Study On Enhanced Treatment Of Mariculture Wastewater By Embedding Immobilized Aerobic Denitrifying Bacteria

In the high-density and intensive circulating recirculating aquaculture system（RAS）,the nitrogen-containing pollutants mainly include ammonia nitrogen（NH₄⁺-N）,nitrite nitrogen（NO₂^--N）and nitrate nitrogen（NO₃^--N）.Biological aerated filter（BAF）in RAS can convert NH₄⁺-N and NO₂^--N with high toxicity into nitrate nitrogen（NO₃^--N）with low toxicity.However,under aeration conditions,the growth and function of common denitrifying bacteria are inhibited,resulting in nitrate accumulation in water,which will still lead to slow growth and reduced survival rate of fish.Therefore,it is of great significance for healthy aquaculture to strengthen the denitrification of microorganisms in BAF and control the concentration of NO₃^--N in RAS at a lower concentration.In order to enhance the denitrification efficiency of the system by adding denitrifying functional bacteria,the following studies were set up in this paper.The compound agent was prepared by separating and purifying high-efficiency salt-tolerant aerobic denitrifying bacteria.The optimum composition was discussed and the growth conditions with excellent denitrification performance were obtained.The compound bacterial agent was embedded by polyvinyl alcohol（PVA）and sodium alginate（SA）.And the optimum preparation conditions and denitrification conditions were determined.The embedded compound bacteria pellets were put into the seawater biofilter to treat mariculture wastewater,which improved the denitrification performance of the system.This study will provide a theoretical basis for the application of immobilized aerobic denitrifying bacteria in seawater biofilter.The main conclusions of this study are as follows:（1）Salt-tolerant compound aerobic microorganisms with denitrification function can promote the reduction of nitrate nitrogen（NO₃^--N）under aerobic conditions.In this study,a salt-tolerant aerobic denitrifier（Zobellella sp.MAD-44）was isolated and combined with two salt-tolerant aerobic denitrifiers（Halomonas alkaliphila HRL-9 and Vibrio spp.AD2）for preparing the compound agent named AHM-M3.When the addition ratio of AD2,HRL-9 and MAD-44 was2:2:3（v/v/v）,the denitrification efficiency of AHM-M3 was the highest.AHM-M3 significantly removed more NO₃^--N than any single strain（i.e.,AD2,HRL-9 or MAD-44）（P<0.05）.Under high salinity（30）conditions,the optimized conditions for NO₃^--N reduction by AHM-M3 were30℃,inoculation amount of 2%（v/v）,shaking speed 120 rpm,and C/N ratio 10.100 mg/L NO₃^--N substrate was completely removed by AHM-M3 in 24 hours without nitrite nitrogen accumulation.Under the optimized conditions,17.07%and 19.80%of NO₃^--N was converted into intracellular nitrogen and other nitrogen-containing products,and 63.13%of NO₃^--N may be converted into gaseous nitrogen.These results revealed that the compound bacteria agent AHM-M3 had great potential in enhancing NO₃^--N reduction in high salinity and aerobic conditions.This study provides a potential application prospect for microbial enhancement technology in seawater pollution control,especially in mariculture wastewater treatment.（2）Based on the aerobic denitrification compound agent AHM-M3 prepared in the previous stage,it was embedded with PVA and SA.The optimum ratio and immobilization conditions of PVA-SA carrier were determined.Meanwhile,the effects of environmental factors on the denitrification performance of immobilized compound bacteria pellets were analyzed,so as to optimize the denitrification conditions.The results showed that under the conditions of PVA 10%,Sa 2%,Ca Cl₂ 2%and crosslinking time of 48 h,the immobilized compound bacteria pellets had excellent spheroidization,mass transfer and stability,and the highest removal efficiency of NO₃^--N（63.47%）was obtained at 24 h.The immobilized pellets have macroporous structure and are suitable for the attachment and growth of strains.The immobilized compound bacteria agent has excellent degradation ability for nitrate simulated aquaculture wastewater.The optimum conditions for NO₃^--N reduction were as follows:the dosage of immobilized pellets was 2%（w/v）,pH 7,30℃.The immobilized compound bacteria completely removed NO₃^--N in 48 h,with no accumulation of NO₂^--N in the system,and the concentration of NH₄⁺-N remained low.The immobilized compound bacteria showed acid and alkali resistance and temperature tolerance.After the immobilized compound bacteria pellets were reused for three times（9 d）,it could still completely remov NO₃^--N from wastewater.It has efficient denitrification ability and stable structure,which provides the possibility of practical engineering application in aquaculture wastewater treatment.（3）In this study,immobilized aerobic denitrifying compound bacteria pellets and biological filter filler were used to form composite filler,and the biological filter group was set as the control group.They were respectively filled into the seawater biofilter to study the effect of the addition of immobilized pellets on the removal efficiency of NO₃^--N and TN in mariculture wastewater.It was found that embedding immobilized strains can enhance the removal efficiency of NO₃^--N and total nitrogen（TN）in aquaculture wastewater,up to 100%and 87.99%respectively.The community structure and relative abundance had changed significantly,especially at the phylum and genus levels.The abundance of Proteobacteria related to denitrification was the highest.The successful enrichment of Halomonas sp.and Zobellella sp.embedded in immobilized pellets may be one of the reasons for the improvement of nitrogen removal efficiency of the system.This may confirm that the embedding immobilization technology enhances the denitrification of the system.