Screening And Optimization Of Pretreatment Methods For Removal Of Typical Tetracycline Resistance Genes In Excess Sludge

With the widespread use of antibiotics,the contamination of resistance genes induced by antibiotics has become one of the great threats to human health.It is estimated that the number of deaths caused by antibiotic resistance genes（ARGs）pollution will be possible to reach 10 million in 2050,which may be more than deaths caused by cancer.At the same time,the production of excess sludge from wastewater treatment plants（WWTPs）will continue to increase with the increasing discharge of municipal domestic sewge.The excess sludge is rich in protein,sugar and other substances,which has the value of potential utilization.However,the abundance of ARGs in excess sludge is much higher than that in natural environment.Therefore,it is important to reduce the abundance of ARGs in excess sludge from WWTPs for protecting the ecological environmental and human health.Alkaline treatment,microwave treatment and fenton treatment are widely used in the pretreatment of excess sludge,whereas the changes of ARGs caused by these treatment processes are rarely studied.In this thesis,the effects of alkaline treatment,microwave treatment and fenton treatment on the abundance of ARGs（tet A,tet C,tet M,tet O,tet Q,tet W,tet X）and class one integrin（int11）were investigated.Among these treatments,the one who had the best removal rate of ARGs（i.e.,fenton treatment）was chosen as the following optimization research.Subsequently,the main influential factors of fenton treatment on removing ARGs and int I1 were explored.Furthemore,the box-behnken response surface design method was used to optimize the conditions of ARGs and int I1 in excess sludge treated by fenton.Finally,16S r RNA high sequencing and apoptosis technology were applied to explore the possible mechanism to reduce ARGs and int I1.According to the above studies,the main findings of this thesis were concluded as follows:（1）Fenton treatment showed a better removal rate of target genes compared with microwave and alkaline treatment.The abundance of ARGs in the excess sludge treated by fenton treatment decreased from 1.32×10⁷～1.14×10⁹ copies/g to 7.06×10⁵～3.10×10⁷copies/g dry sludge.The order of magnitude removed by fenton treatment was about 1.27to 2.23 logs.However,the abundance of target genes increased 0.16～0.75 logs after 10 h of alkaline treated excess sludge,and increased-0.39～0.93 logs by 180 s of microwave treated excess sludge,respectively.（2）During fenton treatment,pH,Fe²⁺concentration and H₂O₂ concentration were found to significantly affect the removal of target genes,but the treatment time showed no significant effect.The optimal treatment condition for removing ARGs and int I1 from the excess sludge was 120 min,pH=3,0.01 mol/L H₂O₂ and 0.005 mol/L Fe²⁺.（3）Based on the results of single factor treatment,the corresponding surface method of Box-Behnken was further used to optimize the fenton reaction conditions and explore the interactive effect of these factors on removing target genes.The most significant influential factor on removing tetA,tetM,tetO and intI1 was pH,the most significant influential factor on removing tet Q,tet W and tet X was Fe²⁺addition concentration,and the greatest effect on removing tet Q was H₂O₂ addition concentration.Fe²⁺concentration and H₂O₂ concentration showed the significantly interactive influence on the removal of tet A,tet M and tet Q,while pH and H₂O₂concentration had the obviously interactive effect on removing tet M and int I1.The optimal condition obtained by response surface analysis was pH 3.00,0.005 mol/L Fe²⁺and 0.0125 mol/L H₂O₂.（4）From the aspect of vertical gene transfer,Firmicutes and Proteobacteria might be potential hosts of the ARGs through the analysis of changes in microbial community structure.In terms of horizontal gene transfer,the optimized Fenton treatment demonstrated good removal effect on the intI1,which was decreased by 9.70×10³ copies/g,decreased by about 4.20 logs’reduction.This result suggested fenton treatment might have a good blocking effect on the horizontal transfer of the ARGs.Besides,the apoptosis data indicated that fenton reagent had damage effect on cells in excess sludge,which leads to ARGs release and removal in sludge.From this stduy,fenton treatment was selected from the abovementioned treatments and then optimized the treatment parameters.Furthermore,the microbial community structure and apoptosis were analyzed to reveal the possible hosts and removal mechanism of fenton treatment.This study provided important information for better removing ARGs from excess sludge.