| The wide application of nanomaterials is bound to cause some harm to the ecological environment.At present,studies have shown that as many as 95%of the nanomaterials scrapped in production and life will eventually enter into sewage,while the amount of sewage produced in China is huge.The traditional activated sludge process finally makes a large number of nanomaterials enriched in the excess sludge,which brings hidden dangers to the follow-up treatment and disposal of excess sludge.Nano-copper oxide（CuONPs）as a typical nano-material,in addition to the toxicity caused by its own nano-properties,the bacteriostatic effect of Cu2+also makes it have a certain impact on the anaerobic digestion of excess sludge.Therefore,how to treat and dispose of the excess sludge polluted by CuONPs is an urgent problem to be solved at present.In this study,vermicomposting was used to treat excess sludge contaminated by CuONPs.Four groups of composting experiments were conducted with four concentration gradients of CuONPs（control group）,500,1000 and 2000mg/kg-TSS（numbered V0,V500,V1000 and V2000）.By investigating the physiological adaptability of earthworms,the physical and chemical properties of composting products and the toxicity reduction of Cu,the feasibility of vermicomposting of excess sludge contaminated by CuONPs was evaluated,and the effects of CuONPs on the composting efficiency of sludge vermicomposting were discussed based on the nutrient elements of nitrogen and phosphorus,the degree of humization and the activity of key enzymes in the substrate.The stress response mechanism of microorganisms and earthworms in sludge earthworm composting to CuONPs toxicity was studied by means of high throughput sequencing and Labelfree macroproteomics technology.the main results are as follows:（1）The average monomer weight of earthworms in V0,V500,V1000 and V2000composting groups increased with the composting process.This shows that earthworms can still grow on organic matter in sludge under the stress of CuONPs.After the end of composting,the increase of the average monomer weight of earthworms and the number of earthworms were negatively correlated with the concentration of CuONPs,that is,the increase of CuONPs concentration could also enhance the toxic effect on the physiological activities of earthworms and reduce the number of individuals,but the survival rate of earthworms in V2000 composting group was still about 68.3%after composting.The above results show that earthworms have a certain tolerance to the toxicity of CuONPs in excess sludge.（2）After the end of composting,TOC and DOC in the composting substrate of the four groups of V0 and V500,V100 and V2000 were degraded obviously.The degradation rates of DOC were 79.7%,71.5%,67.7%and 72.6%,respectively,and the degradation rates of 55.3%,47.0%,43.5%and 45.6%,respectively,which met the requirement that the degradation rate of organic matter in excess sludge treatment should be higher than 40%.The three-dimensional fluorescence spectrum and parallel factor analysis of water-soluble organic matter also showed that the humification degree of substrate after composting was significantly improved.After composting,most of the forms of Cu in the matrix were stable in bound state and residue state.Which indicates that the toxicity of CuONPs is reduced to a great extent.This shows that it is feasible to use vermicomposting to treat excess sludge polluted by a certain concentration of CuONPs.（3）After the end of composting,the contents of total phosphorus,nitrate nitrogen and available phosphorus in the composting substrate of V0meme V500 V100 and V2000 groups increased,and the increases of total nitrogen and available phosphorus in V500 experimental group were 41%and 35.36%,respectively.And the nitrification in the substrate is obvious.In the composting experiment of the four groups,the humus E4/E6 decreased from 5.11 in the initial state to 4.48,4.73,4.72,4.68 after the end of composting,respectively,and the degree of humification increased,but the activity analysis of key enzymes in composting matrix showed that dehydrogenase and urease in V1000 and V2000 composting groups were inhibited to a certain extent.The above results show that in the vermicomposting experiment carried out below the CuONPs concentration of 2000 mg/kg-TSS,the nutrient elements in the matrix and the degree of humification are improved,but in order to achieve a better composting effect,the concentration of Cu ONPs in the substrate should be controlled below500mg/kg-TSS.Within this concentration range,the fertilizer efficiency of vermicomposting products will not be significantly affected by the treatment of excess sludge polluted by Cu ONPs.（4）The presence of CuONPs changed the microbial community structure in the composting substrate.Qualcomm sequencing showed that the abundance and diversity of microbial community increased under CuONPs stress;based on Venn map analysis,it was found that the long-term existence of CuONPs in the substrate changed the species composition of microorganisms in the substrate,and the dominant microorganism in V0 was Proteobacterium,while under higher CuONPs concentration stress（V1000 and V2000）,the dominant microorganism in the substrate evolved into actinomycetes.（5）The stress response of earthworms in composting to Cu ONPs stress was analyzed based on macroproteomics.After composting,there were great differences in the protein expression of earthworms among the four groups.Under the condition of fold-change>2.0,11differential proteins were screened in V500,12 differential proteins in V1000 group and 13differential proteins in V2000 group.Protein differential GO enrichment analysis showed that earthworms changed their molecular function,biological process and cell composition in order to resist the toxicity of CuONPs.The subcellular localization of differential proteins showed that the differential proteins were mainly plasma membrane proteins and mitochondrial proteins,reflecting the defense mechanism of earthworms to the external environment and intracellular stress. |
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