Application Research On Cell Immobilization Technology Based On Spent Mushroom Substrate In Wastewater Treatment

Since the 21st century,on the one hand,due to the accumulation of nitrogen in water,the water pollution problems are becoming more and more serious.Thus,how to find an effective denitrification method is imminent.On the other hand,As an agricultural waste,spent mushroom substrate?SMS?are produced in large quantities every year in the world,which has incurred a serious problem to the loacl environment.Immobilized cell technology has attracted much attention in recent years in the field of nitrogen pollution removal in water with its unique advantages.In this study,Acinetobacter sp.TX5 isolated from a sequencing batch bioreactor?SBR?was used as an experimental strain,and fixed TX5 with SMS to treat piggery wastewater,with the aim of exploring its denitrification performance and nitrogen metabolism pathways.Meanwhile,the preparation method of the immobilized pellets was optimized,so that it has a higher sewage treatment effect while maintaining its own strong stable performance,hopping to lay the foundation for TX5based on the SMS"using pollutants to treat pollutants"treatment method.Firstly,we isolated and purified the aerobic denitrification TX5 from the SBR activated sludge.After 16S r RNA gene amplification and identification,it was found that the strain TX5 has 99%homology with Acinetobacter genus.Then Acinetobacter TX5 was immobilized to treat piggery wastewater based on SMS,through shake flask and fixed bed experiments.They can adapt to the real piggery wastewater environment in a short time.Especially during the fixed bed operation,the removal efficiency?RE?of ammonia nitrogen at 12 h reached 50%,and afterwards increased to 96%after 96 h.The RE of total nitrogen can eventually remain above 90%.Secondly,the important intermediate products of TX5 in the simultaneous nitrification and denitrification processes,such as hydroxylamine and nitrite were detected using the ¹⁵N isotope analysis during the process of nitrogen removal.Then,a nitrite reductase with a molecular weight of 65 k Da was successfully obtained from TX5 by isolation and purification.Combining isotope with enzymatic properties analysis,we can preliminary speculate that the main nitrogen metabolism pathway of TX5 is that ammonia nitrogen forms hydroxylamine under the action of ammonia monooxygenase,then forms nitrite through hydroxylamine oxidase,and then gradually generates gaseous products under the action of nitrite reductase.Subsequently,using sodium alginate?SA?and polyvinyl alcohol?PVA?as the main fixed carrier,the preparation process of immobilized pellets was optimized by response surface test.The final determination of the embedding material mix was:PVA 6.30%,SMS 2.90%and TX5 4.50%.The single-factor optimization resulted in a cross-linking time of 24 hours and a cross-linking agent Ca Cl₂ concentration of 2%.On the basis of optimized conditions,the continuous treatment of actual piggery wastewater through a fixed-bed column was conducted,the final RE of ammonia nitrogen and TN exceeded 96%and 85%,respectively.This showed that the immobilized Acinetobacter TX5 based on SMS has a good application prospect for the treatment of nitrogen-containing wastewater.Finally,through a fixed bed treatment of simulated nitrite-containing wastewater,it was found that the RE of NO₂^--N reached above 90%at 50h,and the RE of TN reached above 85%after 96 h,further illustrating that TX5 has a higher nitrite reductase activity,and TX5 based on SMS has the ability to treat different wastewater.After that,PCR amplification by degenerate primers,gel electrophoresis detection and BLAST analysis showed that TX5 has a nos Z gene,and the nitrous oxide reductase with a molecular weight of 70.05 k Da was successfully isolated and purified from TX5 strain.The denitrification pathway existing in TX5 strain was further improved,that is,the gaseous products produced by NO₂^--N under the action of nitrite reductase will eventually be reduced to N₂ and discharged out of the cell after a series of reactions.