Enhancement Of Antibiotic Resistance Genes Reduction In Sludge Digestive System By Free Nitrous Acid Pretreatment

Sludge digestion is an important way to realize the reduction and recycling of waste activated sludge（WAS）,but it faced the low digestion hydrolysis rate.Pretreatment can effectively strengthen the effect of sludge digestion.Free nitrous acid（FNA）pretreatment has shown excellent performance in promoting residual sludge cell breakage to enhance digestion efficiency.Its main advantages are green,economically and environmental friendly,it can be prepared in situ by anaerobic digestate.Antibiotic resistance genes（ARGs）,as novel contaminants,are widely enriched in WAS and are potentially risky.Studies have confirmed that pretreatment can also promote the removal of ARGs in the digestion process.However,few studies have been conducted to investigate the distribution and removal of ARGs through FNA pretreatment combined digestion process.In this study,FNA was used to pretreat WAS,and the cell breaking conditions of FNA pretreatment were explored based on the change of extracellular polymeric substances（EPS）and ARGs.The distribution and removal of ARGs under different digestion conditions were investigated with the optimal FNA pretreatment combination anaerobic/aerobic digestion.The correlation analysis of ARGs,int I1 and sludge physicochemical properties was performed.The potential hosts of ARGs were analyzed by correlating ARGs with microbial community structure.The main conclusions are as follows:（1）FNA pretreatment conditions:Higher concentration of FNA（lower p H and higher NO₂^--N concentration）,the more obvious WAS cell breaking effect and ARGs removal.In this experiment,p H 5.3 and NO₂^--N 753 mg/L were selected as the FNA pretreatment conditions for subsequent anaerobic and aerobic digestion.Int I1,16Sr DNA,ARGs abundance and FNA concentration were significantly negatively correlated,indicating that FNA pretreatment may control ARGs proliferation by reducing microbial load and inhibiting gene level transfer.（2）The optimization of anaerobic digestion conditions:The influencing factors of temperature>p H>sludge ratio,the best conditions were sludge ratio=2,temperature45°C,p H=10.The EPS of sludge decreased by 13.79%-54.67%from 0 to 24 d.Higher temperature of anaerobic digestion,the better effect of protein decomposition to release NH₄⁺-N,and the better removal of ARGs.The optimal anaerobic digestion conditions resulted in 86.48%and 84.22%reduction of total ARGs and int I1,sul genes（sul1 and sul2）and tet genes of tet A were reduced by 0.86,1.19 and 1.53 log respectively.Pearson correlation analysis showed that nutrient elements indirectly influenced the propagation of ARGs by affecting microbial growth and thus the host composition of ARGs.Horizontal gene transfer plays an important role in the propagation of ARGs during anaerobic digestion.（3）The optimization of aerobic digestion conditions:The influencing factors of temperature>p H>sludge ratio,the best conditions were sludge ratio=2,temperature45°C,p H=10.Microbial death promoted the increase of NH₄⁺-N,the rise of SCOD due to the decomposition of macromolecular organic matter during endogenous metabolism.EPS decreased gradually with time and its proteolysis rate was faster than that of anaerobic digestion.Total ARGs and int I1 were decreased by 99.71%and98.62%,sul genes（sul1 and sul2）,tet genes（tet Q,tet W,and tet A）were reduced by 3.35,2.35,0.63,1.38,and 4.31 logs,respectively.Under optimal conditions,the reduction of ARGs and int I1 by aerobic digestion was better than that by anaerobic digestion.Pearson correlation analysis showed that the changes in int I1 abundance at high temperature and high p H were correlated with nutrient elements.Reduction of ARGs is mainly caused by sul genes.On the one hand,biomass is reduced due to microbial cell lysis due to exhaustion of organic matter;on the other hand,horizontal gene transfer is blocked by aerobic digestion process,which reduces the risk of ARGs proliferation.（4）Analysis of the microbial community structure revealed that the microorganisms that dominated both at the phylum level were Actinobacteriota,Chloroflexi,Patescibacteria,Firmicutes,and Proteobacteria.Sperman correlation showed that int I1,sul1,and sul2 were associated with 10 microorganisms under the class phylum level Patescibacteria,Gemmatimonadota,Nitrospirota,Desulfobacterota,Bacteroidota,NB1-j,Bdellovibrionota,Zixibacteria,Proteobacteria,and Acidobacteriota were significantly and positively correlated.Network covariance analysis showed that 13 genus level under 6 phylum level were identified as potential hosts for the 5 target genes（sul1,sul2,tet W,tet A,int I1）.