Mechanism And Regulation Technology Concerning Nitrogen Transformation And Antibiotic Resistance Genes Transfer During Swine Manure Composting

Nitrogen is a major element in the composting microenvironment,and it is essential for the synthesis of nucleic acids and proteins,thus the nitrogen content in composting products directly determine the quality of composting products.A large amount of antibiotics and heavy metals are excreted in livestock waste due to the overuse of antibiotics and heavy metals in feed.The existence of antibiotics and heavy metals probably could influence the nitrogen transformation process,which is unknown.Nitrogen transformation is closely related to nitrogen transformation functional genes,but few literatures have systematically analyzed nitrogen transformation process in composting process through nitrogen transformation functional genes.The widely use of antibiotics also leads to the generation of antibiotic resistance genes（ARGs）,and antibiotics and heavy metals in livestock waste probably impact the ARGs variation in composting process.However,up to now,the mechanism concerning the effects of antibiotics and heavy metals on ARGs variation is still unclear.This research studied the variations of nitrogen transformation functional genes and ARGs during the composting of swine manure containing antibiotics and heavy metals,to reveal the biological mechanism concerning the nitrogen transformation and ARGs variation under co-exposure of antibiotics and heavy metals.Wood ash,coal fly ash and ozone was used to control the emissions of NH₃ and N₂O,and the spread of ARGs in swine manure composting process in the condition of antibiotics and heavy metals.Furthermore,the reduction mechanism was revealed.Firstly,the effects of antibiotics on nitrogen transformation and ARGs variations during swine manure composting were studied.The results indicated that when swine manure contained 5 mg kg^-1DW（dry matter）sulfamethoxazole,5 mg kg^-1DW norfloxacin,as well as 5 mg kg^-1DW sulfamethoxazole and 5 mg kg^-1DW norfloxacin,the composting process finished successfully.Sulfamethoxazole and norfloxacin both increased the NO₂^--N content in the first 13 days,decreased the abundances of amo A and nxr A（nitrification genes）on Day-2,and elevated the ratio of nos Z/nir K（denitrification genes）on Day-21.Individual existence of sulfamethoxazole and norfloxacin enhanced the NH₃ cumulative emission,which was due to the reduction in amo A.Simultaneous existence of sulfamethoxazole and norfloxacin decreased the NH₃cumulative emission,which was related to NH₄⁺-N and p H.Two N₂O emission periods emerged in composting process.Individual existence of sulfamethoxazole increased the N₂O emission rate in the first N₂O emission period,which was due to the decline in nxr A.Sulfamethoxazole and norfloxacin both decreased the N₂O emission rate in the cooling phase,and the N₂O emission rate in the treatment with sulfamethoxazole and norfloxacin simultaneously was lowest,which was related to nos Z/nir K ratio.Sulfamethoxazole and norfloxacin both increased the abundances of ARGs and mobile genetic elements（MGEs）,and the abundances of ARGs and MGEs in the treatment with sulfamethoxazole and norfloxacin simultaneously was higher than the treatments with sulfamethoxazole and norfloxacin alone.In the condition of antibiotics,ARGs was significantly correlated to physciochemical characteristics of composting materials and MGEs,indicating that the effects of antibiotics on ARGs variations were mainly through altering the physciochemical characteristics of composting materials and influencing the horizontal gene transfer.Co-existence of antibiotics and heavy metals always existed in swine manure,thus based on the study about the effects of antibiotics on nitrogen transformation and ARGs in composting process,the effects of antibiotics and heavy metals on nitrogen transformation and ARGs variation in composting process were studied.Individual existence of 5 mg kg^-1DW sulfamethoxazole,individual existence of 2000 mg kg^-1DW copper,as well as co-existence of 5 mg kg^-1DW sulfamethoxazole and 2000 mg kg^-1DW copper both decreased the NO₃^--N content,increased the NO₂^--N content,reduced the abundances of amo A,hao and nxr A in the first 13 days,and enhanced the nar G（denitrification gene）abundance on Day-2 and the nos Z/nir K ratio on Day-21.Individual existence of sulfamethoxazole,individual existence of copper,and simultaneous existence of sulfamethoxazole and copper elevated the NH₃ cumulative emission,and the NH₃ cumulative emission in the treatment with sulfamethoxazole and copper simultaneously was highest among them,which was because that amo A was inhibited.Two N₂O emission periods emerged in composting process.Individual existence of copper and simultaneous existence of sulfamethoxazole and copper increased the N₂O emission rate in the first emission period,which was on account of the rise in nar G.However,individual existence of copper and simultaneous existence of sulfamethoxazole and copper declined the N₂O emission rate in the second emission period,which was related to nos Z/nir K ratio.Sulfamethoxazole and copper both increased the total abundances of ARGs and MGEs,and the total abundances of ARGs and MGEs were highest in the treatment with sulfamethoxazole and copper simultaneously.Individual existence of copper and simultaneous existence of sulfamethoxazole and copper also elevated the abundances of heavy metal resistance genes（MRGs）.In the conditions of antibiotics and heavy metals co-existence,the key factors influencing ARGs abundance were potential host bacteria,horizontal gene transfer and heavy metals induced co-resistance.The impacts of coal fly ash and wood ash on the nitrogen transformation and ARGs during the composting process were studied,and the reduction efficiency of ozone in ARGs in composting products was evaluated.The results suggested that the addition of10%（based on the weight of swine manure）coal fly ash and 10%wood ash could increase the p H,and decrease the bio-available copper content and NH₄⁺-N content.Wood ash elevated the abundance of hao and NO₂^--N content in the intial composting,declined the nos Z/nir K ratio,and reduced the abundance of hao and NO₂^--N content in the cooling and maturing phase.Coal fly ash decreased amo A,hao and NO₂^--N,increased the nos Z/nir K ratio on Day-5.The addition of wood ash decreased the NH₄⁺-N content and caused the reduction in cumulative NH₃ emission.Coal fly ash reduced the amo A abundance and enhanced the p H,causing the increase of NH₃emission.Two N₂O emission periods emerged in composting process.Wood ash elevated the N₂O emission rate in the first emission period,while reduced the N₂O emission rate in the second emission period.Coal fly ash simultaneously declined the N₂O emission rate in the two emission period.The N₂O emission was related to hao,nos Z/nir K and NO₂^--N.The addion of coal fly ash and wood ash both reduced the total abundances of ARGs,MGEs and MRGs.The reduction in ARGs induced by coal fly ash and wood ash was probably through decreasing the co-selective pressure imposed by bio-available heavy metals,reducing the horizontal gene transfer of ARGs,and declining the abundances of potential host bacteria.Ozone technology was put forward to remove the ARGs in composting products.The results suggest that ozone treatment significantly removed the total ARGs,toal MGEs,intracellular ARGs and intracellular MGEs.The reductions in intracellular 16S r RNA contributed to intracellular tet Q and tet W removal,while the variations of other ARGs related to MGEs abundance.Additionally,the reduction in potential host bacteria also resulted in the intracellular ARGs removal.