不同菌剂处理对猪粪堆肥效果的影响及PCR-DGGE研究堆肥微生物群落变化
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摘要
为研究接种菌剂对堆肥效果的影响及其在堆肥中的变化规律,以及对堆肥微生物群落变化的影响,并探讨动物微生态制剂用于堆肥的可行性,本研究从猪粪腐熟堆肥中筛选优势微生物并制作成复合菌剂,研究复合菌剂和微生态制剂对堆肥效果的影响,并采用PCR-DGGE技术研究堆肥微生物的群落变化,结果如下:
1.复合菌剂和微生态制剂对堆肥效果的影响
利用选择性平板培养和高温筛选从猪粪腐熟堆肥中分离微生物,从中筛选10株堆肥微生物,3株能够耐受80℃高温的芽孢杆菌,3株中温放线菌,3株霉菌和1株酵母菌,用以上菌株制备复合菌剂。将复合菌剂和微生态制剂分别按0.1%比例接种猪粪堆肥,共分成4组:T1是接种堆肥分离的复合菌剂组,T2是微生态菌剂组,T3是微生态菌剂组(接种菌剂浓度是T2的1倍),设不接种菌剂组为对照组。通过测定堆肥30d内温度、pH、水分、全碳、全氮和C/N,评价接种菌剂对堆肥效果的影响。结果表明:整个堆肥过程中,接种堆肥分离微生物和微生态菌剂组的温度显著高于对照组,最高温度也显著高于对照组,T1组最高温度达到73℃,T2为74℃,T3为72℃,对照组为71.5℃。接种菌剂组堆肥高温期持续时间显著比对照组长,堆肥温度在50℃以上维持的时间T2、T3最长,都是23天,T1为21天,而对照组C只有10天。各组水分变化趋势相似,随着堆肥的进行,堆肥水分逐渐下降。各组pH值变化趋势基本一致,堆肥初期呈逐渐上升趋势,堆肥后期则逐渐下降。堆肥结束时,接种菌剂组对碳素的利用率显著高于对照组,T1的全碳含量下降了11.88%,T2下降了5.75%,T3下降了12.96%,而对照组只下降了4.73%。堆肥结束时,T1组和T3组C/N下降最多。综合各项物理化学指标来看,接种堆肥分离微生物的处理组T1效果最好,接种动物微生态制剂的处理组T2和T3的效果也较好,表明动物微生态制剂用于堆肥是可行的。
2.PCR-DGGE技术研究接种菌剂对堆肥微生物的群落变化的影响
通过抽提不同时期堆肥细菌总DNA,PCR扩增细菌16S rDNA V3区和DGGE电泳,进行DGGE图谱聚类分析和条带多样性的分析,结果表明:堆肥过程中细菌种类数量随着堆肥的升温期、高温维持期和降温期的变化呈现出“升高-降低-升高”的变化趋势。各阶段出现不同的菌群组成,接种菌剂堆肥细菌群落变化较对照组慢。堆肥初始,菌群组成主要是土著微生物,各组相似性为93%-100%;与其他时期的堆肥
DGGE条带组成相似度仅为50%。第0-3天,此阶段温度上升较快,细菌群落多样性开始下降,不耐受高温的菌群逐渐消失并伴随着优势菌群的出现。接种的堆肥分离微生物和动物微生态制剂在DGGE图谱中检测出来,其中芽孢杆菌与高温期的优势条带相对应。第9-21天,各组DGGE图谱的相似性为68%,对照组菌群多样性较接种菌剂组高,但接种菌剂组优势菌群明显,且维持时间较长,表明堆肥高温阶段菌群的变化与堆肥温度的变化有关,对照组温度低,更利于不耐受高温菌群的生长;接种菌剂增加了高温期优势菌群的数量。第21-30天为堆肥降温腐熟期,堆肥细菌组成逐渐趋于稳定,各组DGGE图谱相似性为93%-100%,细菌菌落多样性高于堆肥高温期。此阶段接种菌剂组菌群多样性高于对照组,表明在堆肥后期,接种菌剂对堆肥菌群变化有影响,主要是增加了菌群多样性,并且通过前期对堆肥基质的分解,有利于后期的菌群生长。
本研究结果表明,接种菌剂能显著提高堆肥温度,延长高温持续期,加速堆肥基质的分解。通过接种菌剂改变了堆肥微生物的菌落变化,增加了优势菌群的数量和堆肥腐熟期微生物群落的多样性。
To research on effects of defferent inoculants used in pig manure,changes of bacterias in compost,and to approach the feasibility of animal microecological bacteria agents used in composting.In this experiment,ascendant microbials were screened from decomposed pig manure and were made into compound bacteria agents.Effects of inoculating different microorganism agents on composting of swine manure and research on microbial community changes by PCR-DGGE were done.
Screening from decomposed pig manure compost by using selective plate and high temperature,10 composting microbes were used to making inoculants,3 Bacillus strains which could endure 80℃high temperature and a bacilli,3 Actinomycosis,3 mould and 1 yeast fungus.Complex microorganism agents were made with these metioned above.
The agents were inoculated in swine manure by 0.1%respectively,divided into 4 groups:T1 was group of isolated bacterias of compost,T2 was microecological bacteria group,T3 was microecological bacteria too(add a concentration of bacteria one times of the T2),a group without adding bacteria as the control group.The effects of compost was evaluated by determination of index in 30d of composting:temperature,pH,moisture,the entire carbon,nitrogen and C/N.The results showed that:The maximum temperature of T1、T2 and T3 were higher than control group,and sustained period of high-temperature were longer than control group.Time of compost temperature which was above 50℃,T2 and T3 were 23 days which were the longest,T1 for 21 days,while the control group C only 10 days.Water changes of this fout groups were similar trend,with the compost, compost moisture gradually decline,pH value trends were consistent with each group, early were gradually rising trend and later gradually decline.Carbon utilization of T1、T2 and T3 were higher than the control group,at the end of composting,T1 carbon content dropped by 11.88%,T2 dropped by 5.75%,T3 dropped by 12.96%,while the control group only dropped by 4.73%.At the end of composting,C/N of T1 and T3 Group declined the most.When the physical and chemical indicators were integrated considered,the treatment group(T1)which microbial from compost were added was the best,followed by the groups treated addition of animal microecological bacterias(T2 and T3).T2 was better than T3,it showed that how much the addition quantity affected composting effects,but taking into account the actual cost of production,0.1%of bacteria content was feasible.
DNA of bacterias in different periods of compost was extracted,and 16 S rDNA V3 District of bacterias were got by PCR amplification.DGGE patterns showed that:the composting process in the quantities and types of bacteria change in the trend was "higher-lower-higher." Initial composting,the flora of composition of each groups were highly similar,93%-100%;compared to other periods of composting,the similarity was only 50%,the flora changes in the control group was faster than inoculated bacterias Groups.In 3-9 days,composition of flora of the control group and the other three groups were very different,the similarity of PCR-DGGE DNA profiles was 68%.The bacteria community of composting changed greatly,when composting was in a high temperature period,dominant flora and flora which tolerate high temperatures appeared in the stage, and some flora which can not tolerate the high temperature would disappear or weakened. In 21-30 days,it was cooling-maturity period of composting,composition of bacteria in compost gradually became stable and the similarity was 93%-100%,the diversity was higher than high-temperature period,the control group with the diversity of the group emerged early than inoculated bacterias groups.As composting experienced warming period,the maintenance of high temperature,cooling down period and the maturity period, the various stages of composting environment adapted to different microbial survival and growth.Initial composting the variety of bacterias were mainly indigenous bacterias which were larger quantities and types,and the presence of pathogens,such as Treponema brennaborense and Escherichia coli which could cause pig dysentery.In high temperature phase,the bands reduced and advantage zone emerged,which was mainly Bacillus sp.The pathogens bands which could detected in the early composting gradually weakened or disappeared.Bands increased in cooling down period also.Microorganisms and bacterias from compost and animal microecological agents were detected in DGGE profiles. Bacillus was correspondence with the predominant band appeared in high-temperature phase,and the existence of the band was longer than the control goup.
The results showed that inculated bacterias could adapt to composting environment, and became the predominant flora.They could made composting temperature increased significantly and prolonged the duration of high temperature and reduce the smelly odor of compost,and accelerated the decomposition.Animal microecological agents used in composting were feasible.
1.复合菌剂和微生态制剂对堆肥效果的影响
利用选择性平板培养和高温筛选从猪粪腐熟堆肥中分离微生物,从中筛选10株堆肥微生物,3株能够耐受80℃高温的芽孢杆菌,3株中温放线菌,3株霉菌和1株酵母菌,用以上菌株制备复合菌剂。将复合菌剂和微生态制剂分别按0.1%比例接种猪粪堆肥,共分成4组:T1是接种堆肥分离的复合菌剂组,T2是微生态菌剂组,T3是微生态菌剂组(接种菌剂浓度是T2的1倍),设不接种菌剂组为对照组。通过测定堆肥30d内温度、pH、水分、全碳、全氮和C/N,评价接种菌剂对堆肥效果的影响。结果表明:整个堆肥过程中,接种堆肥分离微生物和微生态菌剂组的温度显著高于对照组,最高温度也显著高于对照组,T1组最高温度达到73℃,T2为74℃,T3为72℃,对照组为71.5℃。接种菌剂组堆肥高温期持续时间显著比对照组长,堆肥温度在50℃以上维持的时间T2、T3最长,都是23天,T1为21天,而对照组C只有10天。各组水分变化趋势相似,随着堆肥的进行,堆肥水分逐渐下降。各组pH值变化趋势基本一致,堆肥初期呈逐渐上升趋势,堆肥后期则逐渐下降。堆肥结束时,接种菌剂组对碳素的利用率显著高于对照组,T1的全碳含量下降了11.88%,T2下降了5.75%,T3下降了12.96%,而对照组只下降了4.73%。堆肥结束时,T1组和T3组C/N下降最多。综合各项物理化学指标来看,接种堆肥分离微生物的处理组T1效果最好,接种动物微生态制剂的处理组T2和T3的效果也较好,表明动物微生态制剂用于堆肥是可行的。
2.PCR-DGGE技术研究接种菌剂对堆肥微生物的群落变化的影响
通过抽提不同时期堆肥细菌总DNA,PCR扩增细菌16S rDNA V3区和DGGE电泳,进行DGGE图谱聚类分析和条带多样性的分析,结果表明:堆肥过程中细菌种类数量随着堆肥的升温期、高温维持期和降温期的变化呈现出“升高-降低-升高”的变化趋势。各阶段出现不同的菌群组成,接种菌剂堆肥细菌群落变化较对照组慢。堆肥初始,菌群组成主要是土著微生物,各组相似性为93%-100%;与其他时期的堆肥
DGGE条带组成相似度仅为50%。第0-3天,此阶段温度上升较快,细菌群落多样性开始下降,不耐受高温的菌群逐渐消失并伴随着优势菌群的出现。接种的堆肥分离微生物和动物微生态制剂在DGGE图谱中检测出来,其中芽孢杆菌与高温期的优势条带相对应。第9-21天,各组DGGE图谱的相似性为68%,对照组菌群多样性较接种菌剂组高,但接种菌剂组优势菌群明显,且维持时间较长,表明堆肥高温阶段菌群的变化与堆肥温度的变化有关,对照组温度低,更利于不耐受高温菌群的生长;接种菌剂增加了高温期优势菌群的数量。第21-30天为堆肥降温腐熟期,堆肥细菌组成逐渐趋于稳定,各组DGGE图谱相似性为93%-100%,细菌菌落多样性高于堆肥高温期。此阶段接种菌剂组菌群多样性高于对照组,表明在堆肥后期,接种菌剂对堆肥菌群变化有影响,主要是增加了菌群多样性,并且通过前期对堆肥基质的分解,有利于后期的菌群生长。
本研究结果表明,接种菌剂能显著提高堆肥温度,延长高温持续期,加速堆肥基质的分解。通过接种菌剂改变了堆肥微生物的菌落变化,增加了优势菌群的数量和堆肥腐熟期微生物群落的多样性。
To research on effects of defferent inoculants used in pig manure,changes of bacterias in compost,and to approach the feasibility of animal microecological bacteria agents used in composting.In this experiment,ascendant microbials were screened from decomposed pig manure and were made into compound bacteria agents.Effects of inoculating different microorganism agents on composting of swine manure and research on microbial community changes by PCR-DGGE were done.
Screening from decomposed pig manure compost by using selective plate and high temperature,10 composting microbes were used to making inoculants,3 Bacillus strains which could endure 80℃high temperature and a bacilli,3 Actinomycosis,3 mould and 1 yeast fungus.Complex microorganism agents were made with these metioned above.
The agents were inoculated in swine manure by 0.1%respectively,divided into 4 groups:T1 was group of isolated bacterias of compost,T2 was microecological bacteria group,T3 was microecological bacteria too(add a concentration of bacteria one times of the T2),a group without adding bacteria as the control group.The effects of compost was evaluated by determination of index in 30d of composting:temperature,pH,moisture,the entire carbon,nitrogen and C/N.The results showed that:The maximum temperature of T1、T2 and T3 were higher than control group,and sustained period of high-temperature were longer than control group.Time of compost temperature which was above 50℃,T2 and T3 were 23 days which were the longest,T1 for 21 days,while the control group C only 10 days.Water changes of this fout groups were similar trend,with the compost, compost moisture gradually decline,pH value trends were consistent with each group, early were gradually rising trend and later gradually decline.Carbon utilization of T1、T2 and T3 were higher than the control group,at the end of composting,T1 carbon content dropped by 11.88%,T2 dropped by 5.75%,T3 dropped by 12.96%,while the control group only dropped by 4.73%.At the end of composting,C/N of T1 and T3 Group declined the most.When the physical and chemical indicators were integrated considered,the treatment group(T1)which microbial from compost were added was the best,followed by the groups treated addition of animal microecological bacterias(T2 and T3).T2 was better than T3,it showed that how much the addition quantity affected composting effects,but taking into account the actual cost of production,0.1%of bacteria content was feasible.
DNA of bacterias in different periods of compost was extracted,and 16 S rDNA V3 District of bacterias were got by PCR amplification.DGGE patterns showed that:the composting process in the quantities and types of bacteria change in the trend was "higher-lower-higher." Initial composting,the flora of composition of each groups were highly similar,93%-100%;compared to other periods of composting,the similarity was only 50%,the flora changes in the control group was faster than inoculated bacterias Groups.In 3-9 days,composition of flora of the control group and the other three groups were very different,the similarity of PCR-DGGE DNA profiles was 68%.The bacteria community of composting changed greatly,when composting was in a high temperature period,dominant flora and flora which tolerate high temperatures appeared in the stage, and some flora which can not tolerate the high temperature would disappear or weakened. In 21-30 days,it was cooling-maturity period of composting,composition of bacteria in compost gradually became stable and the similarity was 93%-100%,the diversity was higher than high-temperature period,the control group with the diversity of the group emerged early than inoculated bacterias groups.As composting experienced warming period,the maintenance of high temperature,cooling down period and the maturity period, the various stages of composting environment adapted to different microbial survival and growth.Initial composting the variety of bacterias were mainly indigenous bacterias which were larger quantities and types,and the presence of pathogens,such as Treponema brennaborense and Escherichia coli which could cause pig dysentery.In high temperature phase,the bands reduced and advantage zone emerged,which was mainly Bacillus sp.The pathogens bands which could detected in the early composting gradually weakened or disappeared.Bands increased in cooling down period also.Microorganisms and bacterias from compost and animal microecological agents were detected in DGGE profiles. Bacillus was correspondence with the predominant band appeared in high-temperature phase,and the existence of the band was longer than the control goup.
The results showed that inculated bacterias could adapt to composting environment, and became the predominant flora.They could made composting temperature increased significantly and prolonged the duration of high temperature and reduce the smelly odor of compost,and accelerated the decomposition.Animal microecological agents used in composting were feasible.
引文
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