Application Of Bacteroidales Gene Marker In Tracking Swine Wastewater

Microbial Source Tracking (MST)， a new monitoring technology for identifying sources of fecalpollution in water，is based on the differences of indicators between environmental samples and thepotential production source, or/and the existence of host-specific biomakers. In this paper, we took theswine wastewater as the object, and the method to quantitative detect of pig-specific biomarker in water,stability of the biomarker Pig-2-Bac, relevances between Pig-2-Bac and pollution indicators in watermicrocosms and surface runoff were stuied respectively.Simulated water with pig excrement was used to detect the degree of water pollution dischargedfrom piggery. The extent of pollution was investigated by qPCR with pig-specific Bacteroidales16SrRNA gene markers. The improved CTAB method was suitable for extracting total DNA from piggerywastewater comparing with the other two methods based on our experimental results. The specificity ofprimer1（Bac41F/PS183R）, primer2（Bac41F/Bac163R）and primer3（Bac32F/Bac108R）were testedrespectively. And the primer3was selected for qPCR in this research because it showed higherspecificity and lower detection limit (4.09E+02~1.60E+03copies) than others. As a result, this primercan be used to detect the pollution from the source of piggery wastewater and discriminate othersproperly.The persistence of Pig-2-Bac was affected by temperature sharply. The copies of Pig-2-Bac fellbelow to the detected limit within23days at35℃, but Pig-2-Bac still could be measured until the endof the experiment (23days) at4℃,15℃and25℃. Furthermore, we investigated the effects of light onthe persistence of Pig-2-Bac, the decay of Pig-2-Bac kept the same trend in light or darkness. The resultrevealed that Pig-2-Bac persisted longer at lower temperatures and light had no significant effect on itspersistenceExplore the correlations between Pig-2-Bac and pollutants are the prerequisite whether Pig-2-Baccan be used to indicate the pollutants from swine wastewater. The water microcosms with pig excrementwere simulated, and the variation of total nitrogen (TN), ammonia (NH3-N), nitrate (NO3-N), totalphosphorous (TP), chemical oxygen demand (COD), total number of bacterial and fungus colony insimulated microcosms were investigated at different incubation temperatures. Meanwhile, we analyzedthe correlation between Pig-2-Bac and different pollution parameters.We conducted preliminary outdoorexperiments in Hefei, Anhui Province. To study the variation of Pig-2-Bac and pollution parameters inswine wastewater runoff process, and also conducted a preliminary analysis of the correlation. Themicrocosm trials indicated that Pig-2-Bac was positively correlated with TN, TP and COD, andnegatively correlated with the total number of bacteria (TB). This marker did not have significantcorrelation with other chemical parameters. Due to many factors outdoor experiments did not showconsistent results with microcosms experiments, the correlations between Pig-2-Bac and pollutionparameters are not ideal.These findings indicated that pig-specific genetic marker is a potential alternative way to accurate quantitative monitor pollutant from pig waste by detecting the pig-specific Bacteroides marker usingqPCR.But there are many factors that restrict their application, such as stability of Pig-2-Bac, theenvironmental behavior, the correlation with instructed pollutants and so on, these still need furtherstudies, so that MST can be better applied to environmental monitoring.