Study On Biological Nitrogen Removal Characteristics Of Bacteria Capable Of Denitrification In Low Carbon Nitrogen Ratio Condition

In this study,a denitrifying strain capable of high-efficiency biological denitrification under low carbon to nitrogen ratio（C/N）conditions was screened and isolated from the sediments of a landscape water body in Baoji City.The biological nitrogen removal characteristics under low carbon-nitrogen ratio conditions were identified and studied.At the same time,the film-forming products on the effect of nitrogen removal were studied in combination with the characteristics of bacteria’s aggregation under low carbon nitrogen ratio conditions.The 16Sr DNA strain identification results showed that the bacteria belonged to the genus Comamonas sp.And it was named YSF15.Under the condition of C/N=2.5,the strain YSF15 showed good biological nitrogen removal ability,and it could remove94.01%of nitrate within 18 h without the accumulation of nitrite.Among the three common carbon sources,glucose,sodium acetate and sodium succinate,the best carbon source suitable for the biological denitrification of strain YSF15 is sodium acetate;setting different pH gradients to investigate the effect of environmental acidity and alkalinity on the nitrogen removal of the strain,The results showed that during the whole denitrification cycle,the removal rate of nitrate in alkaline conditions was higher than that in acidic conditions;three inoculation gradients of 1%,5%and 10%（V/V）were selected.To investigate the optimal denitrification rate,the corresponding nitrate removal rates were 40.12%,61.73%,and 93.83%.Different temperatures were set to evaluate the effect of environmental temperature on the biological nitrogen removal of the strains.The results showed that 30℃ It is closer to the optimal reaction temperature of YSF15.A response surface（RSM）experiment was performed to further optimize the biological denitrification conditions of the strain.Three variable levels were designed:initial hydrochloride concentration（15,25,35 mg/L）;temperature（20.00,30.00,40.00℃）and pH（5.00,7.00,9.00）,with the nitrate removal rate as the response,the final analysis shows that under the low carbon-nitrogen ratio environment,the optimal denitrification condition of strain YSF15 is:initial nitrate concentration of 21.92 mg/L,temperature 29.98℃,pH=8.34.A nitrogen balance experiment under low carbon-nitrogen ratio was conducted to study the nitrogen removal pathway of strain YSF15.The results showed that the intracellular nitrogen content before and after the reaction was 1.07±0.05 mg/L and1.12±0.02 mg/L,respectively,without significant changes.NH₄⁺-N was not observed at the same time,indicating that cell assimilation is not the main denitrification pathway of YSF15.Therefore,three key genes in the process of denitrification such as nitrite reductase（Ni R）,nitric oxide reductase（No R）and nitrous oxide reductase（No S）were selected for PCR amplification experiments.The PCR amplification results showed that there were three genes,nir S,nor B and nos Z,in strain YSF15,which could preliminary prove that the biological nitrogen removal of strain YSF15 follows the NO₃^--N→NO₂^--N→NO→N₂O→N₂ pathway.Subsequently,the role of bacterial extracellular polymers（EPS）in bacterial low-carbon-to-nitrogen biological denitrification was discussed.The results showed that the main components of bacterial extracellular polymer（EPS）released by strain YSF15 were polysaccharides and proteins.In the process of bacterial biological denitrification,the release of EPS content is inversely proportional to the concentration of nitrate,and lags behind the change in nitrite content,which promotes the reduction of nitrite.Subsequently,different nutrient gradient conditions（nitrate concentration and C/N）were set to explore the changing rules of EPS under different nutritional conditions.The results showed that when the nutrients were continuously enriched,the polysaccharide and protein content showed a downward trend.The polysaccharide content decreased from 10.44 mg/L to 4.72 mg/L,and the protein content decreased from 3.12 mg/L to 1.79 mg/L.With the gradual decrease of C/N（from 5:1 to 1:1）,the polysaccharide content in EPS increased from 3.58 mg/L to17.09 mg/L,and the protein content increased from 2.31 mg/L to 9.26 mg/L.Afterwards,in order to prove that the EPS released by the strain can indeed be used as an alternative electron donor to help denitrification,a denitrification experiment was performed using EPS extract as a carbon source（without adding external organic carbon sources）,and the results showed that the nitrate concentration was from 23.56 mg/L will reach 13.12mg/L,confirming the role of EPS in replacing carbon sources.At the same time,the scanning electron microscope and fluorescence spectrophotometer were also used to observe the changes in EPS content during the above experiments.During the cultivation of strain YSF15,it was observed that a film-like substance with a certain mechanical strength and capable of moving back and forth in solution appeared in the culture device.It is preliminarily judged that the substance may be a bacterial aggregate produced by strain YSF15 releasing EPS in large quantities.First,the nitrate removal performance of this film-like substance was studied,and the results showed that the nitrate content gradually decreased from the initial 17.46 mg/L to 6.78mg/L,and the removal rate reached 61.17%,confirming the denitrification effect.Subsequently,the aggregation membrane was collected into a culture flask,and three influencing factors were set.The C/N gradient was 2:1 and 2.5:1 and 3:1;the HRT gradient was 2 h,4 h,and 6 h;the nitrate concentration gradient for 15,20,25 mg/L,run for 27 cycles to observe its nitrogen removal performance.The results show that the reactor is in good operating conditions throughout the cycle.With C/N=2 and a nitrate concentration of 20 mg/L,more than 90%of nitrate removal can be achieved in 4 hours.When HRT=6 h,effective removal of nitrate can be achieved under various working conditions;but when HRT is reduced to 2 h,the removal rate drops to about 70%.And there was no significant accumulation of nitrite throughout the entire operating cycle.Subsequently,the thirteenth cycle was selected as the steady-state condition for operation.The setting conditions were:carbon-to-nitrogen ratio=2.5:1,initial nitrate concentration of 20 mg/L,and HRT=6 h.During steady-state operation,the average nitrate removal rate was 95.22%,the average nitrite conversion rate was 1.56%,and the maximum nitrate removal rate was 95.97%(3.42 mg·L^-1·h^-1).The average nitrite content in the effluent throughout the stabilization period was 0.32 mg/L,and no nitrite accumulation occurred,achieving stable operation under low carbon-nitrogen ratio conditions.High-throughput sequencing analysis showed that,with the continuous operation of the reactor,the core strain YSF15 represented Comamonas sp.,showed good adaptability and increased content,thus supporting the removal of nitrate in the reactor under low C/N conditions.