养猪发酵床不同发酵程度垫料微生物群落结构特征的PLFA分析
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摘要
发酵床养猪是一种新型的养殖技术,可有效缓解养猪的环境污染问题,微生物在其中起关键作用。为明确养猪发酵床发酵过程微生物群落的变化规律,为发酵床的科学管理提供依据,本研究采用磷脂脂肪酸生物标记(phospholipid fatty acids,PLFA)法分析养猪发酵床不同发酵等级垫料的微生物群落结构特征。采用色差法将垫料分为3个发酵程度等级:1级、2级和3级,采集不同发酵等级表层(0～15 cm)和里层(30～45 cm)垫料样本,测定各样本的PLFA。结果表明,共检测到61种PLFA,发酵2级垫料的PLFA种类最多,发酵3级垫料的PLFA种类最少。在各垫料中,PLFA分布量均表现为细菌>真菌>放线菌。指示细菌、真菌、放线菌、革兰氏阳性细菌(G~+)、革兰氏阴性细菌(G~-)的PLFA及总PLFA在各发酵等级表层垫料的分布量均显著大于其在里层垫料的分布量,最大值出现在发酵1级表层垫料中。与对照(未发酵垫料)相比,发酵垫料总PLFA含量均显著增加(P<0.05)。发酵3级表层垫料的真菌/细菌值最大,发酵2级表层垫料的G~+/G~-值最大。多样性分析表明,Shannon指数和Pielou指数最大值出现在发酵2级垫料中,而Simpson指数最大值出现在发酵3级表层垫料中。聚类分析表明,当欧氏距离为233.15时,可将不同发酵等级垫料聚为3个类群,同一发酵级别的垫料聚在相同类群中;主成分分析表明,发酵1级表层和里层垫料单独归一类群,其他发酵等级垫料和对照垫料归另一类群中。综上,不同发酵等级垫料的微生物种群结构不同,发酵1级表层垫料微生物分布量最大,发酵2级垫料的微生物种类最多,相同发酵级别表层和里层垫料微生物群落结构相似。
Pig-on-litter system is a new pig-raising technology that can reduce environmental pollution.In this system,micro-organisms are generally considered as the key factor.In order to determine the change in microbial community during fermentation and to set up basic data for scientific management of pig-on-litter system,microbial community of litter with different fermentation levels was analyzed using phospholipid fatty acid(PLFA)biomarkers.Fermentation levels of litters were divided into three grades(1~(st),2~(nd) and 3~(rd))using the chromatic aberration(?E)method.Both surface(0-15 cm)and inner layer(30-45 cm)litters of each fermentation level were sampled.PLFA composition of each sample was determined by the Sherlock MIS 4.5 system.The results showed that the method used was sufficient to detect total 61 kinds of PLFA biomarkers.The most and least kinds of PLFA biomarkers occurred in litters with the 2~(nd) and 3~(rd) fermentation levels,respectively.PLFA biomarkers displayed the same order of distribution abundance in all samples—bacteria>fungi>actinomycetes.The contents of PLFAs that were referable to bacteria,fungi,actinomycetes,G~+,G~-and total PLFA in the surface layer samples were all higher than those in the inner layer samples;being highest in surface layer litters with the 1~(st) fermentation level.Fermented litter had significantly higher content of total PLFA than unfermented litter(CK)(P<0.05).The highest fungi/bacteria and G~+/G~-ratios were for surface layer litter with the 3~(rd) and 2~(nd) fermentation levels,respectively.Diversity analyses indicated that the maximum values of the Shannon index and Pielou index were for litter of the 2~(nd) fermentation level and the maximum values of the Simpson index were for surface layer litter of the 3~(rd) fermentation level.Based on cluster analysis,the samples were clustered into three groups for Euclidean-distance of 223.15.Samples with the same fermentation level were clustered together.Also based on principal component analysis,surface layer and inner layer samples of the 1~(st) fermentation level were clustered into one lone group,while the other samples were clustered into other several groups.Put together,litters with different fermentation levels had different microbial community structures.The maximum values of microbial content and species were in surface layer litter with the 1~(st) and 2~(nd) fermentation levels,respectively.Moreover,surface layer and inner layer litters of the same fermentation level had similar microbial communities.
Pig-on-litter system is a new pig-raising technology that can reduce environmental pollution.In this system,micro-organisms are generally considered as the key factor.In order to determine the change in microbial community during fermentation and to set up basic data for scientific management of pig-on-litter system,microbial community of litter with different fermentation levels was analyzed using phospholipid fatty acid(PLFA)biomarkers.Fermentation levels of litters were divided into three grades(1~(st),2~(nd) and 3~(rd))using the chromatic aberration(?E)method.Both surface(0-15 cm)and inner layer(30-45 cm)litters of each fermentation level were sampled.PLFA composition of each sample was determined by the Sherlock MIS 4.5 system.The results showed that the method used was sufficient to detect total 61 kinds of PLFA biomarkers.The most and least kinds of PLFA biomarkers occurred in litters with the 2~(nd) and 3~(rd) fermentation levels,respectively.PLFA biomarkers displayed the same order of distribution abundance in all samples—bacteria>fungi>actinomycetes.The contents of PLFAs that were referable to bacteria,fungi,actinomycetes,G~+,G~-and total PLFA in the surface layer samples were all higher than those in the inner layer samples;being highest in surface layer litters with the 1~(st) fermentation level.Fermented litter had significantly higher content of total PLFA than unfermented litter(CK)(P<0.05).The highest fungi/bacteria and G~+/G~-ratios were for surface layer litter with the 3~(rd) and 2~(nd) fermentation levels,respectively.Diversity analyses indicated that the maximum values of the Shannon index and Pielou index were for litter of the 2~(nd) fermentation level and the maximum values of the Simpson index were for surface layer litter of the 3~(rd) fermentation level.Based on cluster analysis,the samples were clustered into three groups for Euclidean-distance of 223.15.Samples with the same fermentation level were clustered together.Also based on principal component analysis,surface layer and inner layer samples of the 1~(st) fermentation level were clustered into one lone group,while the other samples were clustered into other several groups.Put together,litters with different fermentation levels had different microbial community structures.The maximum values of microbial content and species were in surface layer litter with the 1~(st) and 2~(nd) fermentation levels,respectively.Moreover,surface layer and inner layer litters of the same fermentation level had similar microbial communities.
引文
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[3]CHEN Q Q,LIU B,WANG J P,et al.Diversity and dynamics of the bacterial community involved in pig manure biodegradation in a microbial fermentation bed system[J].Annals of Microbiology,2017,67(7):491-500
[4]GROENESTEIN C M,OOSTHOEK J,VAN FAASSEN H G.Microbial processes in deep-litter systems for fattening pigs and emission of ammonia,nitrous oxide and nitric oxide[C]//VERSTEGEN M W A,DEN HARTOG L A,VANKEMPEN G J M,et al.Nitrogen Flow in Pig Production and Environmental Consequences.Proceedings of the First International Symposium.Wageningen:Pudoc Scientific Publishers,1993:168-176
[5]TAM N F Y,VRIJMOED L L P.Effects of the inoculum size of a commercial bacterial product and the age of sawdust bedding on pig waste decomposition in a pig-on-litter system[J].Waste Management&Research,1993,11(2):107-115
[6]陈倩倩,刘波,王阶平,等.微生物发酵床猪舍不同发酵等级垫料中大肠杆菌的分离鉴定[J].中国畜牧兽医,2017,44(1):268-274CHEN Q Q,LIU B,WANG J P,et al.Isolation and identification of E.coli in the microbial-fermentation bed of piggery[J].China Animal Husbandry&Veterinary Medicine,2017,44(1):268-274
[7]刘波,王阶平,陈倩倩,等.养猪发酵床微生物宏基因组基本分析方法[J].福建农业学报,2016,31(6):630-648LIU B,WANG J P,CHEN Q Q,et al.Metagenomic analysis of microbial community in a microbial fermentation-bed for pig raising[J].Fujian Journal of Agricultural Sciences,2016,31(6):630-648
[8]熊宽荣,张益焘,曹珍,等.翻耙频率和发酵菌剂对猪舍外发酵床垫料微生物群落的影响[J].家畜生态学报,2016,37(6):43-50XIONG K R,ZHANG Y T,CAO Z,et al.Effects of microorganism inoculants and turning frequency on the litter microbial community of noncontact deep-litter-system[J].Acta Ecologae Animalis Domastici,2016,37(6):43-50
[9]张学峰,周贤文,陈群,等.不同深度垫料对养猪土著微生物发酵床稳定期微生物菌群的影响[J].中国兽医学报,2013,33(9):1458-1462ZHANG X F,ZHOU X W,GHEN Q,et al.The influence of padding of different depth in native microorganism fermentation bed of pigs on microbial flora during steady period[J].Chinese Journal of Veterinary Science,2013,33(9):1458-1462
[10]郑雪芳,刘波,朱育菁,等.磷脂脂肪酸生物标记法分析养猪发酵床微生物群落结构的空间分布[J].农业环境科学学报,2018,37(4):804-812ZHENG X F,LIU B,ZHU Y J,et al.Spatial distribution of microbial communities in a fermentation bed based on phospholipid fatty acid biomarkers[J].Journal of Agro-Environment Science,2018,37(4):804-812
[11]郑雪芳,刘波,林营志,等.利用磷脂脂肪酸生物标记分析猪舍基质垫层微生物亚群落的分化[J].环境科学学报,2009,29(11):2306-2317ZHENG X F,LIU B,LIN Y Z,et al.Using phospholipid fatty acid biomarkers for analysis of the microbial subcommunity in pigsty litter[J].Acta Scientiae Circumstantiae,2009,29(11):2306-2317
[12]文倩,林启美,赵小蓉,等.北方农牧交错带林地、耕地和草地土壤微生物群落结构特征的PLFA分析[J].土壤学报,2008,45(2):321-327WEN Q,LIN Q M,ZHAO X R,et al.Application of PLFAanalysis in determination of soil microbial community structure in woodland,cropland and grassland in farmland-pasture interleaving zone of north China[J].Acta Pedologica Sinica,2008,45(2):321-327
[13]DENG B,SHEN C H,SHEN X H,et al.PCR-DGGE analysis on microbial communities in pit mud of cellars used for different periods of time[J].Journal of the Institute of Brewing,2012,118(1):120-126
[14]XUE D,YAO H Y,GE D Y,et al.Soil microbial community structure in diverse land use systems:A comparative study using biolog,DGGE,and PLFA analyses[J].Pedosphere,2008,18(5):653-663
[15]LAUBER C L,STRICKLAND M S,BRADFORD M A,et al.The influence of soil properties on the structure of bacterial and fungal communities across land-use types[J].Soil Biology and Biochemistry,2008,40(9):2407-2415
[16]SAETRE P,B??TH E.Spatial variation and patterns of soil microbial community structure in a mixed spruce-birch stand[J].Soil Biology and Biochemistry,2000,32(7):909-917
[17]宋泽琼,蓝江林,刘波,等.养猪微生物发酵床垫料发酵指数的研究[J].福建农业学报,2011,26(6):1069-1075SONG Z Q,LAN J L,LIU B,et al.Study on the fermentation index for the pig litter in the piggery of microbial fermentation bed[J].Fujian Journal of Agricultural Sciences,2011,26(6):1069-1075
[18]FROSTEG?RD A,TUNLID A,B??TH E.Phospholipid fatty acid composition,biomass,and activity of microbial communities from two soil types experimentally exposed to different heavy metals[J].Applied and Environmental Microbiology,1993,59(11):3605-3617
[19]KOURTEV P S,EHRENFELD J G,H?GGBLOM M.Exotic plant species alter the microbial community structure and function in the soil[J].Ecology,2002,83(11):3152-3166
[20]WILKINSON S G.Gram-negative bacteria[M]//RATLEDGEC,WILKINSON S G.Microbial Lipids.London:Academic Press,1988:299-488
[21]O’LEARY W M,WILKINSON S G.Gram-positive bacteria[M]//RATLEDGE C,WILKINSON S G.Microbial Lipids.London:Academic Press,1988:117-202
[22]ZELLES L.Community structure of soil microorganisms[M]//SCHINNER F,?HLINGER R,KANDELER E,et al.Methods in Soil Biology.London:Springer Press,1996:77-92
[23]GARLAND J L,MILLS A L.Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level sole-carbo-source utilization[J].Applied and Environmental Microbiology,1991,57(8):2351-2359
[24]唐启义,冯明光.实用统计分析及其DPS数据处理系统[M].北京:科学出版社,2002TANG Q Y,FENG M G.DPS Data Processing System for Practical Statistics[M].Beijing:Science Press,2002
[25]蓝江林,刘波,宋泽琼,等.微生物发酵床养猪技术研究进展[J].生物技术进展,2012,2(6):411-416LAN J L,LIU B,SONG Z Q,et al.Advances in research of microbial fermentation bed in pig farming[J].Current Biotechnology,2012,2(6):411-416
[26]BARDGETT R D,HOBBS P J,FROSTEG?RD?.Changes in soil fungal:Bacterial biomass ratios following reductions in the intensity of management of an upland grassland[J].Biology and Fertility of Soils,1996,22(3):261-264
[27]KLAMER M,B??TH E.Microbial community dynamics during composting of straw material studied using phospholipid fatty acid analysis[J].FEMS Microbiology Ecology,1998,27(1):9-20
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