Nitrogen Removal Process And The Effect Of Metals On Its Nitrogen Removal Capacity Of Arthrobacter Arilaitensis Y-10

The microbial remediation is a green,efficient and economical technology in the nitrogen pollution wastewater treatment strategies.The microbial remediation technology for nitrogen-contaminated wastewater has been rapidly developed after the discovery of aerobic denitrifying bacteria.The aerobic denitrifying microorganisms are used for biological nitrogen removal in wastewater treatment,which has many advantages,such as:?1?fast growth and reproduction,short generation time and low culture cost;?2?no need to add additional acid-base regulator due to simultaneous nitrification and aerobic denitrification;?3?simultaneous removal of various inorganic nitrogen from wastewater system.However,the denitrification pathways of microorganisms are complicated.At present,few of studies focused on the denitrification pathways of the cold-tolerant microorganisms.Moreover,microbial denitrification could be affected by many environmental factors.The metal ions are ubiquitous in wastewater enviroments,which maybe affect the nitrogen removal efficiency by microorganisms.In the wastewater treatment systems,the effects of different metal ions on the biological nitrogen removal process are less systematically studied,especially for the cold-tolerant microorganisms.Based on the cold-tolerant aerobic denitrification strain Arthrobacter arilaitensis Y-10 obtained by separation and screening,the characteristics and pathways of nitrogen transformation strain Y-10 were invesitigated.The denitrification efficiencies under different metal ions shocks and the effect mechanisms of mental ions on nitrogen removal process of strain Y-10 were studied.The main results are as follows:1.The ability of Arthrobacter arilaitensis Y-10 for nitrogen removal from simulated wastewater was studied.Results showed that ammonium was the best inorganic nitrogen for strain Y-10's cell growth,which could also promote nitrate reduction.Approximately 100.0%of ammonium was removed in the nitrogen removal experiments.The nitrate removal efficiency was 73.3%with nitrate as sole nitrogen source,and then the nitrate efficiency was increased to 85.3%and 100.0%with ammonium and nitrate?both about 5 or 100 mg/L?as the mixed nitrogen sources.About 0.02 mg/L and 122.23 mg/L of nitrite accumulation were observed in presence of low and high mixed ammonium and nitrate medium.When the concentration of sole nitrite nitrogen was 10.31 mg/L,the nitrite removal efficiency was 100.0%.Neither ammonium nor nitrate was accumulated during the whole experimental process.All experimental results indicated that A.arilaitensis Y-10 could remove ammonium,nitrate and nitrite at 15?C from wastewater,and could also perform simultaneous nitrification and denitrification under aerobic condition.2.The enzymes activities for nitrogen transformation of the strain Y-10,total nitrogen removal,extracellular and intracellular nitrite nitrogen accumulation were evaluated.The results showed that no extracellular nitrite was detectable during ammonium oxidation process.When hydroxylamine was selected as sole nitrogen source,only 0.65 mg/L of extracellular nitrite and 1.89 mg/L of extracellular ammonium could be detected.In aerobic denitrification process,43.66 mg/L of extracellular nitrite was generated with initial 101.04 mg/L nitrate.About 2.15 mg/L of extracellular ammonium nitrogen was produced with initial 100.03 mg/L nitrite.with the enzymes assay,the enzyme activities of Ammonia monooxygenase?AMO?,Hydroxylamine oxidase?HAO?and nitrite reductase?NR?were 0.42,0.0014 and0.0049 U/mg protein.Additionally,the nitrite reductase could be completely inhibited by diethyldithiocarbamate?DDC?.Intriguingly,a large amount of nitrite was detected in the intracellular fluid by strain Y-10 whether inorganic or organic nitrogen used as sole nitrogen source.The concentration of intracellular nitrite was 43.0,42.26,39.94and 35.01 mg/L,and the corresponding bacterial protein concentrations were 532.30,537.15,741.54 and 718.54 mg/L,which were incubated by the nitrogen source of organic?Luria-Bertani medium,LB?,ammonium,nitrate and nitrite,respectively.The percentages of intracellular nitrite to bacterial protein were 8.08,7.37,5.38 and 4.87,respectively.These results confirmed that the highest concentration of intracellular nitrite was accumulated with the organic nitrogen addition,followed by ammonium and nitrate,then the nitrite was the least.Hitherto,no known biochemical mechanism has been able to explain the high concentration of nitrite was present in intracellular.The greenhouse of CO₂ and N₂O emission flux were successfully determined during heterotrophic nitrification and aerobic denitrification process at 15?.The results showed that the flux of CO₂ were 76.69,74.78 and 67.18 mg/L,whereas the flux of N₂O were only 0.06,0.08 and 0.84 mg/L in ammonium,nitrate and nitrite nitrogen treatments.These results demonstrated that the greenhouse of N₂O was far less than CO₂ during nitrogen removal process.Additionally,strain Y-10 has a low greenhouse gases contribution and possesd a potential for practical applications.Combined with the denitrification intermediates,nitrification and denitrification inhibitors and the production of N₂O,it is speculated that the heterotrophic nitrification pathway of strain Y-10 is ammonium nitrogen?hydroxyammonia?nitrite nitrogen?gaseous nitrogen.The aerobic denitrification pathway is nitrate Nitrogen?nitrite nitrogen?gaseous nitrogen.It is also clear that N₂O is not the main product of biological nitrogen removal by strain Y-10.3.This study investigated the impacts of alkaline-earth metals[Ca???,Mg???]and heavy metals[Zn???,Ni???]on nitrogen removal capacity of Arthrobacter arilaitensis Y-10.StrainY-10 was able to tolerate to 20 mg/L of Ca???and its ammonium removal efficiency was 100%.A Ca???concentration of 0.5 mg/L effectively promoted the total nitrogen removal when the wastewater contained nitrite.Mg???supplementation substantially enhanced the bacterial growth and nitrogen reduction.As Mg???concentrations increased from 0 to 2 mg/L,the ammonium,nitrate and nitrite removal efficiencies increased by 40.62%,69.91%and 64.68%,respectively.However,the nitrogen removal ability of strain Y-10 was sharply hindered by Zn???and Ni???.Nonetheless,the nitrogen removal was continuously conducted even when the Zn???concentration reached to 30 mg/L.Moreover,the ammonium and total nitrogen removal almost stopped at 8 mg/L of Ni???,and the denitrification capacity was lost when the Ni???concentration exceeded 1 mg/L.Our results demonstrated that Ca???and Mg???,especially Mg???,could significantly enhance the nitrogen removal capacity of Arthrobacter arilaitensis compared to Zn???and Ni???.4.The objective of this study was to determine the effect of different copper concentrations on ammonium,nitrate and nitrite removal performances of pure simultaneous nitrification and denitrification strain:Arthrobacter arilaitensis Y-10.The non-competitive inhibition model was employed to evaluate the 50%inhibition concentration(IC₅₀)of copper ion on the strain Y-10 for removing nitrogen.The experimental results indicated that the effects of the identical copper concentration on nitrification with ammonium and denitrification with nitrate or nitrite are almost the same for strain Y-10.Addition of 0.25 mg/L of copper ion could extremely inhibit the ammonium,nitrate and nitrite nitrogen removal ability for strain Y-10.Moreover,The IC₅₀ of copper ion on nitrate and nitrite removal efficiencies of strain Y-10 were 0.195and 0.090 mg/L.Additionally,it was found that trace amounts of magnesium ion could improve the cell growth,nitrogen removal efficiencies and copper ion resistance for strain Y-10.5.The objective of this study was to determine the effect of Cd???,Co???and Mn???on heterotrophic nitrification and aerobic denitrification of Arthrobacter arilaitensis Y-10.Both nitrogen removal efficiency and the 50%inhibition concentration of heavy metals were taken into account,and the order of the inhibitory effect was Co???>Cd???>Mn???.A 100%of ammonium removal efficiency was observed when the Cd???concentration was lower than 2.5 mg/L.The nitrate and nitrite removal nitrogen efficiencies were enhanced by 0.1 and 0.25 mg/L of Cd???,but hindered by more than 0.25 and 0.5 mg/L of Cd???,respectively.Conversely,Co???could strongly inhibit the biological nitrogen removal process,especially for the aerobic denitrification.The cell growth and nitrogen removal ceased immediately once the Co???was supplemented.In terms of Mn???,no conspicuous inhibition effect in nitrification process was obtained even when the concentration of Mn???supplemented as high as 30 mg/L.The nitrate and nitrite removal efficiencies were significantly improved by 0.5 mg/L of Mn???,and then dropped sharply along with the increase of Mn???.All results highlighted that the aerobic denitrification was more sensitive to the inhibition effects of Cd???,Co???and Mn???than heterotrophic nitrification.6.The transcriptome sequencing analysis of strain Y-10 was carried out with no manganese ions as the experimental control treatment.Compared with the control treatment,whether ammonium or nitrate nitrogen was used as the sole nitrogen source,after adding manganese ions,a large number of differential expressions were observed in the genes.In the GO annotation database,the six genes related to nitrogen metabolism,such as sdaA,sugE,glnA,NirS,nirK and nirB,were up-regulated by 0.77,0.48,0.35,5.17,0.90 and 0.88 times,respectively.Under the same conditions,ten genes related to nitrogen metabolism,such as hutH,aspA,ilvA,amt,nadE,narG,narH,narJ,narI and nirD,were down-regulated by 1.77,1.24,1.06,1.02,0.52,2.01,1.80,1.41,1.36,1.33 and 1.00 times,respectively.In the KEGG metabolic pathway,only the annotated NxrAB gene associated with nitrification showed a down-regulation effect,and the remaining genes involved in the nitrification process were not significantly affected.In the denitrification process,nitrate nitrogen was used as the nitrogen source.According to the control treatment?without adding manganese ions addition?,the expression levels of many genes in strain Y-10 occurred after the addition of manganese ions.The up-regulated gene types and gene numbers associated with nitrogen metabolism are higher than the down-regulated gene types and numbers.In the GO annotation database,ten genes of amt,glnA,hutH,nadE,sdaA,sugE,nirB,nirD,nirS and NarK were up-regulated by 3.07,1.82,1.73,1.72,0.67,0.38,3.25,1.84,0.86 and 0.83 times,respectively.The seven genes associated with nitrogen metabolism,such as aspA,glnA,ilvA,narI,narG,narH,and narJ,were down-regulated by 1.98,0.73,0.68,0.79,0.67,0.41,and 0.34,respectively.In the KEGG metabolic pathway,all genes involved in ammonium oxidation,including AmoCAB,Hao,and NxrAB,were unaffected.Then genes of NasB,NirBD,NRT,narK,nrtP,and nasA associated with nitrate metabolism were up-regulated.In the experiment of studying the removal efficiency of nitrite nitrogen,it was found that nitrite could be continuously transformed to gaseous nitrogen by strain Y-10 in the process of nitrate nitrogen conversion process.In this experiment,the nitrite reductase gene of nirB also showed an up-regulation effect.In addition,the glutamate synthase genes of gltB and gltD also showed an up-regulation effect.The effect of manganese ions on the biological nitrogen removal process of strain Y-10 is mainly achieved by affecting the transcriptional levels of genes during nitrification and denitrification process.The type and quantity of affected genes in the denitrification process are greater than those in the nitrification process.Moreover,these results also indicated that the denitrification process of strain Y-10 is more sensitive to the promotion or inhibition of heavy metal manganese ions than the nitrification process by gene levels.