再生固体牛粪垫料中细菌多样性分析及评价

Analysis of Bacterial Diversity and Safety Evaluation of the Recycled Manure Solids

【目的】评估牛粪垫料再生系统(Bedding Recovery Unit,BRU)处理的再生固体牛粪垫料(Recycled Manure Solids,RMS)的安全性,为养殖场安全合理应用RMS提供理论依据和参考。【方法】采用可培养法对BRU系统处理前、处理后及堆存30 d的RMS样品中细菌总数、大肠杆菌、金黄色葡萄球菌、无乳链球菌和沙门氏菌的数量进行检测,并利用高通量测序方法分析样品中致病细菌群落组成和多样性。【结果】牛粪经BRU系统好氧发酵16~18 h后,细菌总数和大肠杆菌数量均显著下降,且都下降了一个数量级;金黄色葡萄球菌数量未发生显著变化(P>0.05);RMS在堆放30 d后,物料中的金黄色葡萄球菌数量显著下降;而细菌总数和大肠杆菌数量又呈现出了显著增加的趋势(P<0.05),在3种样品中均未检测到无乳链球菌和沙门氏菌。利用分子生物学方法仅在未经BRU系统处理的样本中检测到条件致病菌4个属:气球菌属(丰度为1.40%±0.8%)、费克蓝姆氏菌属(20.73%±3.97%)、厌氧梭菌属(7.20%±2.35%)和肉杆菌属(0.27%±0.15%)。经BRU系统处理后样品中细菌Alpha多样性指数呈现出极显著的下降趋势(P<0.01),表现在细菌OTU数量上减少了57.73%,细菌丰富度指数(ACE指数)降低了57.47%,香农-威纳多样性指数降低了81.63%。而RMS经过堆存30 d后,香农-威纳多样性指数显著增加了21.76%。经BRU系统处理显著的改变了细菌群落组成,并且其群落组成在堆存30 d后出现显著增加(P<0.01)。【结论】牛粪经BRU系统处理16~18 h后能够显著降低RMS中细菌多样性,改变群落组成,但其对除大肠杆菌外其他致病菌的杀灭作用有限,可通过延长堆放时间来降低其使用风险。

【Objective】 To evaluate the safety of recycled manure solids(RMS)treated by bedding recovery unit(BRU)in the hope of providing theoretical basis and reference for safe and reasonable application RMS on breeding farms.【Methods】 Bacterial cultivation methods were used to detect the counts of the total bacteria,Escherichia coli,Salmonella,Staphylococcus aureus and parasitic eggs in RMS before and after the treatments by BRU.Besides,those detecting items in samples of the BRU treated by RMS that were stacked for a month were also determined and high-throughput sequencing method was used to detect the composition and diversity of pathogenic bacteria.【Results】 After 16-18 h of aerobically fermentation,the counts of total bacterial and Escherichia coli in manure samples were decreased tenfold compared with fresh manure,but the counts of Staphylococcus aureus and parasitic eggs were not significantly changed(P>0.05).The counts of total bacterial number and Escherichia coli were significantly increased(P<0.05),but the counts of parasitic eggs were on the contrary(P<0.01).The pathogenic bacteria were only detected in manure samples untreated by BRU and they were divided into 4 genera:Aerococcus(1.40%±0.8%),Facklamia(20.73%±3.97%),Paeniclostridium(7.20%±2.35%) and Carnobacterium(0.27%±0.15%).Bacterial alpha-diversity indexes presented an significant decreasing trend after BRU treatment(P<0.01),mainly in terms of the bacterial OTU numbers,the bacterial richness(ACE index),and the Shannon-Weiner diversity index decreased 57.73%,57.47% and 81.63%,respectively.While,the Shannon-Weiner diversity index of samples in RMS stacked for a month was significantly increased(21.76%) compared with the index in RMS treated by BRU.Furthermore,the bacterial beta-diversity analysis showed that the bacterial community structures were significantly changed after BRU treatment,and its communities were significantly increased in RMS stacked for a month(P<0.01).After 16-18 h of aerobically fermentation,the counts of total bacterial number,bacterial diversity and community structures were significantly changed.However,its killing effect on most pathogenic bacteria(apart from Escherichia coli) and parasitic eggs were still weak.【Conclusion】 When RMS is treated with BRU system for 16-18 h,bovine manure can significantly reduce bacterial diversity and change community composition,but its killing effect on pathogenic bacteria and parasite eggs other than Escherichia coli is limited.Therefore,the risk can be reduced by prolonging stacking time.