Research On The Mechanism Of Copper And Sulfonamides In Livestock Wastewater Treated By Microalgal-Fungal Co-Cultivation

The large-scale and intensive way of livestock and poultry produced a large amount of wastewater.The wastewater contained high concentrations of chemical oxygen demand（COD）,ammonia nitrogen（NH₄⁺-N）,total nitrogen（TN）,total phosphorus（TP）,heavy metals and antibiotics.Most of the biological treatment technologies based on microalgae were aimed at the removal of C,N and P from livestock wastewater,while there were few studies on heavy metals and antibiotics.Therefore,three common sulfonamides（SAs）and Cu（Ⅱ）in livestock wastewater were selected as the main research objects.The microalgae single culture system,microalgal-fungal pellets and microalgal-fungal spores co-cultivation system were used to explore the removal performance of SAs and Cu（Ⅱ）,and to further study the removal mechanism and biodegradation pathway of SAs.The main results of this study were as follows:（1）The effects of different concentrations of SAs and Cu（Ⅱ）on Chlorella vulgaris were studied.It was found that SAs at concentrations of 100-400μg/L inhibited the growth of C.vulgaris with the inhibition rate of 3.98%-17.08%.When combined with Cu（Ⅱ）,the inhibition rate was obviously increased.SAs and Cu（Ⅱ）affected the growth and photosynthetic efficiency of C.vulgaris,resulting in changes of protein and polysaccharide contents.Under the stress of SAs and Cu（Ⅱ）,the antioxidant enzymes could alleviate the oxidative damage of cells by increasing the enzymatic activities of SOD and CAT.After 14-day treatment,the removal efficiencies of C.vulgaris to 100μg/L SMX,SMZ and SMM were 54.34%,49.77%and 36.41%,respectively.The addition of 200μg/L Cu（Ⅱ）increased the removal efficiencies of SMX,SMZ and SMM,most of which（>90%）were removed by biodegradation.（2）The adsorption effect and mechanism of microalgal-fungal pellets on Cu（Ⅱ）and SAs were studied.The filamentous fungus A.oryzae could form fungal pellets within 48 hours.After the condition optimization experiment,the fungal pellets could completely combine to C.vulgaris within 5 hours,and the maximum combineation efficiency was 98.65%.Through the adsorption kinetic model analysis,the adsorption of SAs by the microalgae-fungal conformed to a nonlinear pseudo-first-order kinetic model.The equilibrium adsorption of SMZ,SMM and SMX by microalgae-fungal pellets increased from 1.07 to 2.30 mg/g,0.94 to 1.82 mg/g,and 1.67 to 2.93 mg/g,respectively.Through Fourier transform infrared spectroscopy analysis and scanning electron microscope（SEM）images,it was speculated that the presence of Cu（Ⅱ）enhanced the adsorption of SAs by microalgae-fungal pellets mainly through ion exchange and adsorption bridging.Through excitation-emission matrix spectroscopy-parallel factor analysis（EEM-PARAFAC）,the dissolved organic matter（DOMs）released by the microalgal-fungal pellets were mainly protein-like substances,humic-like substances and NADH.The increase of Cu（Ⅱ）concentration distinctly increased the fluorescence intensity of protein-like substances and NADH,which formed complexes with Cu（Ⅱ）to relieve the damage of Cu（Ⅱ）to organisms.（3）The effect of microalgal-fungal spores co-cultivation system on the removal of pollutants in simulated livestock wastewater was studied.In comparison to the single system of C.vulgaris,the removal efficiencies of co-cultivation system were 71.19%,72.51%,92.23%and 91.47%for NH₄⁺-N,TN,TP and COD,respectively.In addition,the removal efficiencies for SMZ,SMM,SMX and Cu（Ⅱ）were 57.60%,50.48%,58.31%and 90.37%,respectively.The maximum harvesting efficiency of C.vulgaris reached 76.91%when the inoculation ratio of C.vulgaris and A.oryzae spores was 10:1.From SEM,it was found that the hyphae of C.vulgaris and A.oryzae were entangled with each other to form a complex spatial three-dimensional aggregate,which provided the basis for gas and nutrient exchange.The co-cultivation system not only increased the total biomass,the maximum biomass was up to 1.26g/L,but also increased the lipid content,up to 258 mg/g.The fatty acid components in C.vulguris were mainly palmitic acid（C16:0）,oleic acid（C18:1）and linoleic acid（C18:2）,while the proportion of total saturated fatty acids increased obviously in the co-cultivation system.（4）The effect of Cu（Ⅱ）on the removal of NH₄⁺-N,TN,TP,COD and SAs in simulated livestock wastewater by microalgal-fungal spores co-cultivation system was studied.When the concentration of Cu（Ⅱ）was 0.2 mg/L,the maximum removal efficiencies of NH₄⁺-N,TN,TP and COD were 79.19%,76.18%,92.92%and 93.29%,respectively.At the same time,the removal efficiency of SMZ,SMM and SMX in the co-cultivation system was also evidently improved,reaching 63.9%,58.76%and 63.51%,respectively.Based on the EEM-PARAFAC,the DOMs released from the co-cultivation system were analyzed,and there were three main components（humic-like substances,terrestrial humic-like substances and protein-like substances）.Through the analysis of fluorescence parameters,Cu（Ⅱ）had litter effect on HIX,indicating that the microorganisms in the co-cultivation system still maintained a high biological activity.Based on high performance liquid chromatography tandem mass spectrometry（HPLC-MS/MS）technology,the biodegradation products of SMZ,SMM and SMX in the co-cultivation system were analyzed and the degradation pathways were speculated.There were 8 kinds of degradation products of SMZ with 3 degradation pathways inferred.As for SMM,there were 5 kinds of degradation products and 2 degradation pathways were speculated.Meanwhile,there are 6 kinds of degradation products of SMX,and 2 degradation pathways were inferred.（5）The molecular response mechanism of C.vulgaris and A.oryzae in the co-cultivation system was studied under the single effect of SAs and the combined effect of SAs+Cu（Ⅱ）.Compared with the control,there were a total of 251 differentially expressed genes（DEGs）of C.vulgaris under SAs alone,of which 228 were up-regulated and 23 were down-regulated.Meanwhile there were a total of 2653 DEGs of C.vulgaris under SAs+Cu（Ⅱ）combined effect,of which 1394 were up-regulated and 1259 were down-regulated.Through GO and KEGG enrichment analysis,it was found that SAs ovbiously affected the amino acid metabolism,lipid metabolism and sugar metabolism of C.vulgaris.It was speculated that the toxicity mechanism of SAs to C.vulgaris was through affecting the metabolism and synthesis of basic substances.Furthermore,the synthesis and metabolism of nutrients such as carbohydrates,lipids,and proteins,as well as genetic information in cells were also affected.Under the combined effect of SAs+Cu（Ⅱ）,DEGs in the photosynthesis metabolic pathway were clearly enriched and most genes were up-regulated,indicating a significant effect on C.vulgaris photosynthesis.In addition,through the analysis of peroxisome metabolic pathway,the protective mechanism of C.vulgaris in response to SAs+Cu（Ⅱ）stress was related to the up-regulated expression of SOD synthesis-related genes.Transcriptomic analysis of A.oryzae found that there was a total of 196 DEGs under SAs alone,of which 109 were up-regulated and 87 were down-regulated in comparison to the control.Meanwhile there was a total of 2162 DEGs of A.oryzae under SAs+Cu（Ⅱ）combined effect,of which 811 were up-regulated and 1351 were down-regulated.In the GO enrichment analysis,significant upregulation of monooxygenase and oxidoreductase-related genes was found,which was involved in the biodegradation of SAs by A.oryzae.Through KEGG enrichment analysis,the significant down-regulation of genes involved in amino acid and fatty acid metabolism,which was the protective or resistance mechanism of A.oryzae in response to SAs stress.The addition of Cu（Ⅱ）significantly increased the expression of genes related to ion transmembrane transport,which affected the uptake and accumulation of Cu（Ⅱ）by membrane transporters.The enrichment analysis of protein processing in the endoplasmic reticulum revealed that 25 DEGs were significantly enriched in this metabolic pathway and 23 were up-regulated,which was related to the accumulation and degradation of SAs+Cu（Ⅱ）cells by A.oryzae.