Characteristics And Mechanism Of Halotolerance And Nitrogen Removal Of A Novel Aerobic Denitrification Bacterium

Due to the excessive discharge of nitrogenous wastewater into the water body,the eutrophication is becoming even more serious.Therefore,nitrogen removal of wastewater has become a hot issue in the field of water pollution control.At present,the most widely used nitrogen removal technology is the traditional biological nitrogen removal technology,which contains two parts of aerobic autotrophic nitrification and anaerobic heterotrophic denitrification.However,due to the different requirements for carbon source and oxygen of aerobic tank and anoxic tank,nitrification and denitrification processes need to be carried out separately.In recent years,the discovery of some novel denitrification bacteria with heterotrophic nitrification and aerobic denitrification capblility provides new ideas for biological nitrogen removal of wastewater in the same aerobic environment,and is expected to solve the disadvantages of traditional biological nitrogen removal technology.However,the heterotrophic nitrification and aerobic denitrification bacteria that have been isolated at present mainly come from the fresh water and low salinity environment,which are difficult to effectively treat the high saline wastewater discharged by pharmaceutical,tannery,and food processing industries etc.Therefore,screening the heterotrophic nitrification and aerobic denitrification bacteria with halotolerant capacity and investigating the nitrogen removal characteristics and mechanism and salt resistance characteristics and mechanism are of great significance to improving the nitrogen removal efficiency of saline organic wastewater at aerobic environment.The main results are as follows:（1）After activation,enrichment,and repeated acclimation,the saline activated sludge samples taken from pharmaceutical wastewater treatment system were used to isolate the halotolerant heterotrophic nitrification and aerobic denitrification bacteria with salt resistance capability up to 15%.Among the isolated bacteria,strain Y5exhibited the highest ammonia nitrogen and nitrate nitrogen removal capability and was selected as the dominant strain for further study.After morphological observation,physiological and biochemical testing,16S r DNA sequencing and homology analysis,strain Y5 was proved to be Halomonas salifodinae.The 16S r DNA sequences of strain Y5 were deposited into the Gen Bank database with Gen Bank ID#OK559719.（2）Single-factor experiments indicated that the optimal heterotrophic nitrification and aerobic denitrification nitrogen removal conditions of strain Y5 were sodium succinate as carbon source,C/N ratio of 15,initial pH value of 7～8,culture temperature of 30℃,and shaking speed of 150～200 rpm.Furthermore,strain Y5 can remove above90%of ammonia nitrogen and nitrate nitrogen at salinity of 0～15%and initial nitrogen source concentration of 50～400 mg/L,exhibiting excellent heterotrophic nitrification and aerobic denitrification capability.Based on the single-factor experiments,response surface methodology（RSM）was used to further analyze the effects of environmental conditions on ammonia nitrogen and nitrate nitrogen removal of strain Y5.The results showed that strain Y5 exhibited the highest ammonia nitrogen removal efficiency of98.45%at C/N ratio of 15.56,initial pH value of 7.57,culture temperature of 28.86℃,and shaking speed of 190 rpm.At C/N ratio of 17.19,initial pH value of 7.53,culture temperature of 33.29℃,and shaking speed of 206 rpm,strain Y5 showed the highest nitrate nitrogen removal efficiency of 97.67%.（3）When ammonia nitrogen,hydroxylamine,nitrite nitrogen,and nitrate nitrogen were used as single nitrogen source,the removal of ammonia nitrogen,hydroxylamine and nitrogen nitrate was relatively fast,corresponding to the maximum nitrogen removal rates of 6.77,6.78 and 5.56 mg/（L·h）,and the removal of nitrite nitrogen was slow,with a maximum nitrogen removal rate of 2.77 mg/（L·h）.When ammonia nitrogen,hydroxylamine,and nitrate nitrogen were used as single nitrogen source,nitrite nitrogen was accumulated and was removed subsequently.When ammonia nitrogen,nitrite nitrogen,and nitrate nitrogen were used as mixed nitrogen,strain Y5 preferentially removed ammonia nitrogen and nitrate nitrogen.When ammonia nitrogen and nitrate nitrogen were almost completely removed,strain Y5 began to remove nitrite nitrogen.（4）the nitrifying and denitrifying functional genes were successfully amplified from the genomic DNA of strain Y5,including amoA gene,hao gene,napA gene,and nirS gene.In addition,the enzyme activity of ammonia monooxygenase（AMO）,hydroxylamine oxidase（HAO）,nitrite reductase（NIR）,and nitrate reductase（NAR）were detected as well.Combined with the utilization of different nitrogen sources by strain Y5,the detection of nitrogen removal functional genes,and the determination of nitrogen removal key enzyme activity,the proposed heterotrophic nitrification and aerobic denitrification nitrogen removal pathway of strain Y5 was:ammonia nitrogen was converted into hydroxylamine under the action of AMO,then hydroxylamine was converted into nitrite nitrogen under the action of HAO,meanwhile,nitrate nitrogen could also be converted to nitrite nitrogen through NAR,finally,nitrite nitrogen was reduced to gaseous nitrogen under the action of NIR,revealing the nitrogen metabolic pathway and mechanism of strain Y5 at high salinity environment.（5）Strain Y5 can effectively remove ammonia nitrogen in the medium containing NaCl,Na₂SO₄,KCl,and K₂SO₄ respectively with the salinity range of 3%～10%.Among the different kinds inorganic salts,the inhibition of nitrogen removal capability of strain Y5 is in the order of Na₂SO₄>K₂SO₄>NaCl>KCl.The heavy metal salts including Cr（Ⅵ）,Mn（Ⅱ）,Ni（Ⅱ）,Cu（Ⅱ）,and Zn（Ⅱ）showed the inhibition effect on nitrogen removal capability of strain Y5,while Fe（II）showed promotion effect on nitrogen removal of strain Y5.The inhibition degree of heavy metal salts on nitrogen removal capability of strain Y5 was in the order of Cu（Ⅱ）>Ni（Ⅱ）>Cr（Ⅵ）>Zn（Ⅱ）>Mn（Ⅱ）.（6）The intracellular metabolites of strain Y5 cultured repeatedly at 0%,5%,10%and 15%salinities were extracted through repeatedly freeze-thaw.Then the intracellular metabolites were detected by gas chromatography-mass spectrometry,and the detection results were analyzed by metabolomics.It can be found that glutamic acid,D-proline,and trehalose are higher after strain Y5 cultured at 5%,10%,and 15%salinities.While3-hydroxybutyric acid,2,4-dihydroxybutanoic acid,and histamine are higher after strain Y5 cultured at 0%salinity.Therefore,it can be proposed that strain Y5 mainly accumulates glutamic acid,D-proline and trehalose as intracellular metabolites to resist the external osmotic pressure in the saline environment,elucidating the salt tolerance mechanism of strain Y5 at high saline environment.