Identification And Nitrogen Removal Characteristics Of Two Heterotrophic Nitrifying-aerobic Denitrifying Bacteria

Two heterotrophic nitrifying-aerobic denitrifying bacteria,isolated from the activated sludge of a coking wastewater treatment plant,were identified as Alcaligenes faecalis C16 and Acinetobacter sp.Y1 by morphological,physiological characteristics and phylogenetic analysis of its 16S rDNA gene sequence.Nitrogen removal characteristics and affecting factors were investigated.A.faecalis C16 converts NH4+-N and produces hydroxylamine during the growing phase and nitrite accumulation is a distinct metabolic feature of A.faecalis C16.However,the nitrification products including hydroxylamine,nitrite and nitrate were hardly detected during heterotrophic nitrification by Acinetobacter sp.Y1.Heterotrophic nitrification could take place only in the presence of certain carbon source.Citrate and acetate are suitable organic substrates for heterotrophic nitrification by them among carbon sources selected for the study.And the suitable carbon source for them is citrate compared to acetate.A.faecalis C16 can utilize ammonium but not nitrite and nitrate as the sole nitrogen source for growth.However,nitrate can be used when ammonium was simultaneously added to the medium.A.faecalis C16 could grow and remove nitrogen at high C/N ratios.Ammonium removal activities varied only slightly with C/N ratios in the range 6 to 30.Nitrite accumulation increased as the C/N ratio was increased and reached the maximum at C/N 14,then gradually decreased as the C/N ratio was increased further.At a fixed ratio of C/N 14,the ammonium removal ratio varied only slightly in the range of NH4+-N 100-300 mg/L,but nitrite accumulation increased with the increase of NH4+-N.A.faecalis C16 was able to tolerate a broad pH range.When the initial pH was set at 6.0 to 11.0,no obvious difference in the decrease in NH4+-N was observed,and the growth resulted in a final pH above 9.2 irrespective of initial pH.The optimal NO2--N production by the bacterium was at pH 6.0.The ammonium removal abilities at 30?,35?,and 40? were almost same,and the most appropriate temperature for ammonium removal and nitrite accumulation by C16 was 30?.The suitable shaking speed for nitrogen removal and nitrite accumulation was 120rpm.Under the optimum conditions(initial pH 6.0,C/N 14,30? and 120 rpm)the maximum accumulation,28.29 mg/L N02--N,was achieved when the organism grew with citrate in 4 days.Furthermore,A.faecalis C16 was shown to have phenol-degrading capacity during ammonium removal.Metabolism of phenol resulted in acidification of the media,which is not favorable for nitrification,whereas many other carbon sources made the medium more alkaline.However,when phenol and acetate were used as mixed carbon source at different phenol/sodium acetate(P/S)ratios and their pH values were all controlled above 9.2 or P/S ratios below 5:5,no inhibitory effect by phenol was observed.The experimental results show that A.faecalis C16 has the potential application in industrial wastewater treatment systems.The nitrogen balance under different conditions shows that approximately 28-45%of the initial ammonium was assimilated into the cells,44-60%was denitrified and several percent was converted to nitrification products.Based on the production of intermediates and the detection of nitrogen gas,a possible pathway for ammonium removal by C16 was suggested:ammonium is initially oxidized to hydroxylamine then to nitrite followed by nitrate which is then reduced to nitrite and eventually to nitrogen gas.TOC removal rate was lower due to the secondary metabolites based on nuclear magnetic resonance spectroscopy.Ammonium,nitrite and nitrate could be removed by Acinetobacter sp.Y1,when they were provided as sole nitrogen source,respectively.The removal rates of ammonium and TN were higher with C/N ratios in the range 15 to 60,and NH4+-N was removed above 95%within 24 h at 100 mg/L NH4+-N concentration.At a fixed ratio of C/N 14,and NH4+-N was removed above 98%within 36 h at NH4+-N 100 mg/L,200 mg/L and 400 mg/L.The removal rates of ammonium was lower only at 800 mg/L and 1600 mg/L.When the initial pH was set at 6.0 to 9.0,no obvious difference in the decrease in NH4+-N was observed.Well growth and high nitrogen removal efficiency were attained from 25? to 40?.In the range of shaking speed of 40 rpm-150 rpm nitrogen removal rate were not significant difference.The activities of hydroxylamine oxidase(HAO),nitrate reductase(NR)and nitrite reductase(NiR)were detected in crude extract prepared from A.faecalis C16.However,in Acinetobacter sp.Y1,only the activities of nitrate reductase and nitrite reductase were detected,and hydroxylamine oxidase activity was very week.Producted of N2 was detectable during the processes of heterotrophic nitrification and aerobic denitrification by two bacteria.The pathway for ammonium removal by Acinetobacter sp.Y1 may be different from A.faecalis C16.