| Due to the continuous acceleration of urbanization in China,the number of new sewage treatment plants is gradually increasing.As the terminal by-product of sewage treatment,dewatered sludge has the dual characteristics of "resource" and "pollution".Its treatment and disposal has become one of the difficult problems in the water treatment industry.Sludge vermicomposting technology is not only simple and economical,low-carbon and environmental protection,but also its vermicomposting products can be used as high-quality bio-organic fertilizer.However,due to the complexity of sludge structure and a large number of drug antibiotic resistance genes(ARGs),it is difficult to remove ARGs in sludge earthworm manure,which has become one of the bottlenecks restricting the development of this technology.Sludge is mainly composed of microbial aggregates encapsulated by extracellular polymer(EPS).The interior of sludge is not only the main place of organic matter degradation and microbial activity,but also the main storage place of free ARGs and mobile genetic element(MGEs).Studies have confirmed that sludge EPS is the key place for horizontal transfer of ARGs,so the study on the effect of vermicomposting on sludge EPS structure and ARGS distribution is of great significance to reveal the mechanism of vermicomposting on 0ARGs reduction.However,there is little research in this area at present.Therefore,in this study,600 and 1200 earthworms were added to 10 kg sludge respectively,and compared with non-earthworms,the changes of ARGs and MGEs abundance in each layer of sludge EPS structure composition machine during vermicomposting were analyzed,and the effect of vermicomposting on sludge EPS structure and associated ARGs distribution was investigated.The results were as follows.(1)Compared with the control group without earthworms,the electrical conductivity of the final sludge earthworm manure in the low-density and high-density earthworm groups increased significantly by 1.64 and 1.82 times,respectively(P < 0.05).At the same time,earthworms could promote the degradation and mineralization rate of proteins and polysaccharides in organic matter(P < 0.05).And accelerate the transformation of aromatic proteins and microbial by-products in organic compounds to humic acids and fulvic acids.The results of high-throughput sequencing showed that compared with the products of the control group,the Shannon index of bacterial 16 S r DNA in low-density and high-density earthworm groups decreased by 11.43% and 7.08% respectively;and β diversity also changed greatly.Vermicomposting could significantly increase the abundance of actinomycetes and Proteus(P< 0.05).The horizontal population structure of microorganisms in the vermicomposting system changed greatly during the ripening stage of vermicomposting,and the abundance of Xanthomonadales,Chitinophagales,Rhodobacterales,Streptomycetales and Propionobacteriales in the vermicomposting system was significantly higher than that in the control group.Compared with the horizontal population structure of microorganisms in the control group,the abundance of Dokdonella and Thermomonas in the vermicomposting system of vermicomposting group increased significantly.(2)By analyzing the EPS properties of the sludge,it was found that at the end of the earthworm degradation period on the 20 th day,the contents of nitrate nitrogen(NO3-),ammonia nitrogen(NH4+)and dissolved organic carbon(DOC)in the sludge EPS mucus layer of the high density earthworm group increased by 4987.33%,275.13% and 21.53% respectively compared with the control group.Three-dimensional fluorescence results showed that with the progress of vermicomposting,the contents of dissolved microbial by-products and aromatic-like proteins in each mucus layer decreased,while those in TB(Tight binding layer)-EPS layer were on the contrary,and humic acids such as soluble microbial by-products,aromatic-like proteins,humic acid and fulvic acid in each layer of sludge EPS increased at the end of ripening period.Compared with the control group,the aromatic protein-like substances in the TB-EPS layer of vermicomposting group were significantly increased.The results showed that earthworms could significantly promote nitrification and organic matter degradation in sludge mucus layer and LB(Loose binding layer)-EPS layer.(3)Compared with the control group,vermicomposting significantly increased the abundance of MGEs(transposon(int I-1)and integron(tnp A-04)),sulfonamide ARGs(sul-1and sul-2)and tetracycline ARGs(tet X and tet G),and significantly decreased the abundance of macrolides(erm F and erm B)and tetracycline ARGs(tet M)(P < 0.05).(3)compared with the control group,vermicomposting significantly increased the abundance of MGEs(tnp A-04 and int I-1),sulfonamide ARGs(sul-1 and sul-2)and tetracycline ARGs(tet M)(P < 0.05).From the distribution of ARGs in sludge EPS,tet M,tet X and erm B were mainly distributed in the mucus layer,tet G,sul-1,sul-2,ere A and erm F were mainly distributed in the LB-EPS layer,and MGEs was mainly distributed in the TB-EPS layer.At the end of the earthworm degradation period,tet M,tet X,sul-2,ere A,erm F and erm B in the vermicomposting group were mainly distributed in the mucus layer,tet G was abundant in the mucus layer and LB-EPS layer,and sul-1 and MGEs were mainly distributed in TB-EPS.At the end of ripening stage,tet M,tet X,sul-2 and erm F in vermicomposting group were mainly distributed in mucus layer,tet G and erm B were abundant in mucus layer and LB-EPS layer,sul-1,ere A,int I-1 and tnp A-04 were mainly distributed in LB-EPS layer.In contrast,the distribution of ARGs(sul-2,tet X,tet G)in the sludge EPS layer in the control group was significantly different from that in the vermicomposting group.The results showed that earthworms changed the EPS structure of sludge by feeding and digging holes,so that the DNA in the tight binding layer diffused outward,while the DNA in the mucus layer and loose binding layer diffused inward,resulting in an increase in the abundance of ARGs(sul-1,sul-2,tet X and tet G)which is easy to bind to MGEs.To sum up,vermicomposting of sludge can significantly increase the number and diversity of functional microflora,increase the content of electrical conductivity,nitrate and ammonia nitrogen in sludge,and transform sludge EPS from aromatic histones to humic acid and fulvic acid.The products of vermicomposting are more stable and have high agricultural potential.In addition,earthworms change the structure of EPS through feeding and biological disturbance behavior,affect the temporal and spatial distribution of ARGs and the transfer law of MGEs,and then regulate the fate of drug resistance genes. |
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