Effect Of Enhanced Anaerobic Digestion On Microbial And Antibiotic Resistance Genes In Waste Actived Sludge

The rapid urbanization in China is bound to be accompanied by the annual increase of municipal sewage discharge,thus producing a large number of wastes activated sludges.The wastes activated sludge poses a serious threat to the ecological environment because it cannot be properly treated.On the other hand,the environmental microbial resistance caused by the abuse of antibiotics also seriously threatens the safety of human drug use.Wastewater treatment plants would absorb and enrich a large number of Antibiotic resistance genes(ARG)in the process of wastes activated treatment and aggravate the resistance of human pathogenic bacteria through gene horizontal transfer.As an important way of sludge treatment and resource utilization,anaerobic digestion(AD)technology can effectively reduce the sludge volume and obtain clean energy(mainly methane).At the same time,it is considered to have the potential to kill pathogenic bacteria and reduce the risk of antibiotic resistance transmission.However,the current AD technology is limited by the hydrolysis-acidification of sludge,and the biogas production efficiency needs to be improved by strengthening AD technology.In addition,there is still a great controversy about whether the AD technology can effectively control the risk of ARGs spread in sludge.It is necessary to conduct in-depth study on the mechanism of resistance gene transformation and pathogenic bacteria acquiring drug resistance in the process of AD.In view of the low efficiency of traditional AD and the unclear transmission mechanism of ARGs in the digested sludge,the AD process was strengthened by 1)introducing additives into the AD system;2)gradually transforming the mesophilic AD system into the thermophilic AD system.This study used metagenomics technology and multivariate mathematical statistics,combined with semi-continuous AD experiments and multi-batch AD experiments,to comprehensively explore ARGs,the mobile genetic elements(MGEs)and antibiotic resistant bacteria during AD process.The molecular ecological network of ARGs and drug-resistant bacteria was constructed to identify the key gene elements and host bacteria carrying ARGs.The mechanism of horizontal and vertical migration of ARGs with MGWs and drug-resistant bacteria was analyzed.The mechanism of the influence of anaerobic digestion technology on the ARGs dynamic changes was identified,and a control method which could effectively cut off the spread of antibiotic resistance was proposed.As a cheap electron donor,nano zero-valent iron(nZVI)can create an anaerobic environment with low redox potential for Fe₃O₄ nanoparticles(NPs)promote direct interspecific electron transfer in the generation of syntrophic methane.These two kinds of iron NPs are therefore used as exogenous additives in the AD system.After 80 days of semi-continuous AD experiment,the F1 with 0.5 g L^-1 Fe₃O₄NPs added and N2 with 1 g L^-1 nZVI added had the maximum cumulative biogas yield,which increased the cumulative biogas yield by 15.70%and 13.44%respectively.The high removal rates of s COD and VS in the F1 and N2 demonstrated the iron NPs can enhance the degradation of organic matter in AD.The results of three-days hydrolyzation-acidification experiments showed that Fe₃O₄ NPs and nZVI could promote the hydrolysis and acid production process,and thus promote the methane production.The fitting results of the kinetic model showed that Fe₃O₄ NPs and nZVI increased the maximum biogas production rate and hydrolysis rate.The Cone model is the most suitable model for semi-continuous AD biogas yield.Both Fe₃O₄ NPs and nZVI significantly reduced the Alpha diversity of microorganisms in AD sludge and changed the microbial community structure in a long-term semi-continuous AD experiment(100 days).However,the concentrations of Fe₃O₄ NPs and nZVI in the AD system did not significantly affect the microbial distribution.Quantitative PCR(q PCR)analysis of the target ARGs showed that two kinds of iron NPs had good effect on the removal of some ARGs,including tetracycline resistance coding genes(tet T,tet E and tet W)and?-lactamase resistance coding genes(bla OXA).Two common genes encoding drug resistance(sul I and sul II)were increased in AD sludge with the addition of iron NPs.Co-occurrence network analysis showed that the three-dominant bacterial phylum(Proteobacteria,firmicutes and Actinomycetes)in AD sludge were also the main potential hosts of ARGs.Fe₃O₄NPs and nZVI reduced the abundance of these three dominant bacteria.Therefore,succession of bacterial community is the main cause of ARGs abundance change.At the same time,different physical and chemical properties of sludge(such as p H,s COD and heavy metals)were significantly correlated with the abundance of ARGs.Combined with metagenomic sequencing technology,the microbial function and ARGs transmission mechanism in sludge were further analyzed.There were significant differences in the number of genes in the Blank group,Fe₃O₄ NPs group and nZVI group.The long-term existence of the two kinds of iron NPs significantly reduced the number of microbial genes in the AD system.At the same time,iron NPs have a certain selection effect on Fe₃O₄ NPs and nZVI create a unique AD condition for sludge so that the Fe₃O₄ NPs group and nZVI group form relatively independent microbial flora compared with the Blank group respectively.However,the results of gene function annotation showed that the metabolic function of sludge AD system would not change substantially in the presence of Fe₃O₄ NPs or nZVI.The main metabolic types of microorganisms in the three groups of samples were carbohydrate metabolism,amino acid metabolism and energy metabolism.The results of antibiotic resistance gene annotation show that Fe₃O₄ NPs and nZVI will increase the number of ARGs in general and enrich the abundance of antibiotic resistance mechanism in particular.Proteobacteria,Bacteroidetes and Firmicutes were the main hosts of ARGs in this study.In addition to nano-iron particles,the other four common engineering NPs(including carbon NPs,Al₂O₃ NPs,Zn O NPs and Cu O NPs)will inevitably enter the urban sewage treatment system due to their wide application in industry and personal life,thus affecting the AD process of sludge.The dosage of the four NPs is 50 mg/g VS_added.Carbon NPs and Al₂O₃ NPs could promote AD,while Zn O NPs and Cu O NPs could inhibit AD.All four NPs reduced the Alpha diversity of the bacteria.Carbon NPs and Al₂O₃ NPs increased the Alpha diversity of archaea,while Zn O NPs and Cu O NPs decreased the Alpha diversity of Archaea.The inhibition of Zn O NPs and Cu O NPs to archaea resulted in the reduction of methane production in the corresponding AD reactor.Compared with Carbon NPs and Al₂O₃ NPs,Zn O NPs and Cu O NPs had more significant influence on the structure change of microbial community.q PCR results showed that the four NPs significantly increased the MGEs abundance in AD reactors.The total absolute abundance of MGEs increased by 1 4 5.0 1%?1 5 9.6 7%?3 5 4.7 0%a n d 1 3 2.8 0%,respectively.The enrichment rate of tnp A-03 inZn O NPs group was the highest,reaching 2854.80%.Co-occurrence network analysis showed that Proteobacteria and Firmicutes were the main potential hosts of MGEs.Redundancy analysis and variation division analysis showed that metabolites(including total polysaccharides,VFA,SCOD and proteins)were the most important parameters affecting bacterial community changes,and the amount of explanation for bacterial community changes was 47.06%.In addition,in this study,the four metabolites showed significant positive correlation with t n p A-0 1?t n p A-0 2?t n p A-0 3?t n p A-0 4?t n p A-0 5?t n p A-0 7 and ISCR1.Although adding NPs can improve the AD performance to some extent,it is not effective in removing ARGs.Therefore,a gradual warming strategy is adopted to transform mesophilic AD into thermophilic AD within 20 days,so as to explore the removal effect of heating process on ARGs.The microorganisms in the reactor were sensitive to the temperature change of the AD system,and the Proteobacteria(10.93-18.95%)of the dominant bacteria were gradually transformed into Firmicutes(18.12-74.89%)under medium temperature.In addition,the metagenome revealed the broad spectral distribution of ARGs during the heating process.The results showed that high temperature could reduce the ARGs subtype by more than 30%.Among them,the high temperature conditions had a better effect on the removal of tetracycline,macrolide,penem,fluoroquinolone,acridine-dye and peptide resistance genes.The decrease in ARGs in the high-temperature anaerobic digestive system may be due to deletion of genes encoding antibiotic efferent pump,rapid decomposition of biomass,loss of microbial diversity,and destruction of multi-drug resistant bacterial cells.Considering that the activated sludge process at high temperature has the advantages of efficient removal of ARGs and higher methane production than the activated sludge process at medium temperature,it is considered that high temperature is an important technology for municipal sludge treatment and reuse.