C, Lamprecht C, et al. PCR-based DGGE fingerprinting and identification of methanogens detected in three different types of UASB granules. Systematic and Applied Microbiology, 2006, 29(1): 77-84.
[24] Malin C, Illmer P, Ability of DNA content and DGGE analysis to reflect the performance condition of an anaerobic biowaste fermenter. Microbiological Research, 2008, 16(3): 503-511.
[25] Liu W, Chan O, Fang H H P. Microbial community dynamics during start-up of acidogenic anaerobic reactors. Water Research, 2002, 36: 3203-3210.
[26]孙寓娇.厌氧产甲烷颗粒污泥微生态学的研究现状[J].中国沼气, 2007, 25(4): 5-7.
[27] Hallin Sorensen B, Nielsen SN, Lansky PF, et al. Oc2currence, fate, and effects of pharmaceuticals in the en2vironment - A review. Chem osphere, 1998, 36: 357 -365.
[28] Bruce J R, Paul K S L, Micheal M. Emerging chemicals of concern:Pharmaceuticals and personal care products(PPCPs) in Asia,with particular reference to Southern China[J]. Marine Pollution Bulletin, 2005, 50: 913-920.
[29]唐礼庆,何成达,王惠民.两种四环素类抗生素厌氧生物毒性试验研究[J].四川环境.2007,6(26):11-13.
[30]李荣平,李秀金.牛粪和厨余废物不同混合比例下厌氧消化的试验研究[J].中国沼气2007, 25(5): 19-20.
[31] AMERICAN PUBLIC HEALTH ASSOC. Standard methods for the examination of water and waste water[M]. Washington DC: Science publisher, 1998.
[32]赵明星,严群,阮文权,邹华,徐岩. pH调控对厨余物厌氧发酵产沼气的影响[J].生物加工过程,2008, 6(4): 45-49.
[33]付胜涛,严晓菊,付英,于水利.污水厂污泥和厨余垃圾的混合中温厌氧消化[J].哈尔滨商业大学学报,2007, 23(1): 32-39.
[34]邓敏,方江敏,周兴求,伍健东.低浓度Fe3+和Cu~(2+)对厌氧颗粒污泥性能的影响[J].中国沼气,2008,26(1):12.
[35] Water J, Wjcik. Anaerobic dry fermentation[J]. Biotechnology Bioengineering Sump, 1980, 10: 43-65.
[36]张波,徐剑波,蔡伟民.有机废物厌氧消化过程中氨氮的抑制性影响[J].中国沼气,2003, 21(3): 26-28.
[37]金仁村,黄冠男,沈李东.厌氧消化工艺的金属抑制现象[J].环境污染与防治,2010, 32 (1): 79-81.
[38] Chang B V, Liao C S, Yuan S Y. Anaerobic degradation of diethy l phtha late, di-n-buty l phtha late, and di-(2-eth-ylhexyl) phthalate from river sediment in Taiwan[J]. Chemosphere, 2005, 58(11): 1601- 1607.
[39] Fischer S L Lerman.DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory[J]. Proc Natl Acad Sci USA, 1983, 80(6): 1579-1583.
[40]师晓爽,刘德立. PCR-DGGE技术在农村户用沼气发酵微生物研究中的初步应用[J].山东师范大学学报:自然科学版,2007, 22(2): 120-122.
[41]刘新春,吴成强,张昱,杨敏,李红岩. PCR-DGGE法用于活性污泥系统中微生物群落结构变化的解析[J].生态学报,2005, 4(25): 842-843.
[42]蒋建林.沼气发酵的物料理化特性及微生物群落多样性分析[D].广西:广西大学,2009: 36-38.
[43]徐成斌,孟雪莲,马溪平,张利红,李法云,付保荣,惠秀娟. 16SrDNA克隆文库方法对制药废水处理系统中为生物多样性的研究[J].生态环境学报,2009, 18(4): 1236-1240.
[2]孙建平.抗生素与重金属对猪场废水厌氧消化的抑制效应及其调控对策[D]. 2009:3-4.
[3]苗蕾,寇渊博,李有,李爱伟,沈连峰.河南省畜禽养殖污染现状分析及对策[J].河南科学,2010, 28(1): 94-95.
[4]周传社,谭支良.集约化畜禽养殖环境风险与技术对策[J].今日畜牧兽医,2009(12).
[5] Poels J, Van Assche P, Verstraete W. Effect of antibiotics and chemotherapeutics on biogas production from piggery waste. Bioenergy, 1984, 84: 422-426.
[6] Masse D I, Lu D, Masse L, et al. Effect of antibiotics on psychrophilic anaerobic digestion of swine manure slurry in sequencing batch reactors. Bioresource Technology, 2000, 75: 205-211.
[7] Van Nevel C J and Demeyer D I. Effect of Monensin on rumen metabolism In vitro.Appl.Environ. Microbiol, 1997, 34: 251-257
[8]郝俊虎,王俊东,张建海.铬在畜禽中的应用研究进展.兽药与饲料添加剂,2003,8(3): 17-19
[9] LIN C Y. Effect of heavy metals on acidogenesis in anaerobic digestion[J]. Water Res.,1993, 27(1): 147-152.
[10] LA WRENCE A W, MCCARTY P L. The role of sulfide in preventing heavy metal toxicity on anaerobic treatment[J]. J.Water Pollut.Control Fed., 1965, 37(3): 392-405.
[11] SHEN C F, KOSARIC N,BLASZCZYK R. The effect of selected heavy metals (Ni,Co,and Fe) on anaerobic granules and their extracellular polymeric substances (EPS) [J]. Water Res., 1993, 27 (1): 25-33.
[12] CALLANDER I J, BARFORD J P. Precipitation, chelation , and the availability of metals as nut rients in anaerobic digestion.I. Applications [J]. Biotechnol.Bioeng., 1983, 25(8): 1947-1957.
[13] Strritt R M, Lester J N. Interaction of heavy metals with bacteria.Sci. Total Environ, 1980, 14(1): 5-17.
[14] Hickey R F, Vanderwielen J,Switzenbaum Ms. The effect of heavy metals on methane production and hydrogen and carbon monoxide level during batch anaerobic sludge digestion.Water Res, 1989, 23, 207-218.
[15] Zayed G and Winter J. Inhibition of methane production from whey by heavy metals-protective effect of sulfide. Appl Microbiol Biotechnol, 2000, 53: 726-731.
[16] Ye Chen, Jay J, Cheng, Kurt S. Creamer. Inhibition of anaerobic digestion process: A review [J]. Bioresource Technology ,2008:4044-4064.
[17]岑承志,陈砺,严宗诚,王红林,邓涛.沼气发酵技术发展及应用现状[J]. 2009, 6(36): 78-79.
[18] Amann RI, Ludwig W Schleifer KH. Phylogenetic identification and in situ detection of individual microbial cells without cultivation[J]. Microbiol.Rev, 1995, 59(1): 143-169.
[19] Fischer SG, Lerman LS. Length-independent separation of DNA reatriction fragments in two-dimensional gel electrophoresis[J]. Cell, 1979(16): 191-200.
[20] Muyzer G, De Waal EC, Uitterlinden AG. Appl Environ Microbio, 1993, 59: 695-700.
[21] Murrell J C, McDonald I R, Bourne D G. Molecular metheds for the study of methanothoph ecology. FEMS Microbiology Ecology, 1998, 27: 103-114.
[22] Dearman B P,Marschner R H,Bentham.Methane production and microbial community structure in single-stage batch and sequential batch systems anaerobically co-digesting food waste and biosolids. Applied Microbiology and Biotechnology, 2006, 69: 589-596.
[23] Keyser M,Witthuhn R C, Lamprecht C, et al. PCR-based DGGE fingerprinting and identification of methanogens detected in three different types of UASB granules. Systematic and Applied Microbiology, 2006, 29(1): 77-84.
[24] Malin C, Illmer P, Ability of DNA content and DGGE analysis to reflect the performance condition of an anaerobic biowaste fermenter. Microbiological Research, 2008, 16(3): 503-511.
[25] Liu W, Chan O, Fang H H P. Microbial community dynamics during start-up of acidogenic anaerobic reactors. Water Research, 2002, 36: 3203-3210.
[26]孙寓娇.厌氧产甲烷颗粒污泥微生态学的研究现状[J].中国沼气, 2007, 25(4): 5-7.
[27] Hallin Sorensen B, Nielsen SN, Lansky PF, et al. Oc2currence, fate, and effects of pharmaceuticals in the en2vironment - A review. Chem osphere, 1998, 36: 357 -365.
[28] Bruce J R, Paul K S L, Micheal M. Emerging chemicals of concern:Pharmaceuticals and personal care products(PPCPs) in Asia,with particular reference to Southern China[J]. Marine Pollution Bulletin, 2005, 50: 913-920.
[29]唐礼庆,何成达,王惠民.两种四环素类抗生素厌氧生物毒性试验研究[J].四川环境.2007,6(26):11-13.
[30]李荣平,李秀金.牛粪和厨余废物不同混合比例下厌氧消化的试验研究[J].中国沼气2007, 25(5): 19-20.
[31] AMERICAN PUBLIC HEALTH ASSOC. Standard methods for the examination of water and waste water[M]. Washington DC: Science publisher, 1998.
[32]赵明星,严群,阮文权,邹华,徐岩. pH调控对厨余物厌氧发酵产沼气的影响[J].生物加工过程,2008, 6(4): 45-49.
[33]付胜涛,严晓菊,付英,于水利.污水厂污泥和厨余垃圾的混合中温厌氧消化[J].哈尔滨商业大学学报,2007, 23(1): 32-39.
[34]邓敏,方江敏,周兴求,伍健东.低浓度Fe3+和Cu~(2+)对厌氧颗粒污泥性能的影响[J].中国沼气,2008,26(1):12.
[35] Water J, Wjcik. Anaerobic dry fermentation[J]. Biotechnology Bioengineering Sump, 1980, 10: 43-65.
[36]张波,徐剑波,蔡伟民.有机废物厌氧消化过程中氨氮的抑制性影响[J].中国沼气,2003, 21(3): 26-28.
[37]金仁村,黄冠男,沈李东.厌氧消化工艺的金属抑制现象[J].环境污染与防治,2010, 32 (1): 79-81.
[38] Chang B V, Liao C S, Yuan S Y. Anaerobic degradation of diethy l phtha late, di-n-buty l phtha late, and di-(2-eth-ylhexyl) phthalate from river sediment in Taiwan[J]. Chemosphere, 2005, 58(11): 1601- 1607.
[39] Fischer S L Lerman.DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory[J]. Proc Natl Acad Sci USA, 1983, 80(6): 1579-1583.
[40]师晓爽,刘德立. PCR-DGGE技术在农村户用沼气发酵微生物研究中的初步应用[J].山东师范大学学报:自然科学版,2007, 22(2): 120-122.
[41]刘新春,吴成强,张昱,杨敏,李红岩. PCR-DGGE法用于活性污泥系统中微生物群落结构变化的解析[J].生态学报,2005, 4(25): 842-843.
[42]蒋建林.沼气发酵的物料理化特性及微生物群落多样性分析[D].广西:广西大学,2009: 36-38.
[43]徐成斌,孟雪莲,马溪平,张利红,李法云,付保荣,惠秀娟. 16SrDNA克隆文库方法对制药废水处理系统中为生物多样性的研究[J].生态环境学报,2009, 18(4): 1236-1240.
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