Study On Variation Of Material Properties In The Process Of Dairy Manure Making Bedding In Dairy Farm And Detection Method Of Viable Pathogens

Nowadays,the demand for meat products and dairy products has increased along with the continuous improvement of people's living standards,which has promoted the rapid development of China's aquaculture industry.Dairy manure is an inevitable pollutant in dairy farm.If a large amount of dairy manure is discharged without treatment,it will cause pollution to the surrounding environment and threaten human health.Therefore,how to handle dairy manure quickly and effectively becomes a key problem that needs to be solved as soon as possible.Dairy manure contains organic components and cellulose that can be used by microorganisms,it has the potential to be converted into bedding material.In recent years,the emerging rotary aerobic fermentation technology-BRU?Bedding Recovery Unit?system has the advantages of less time,small footprint and elimination of pathogens during the process.However,there is still a lack of systematic research on the nature of materials,heavy metals and fecal coliforms in the actual operation of the BRU system at home and abroad,resulting in the inability to widely promote this technology.In order to investigate the annual variation of dairy manure and related pollutants in the process of recover bedding material from liquid manure,a typical large-scale dairy farm in Tianjin was selected as the research object in this study.Continuously sampling was conducted in different stages and different seasons.TN,TP,pH,water content,organic matter,fecal coliform,Cu,Zn and other heavy metals in the samples were all detected and analyzed.The results of testing data showed that there was a significant seasonal difference?P<0.05?in TN,TP,VS,Cu and Zn emissions from dairy manure,and the emissions of TN,TP,Cu,Zn and Cd were the highest in winter.After BRU aerobic fermentation,we found that the moisture content,Zn-content and fecal coliform appeared decreased correspondingly,while TN,TP and Cu-content showed an increasing trend in Recycled Manure Solid.Since qPCR is limited by its inability in differentiating viable bacterial cells from nonviable bacterial cells,a novel detection method of quantitative PCR combined with a DNA intercalating dye propidium monoazide?PMA-qPCR?was developed in this study,which could selectively detect viable bacterial cells with intact cell membrane.The selective functions of PMA depend on the differences among various species.In this study,two stains of Gram-negative bacteria?E.coli?and Gram-positive bacteria?S.aureus?were choosen to analyze the influence of PMA on live and dead cells.Several key factors of PMA effect were optimized,including the photo reaction time,the minimum inhibitory concentration,and the maximum toxic concentration.Experimental results showed that when OD₆₀₀ value of E.coli was 0.5,PMA concentration was 60?g/mL and photoreaction time was 1 minute,the PCR amplification of the membrane-damaging bacterial DNA in the samples could be inhibited.Experimental results showed that when OD₆₀₀ value of S.aureus was 0.5,PMA concentration was 65?g/mL and photoreaction time was 1 minute,the PCR amplification of the membrane-damaging bacterial DNA in the samples could be inhibited.To establish standard curve of PMA-qPCR,we designed and built standard plasmid for targeting the specific sequence of E.coli uidA and S.aureus nuc genes.The cell copy number of uidA and nuc genes and cycle threshould values presented the satisfactory linear dependability and the coeffient of correlation R² approached 0.993 and 0.998.When PMA-qPCR was applied to the samples of dairy manure bedding,we found that using a single layer of sterile gauze to filter the samples can remove most of the particulate matter,and it does not affect the number of viable bacteria in the sample.It is a successful attempt to use PMA-qPCR for quantitative detection of viable pathogens in Recycled Manure Solid.According our research,PMA-qPCR technology can effectively and quickly distinguish live and dead pathogens.PMA-qPCR technology can also applied to the detection of viable bacteria in environmental samples in other environmental samples,which will have broader development prospects in other areas of detection.