Screening Of Denitrifying Bacteria In Low Temperature And Research On Its Application

Denitrifying bacteria are a group of bacteria which perform denitrification with denitrifying enzymes.Denitrification is a key microbial approach to nitrogen regulation and control.Nitrate becomes the most ubiquitous chemical contaminant in groundwater due to excessive utilization of nitrogenous fertilizer in agricultural activity and inappropriate disposal of untreated industrial waste water.Nitrogen removal is the emphasis and difficulty of sewage disposal and purification.Biological denitrification is the most economical and effective among wastewater treatment methods.Therefore,denitrifiers aroused widespread concern and research.Lake eutrophication is quite serious in our country and nitrogen is an important contributing factor to such phenomena.Winter and low temperatures have much influence on biological denitrification.Based on denitrification characteristics of the denitrifying bacteria and low temperature limitation in winter,we obtained a group of microorganisms with high denitrification ability after several liquid enrichment processes,and four strains of denitrifying bacteria were screened and isolated from eutrophic water.Physiological and biochemical properties of the four denitrifying bacteria ere studied and in-depth research was carried out on denitrification capability of these four strains.Denitrification efficiency of denitrifiers after immobilization and under low temperature conditions were investigated from practical engineering application perspectives.Artificially hanged membrane with biocord padding from the obtained strains was studied and was then added to artificial wetlands for practical application research.Main results are as follows:1.Denitrifiers with strong denitrification ability were obtained from eutrophic lakes and the removal rates of nitrate and nitrite by these strains were investigated.2.Four strains of aerobic denitrifiers were isolated from denitrifying community and were named N1,N2,N3,and N4 respectively.According to their culture characteristics,physiological and biochemical properties as well as 16SrDNA sequence homology analysis,N1 and N2 belong to Achromobacter sp.while N3 and N4 are Pseudomonas stutzeri.3.Flask-shaking and stationary culture experiments revealed that the four strains could remove nitrate and nitrite efficiently under anaerobic and aerobic conditions.Their denitrification capability under different C/N ratios,nitrate concentrations and temperatures were also investigated.C/N ratios were set as 4,8 and 12.Nitrate concentrations were set as 20,30,40,50,60,70,80 and 90 mg/L and temperatures were set as 4,10,15,20,30,40,50,and 60?.Results indicated that N1 and N3 can still perform denitrification under temperature as high as 50? and the nitrogen removal rate can reach 100%after 12 hours.Nitrite rarely accumulated during this process and there was no secondary pollution.Denitrification ability of the two strains still exist under temperatures of 4 ? and 60?.The two strains can tolerate some low and high temperatures and therefore can be applied in some extreme environments.4.Immobilization effect was studied with different immobilization carriers and immobilization parameters were optimized.Immobilized aerobic denitrifiers with high mechanical strength and denitrification ability were obtained accordingly.Denitrification performance of immobilized microbes was studied and lab-scale batch sequencing experiments were carried out under low temperatures.Immobilized microorganisms exhibited higher environmental tolerance and denitrification capability compared with free microorganisms.Batch experiments also indicated that immobilized microbial spherules have higher denitrification efficiency and better application prospects even under low temperatures in winter.5.Comprehensive comparison on growth rate and nitrogen removal efficiency of the four strains was performed and N3 was selected in the lab-scale artificially hanged biofilm research.Membrane-hanging effects with various inoculum doses(initial inoculum doses were set as 100%,80%,50%,25%respectively)were studied,showing that 25%is the optimal inoculum dose.6.Artificially hanged membrane with biocord padding was added to artificial wetlands and then field experiments were carried out.Short-term monitoring results showed slight reduction in nitrate concentration and scarce nitrite accumulation.Ammonium nitrogen concentration in the effluent decreased and orthophosphate removal rate reached approximately 50%.In conclusion,denitrifying bacteria attached to artificial biological stuffing can remove nitrogen efficiently through denitrification at low temperatures,thus lowering nitrogen concentration in the water and improving environmental quality.