Study On Performances And Mechanisms In Simultaneous Removal Of Ammonium And Phenol By A Heterotrophic Nitrifier WY-01

Heterotrophic nitrifiers with several advantages including growing fast,tolerating high substrates,and performing simultaneous nitrification and denitrification,have been attracting international attention on the biological denitrification field.Nowadays,heterotrophic nitrifiers mostly use nontoxic organic small molecular substances as carbon sources for nitrogen removal,and the reports about using harmful materials such as phenol as substrates to degrade nitrogen are few in alien.As we all know,some industrial wastewaters usually contain a lot of ammonium nitrogen and phenol,and phenols have an inhibitory effect on nitrification process,which can seriously hinder the nitrogen removal in this kind of wastewater.Therefore,removing ammonium and phenol efficiently has become a crucial issue on biological treatment of phenol-bearing wastewater.In this paper,a heterotrophic nitrifier WY-01,isolated from distilled ammonia wastewater from Taiyuan Iron and Steel?Group?Co.,Ltd.could remove ammonium nitrogen in the wastewater containing phenol.Firstly,the classification and identification of WY-01 were evaluated by morphological observation,physiological and biochemical characteristics,and molecular biology methods.Then,its removal performances and mechanisms of ammonium and phenol were analyzed systematically from the substrate metabolism characteristics,metabolic pathways and physical-chemical factors.Finally,simultaneous biodegradation of phenol and ammonium by WY-01 was successfully carried out through two methods: one was adding external carbon source,the other was constructing an adsorption–desorption–biodegradation system,especially the second method providing an effective and economic strategy for simultaneous removal of ammonium and phenol in phenol-rich wastewater.The main research conclusions are as follows:?1?A heterotrophic nitrifier WY-01,isolated from distilled ammonia wastewater,could remove ammonium in phenol-bearing wastewater,and was identified as Alcaligenes faecalis on the basis of its morphological observation,physiological and biochemical characteristics,and 16 S rDNA sequence analysis.?2?The biodegradation of ammonium was closely related to the growth of strain WY-01,and mainly happened in exponential growth period.When the initial concentration of ammonium was about 400 mg N/L,strain WY-01 exhibited unusual capability for ammonium removal with low accumulated intermediates.Besides,the utilization of nitrification intermediates and the components of gas production were analyzed,and the results indicated that both ammonium and hydroxylamine could be denitrified by strain WY-01 to produce N₂ under aerobic conditions,when they were separately used as single nitrogen source.However,nitrite and nitrate could not be denitrified by strain WY-01 as the sole N-source.?3?Neither NR nor NiR showed enzyme activity in strain WY-01,but HAO did,implying that HAO might be involved in the aerobic conversion of hydroxylamine to N₂.Gene detection results further indicated that the four key enzyme genes,amoA?NirK?Nor B and NosZ were detected in the genome of strain WY-01.From the utilization of nitrogen compounds,enzyme assay,gene detection and gas production characteristics,two possible nitrification and denitrification pathways were proposed for strain WY-01.One was oxidizing ammonium into nitrite followed by denitrification of nitrite to N₂,which was catalyzed by AMO,HAO,NiR,NOR and NOS in turn;the other was oxidizing ammonium to hydroxylamine,which was directly converted into N₂ under the action of HAO.?4?Single-factor experiments were investigated to study the influencing factors such as temperature and salinity on ammonium removal,and results indicated that strain WY-01 exhibited better ammonium removal ability in the following culture conditions: temperature as low as 10°C;salinity below 60 g/L;[Cu²⁺]?8 mmol/L or [Zn²⁺]?0.5 mmol/L or [Cu²⁺-Zn²⁺]?1 mmol/L;pH from 5 to 10;DO?4.67 mg/L;Meanwhile,strain WY-01 performed strong resistance to high ammonium concentration,and it could still grow when the concentration of ammonium was as high as 2500 mg/L.?5?The biodegradation of phenol was closely related to the growth of strain WY-01,and phenol had an influence on the accumulation of intermediates during WY-01's ammonium biodegradation process.WY-01's maximum tolerable concentration of phenol was 1200 mg/L,and phenol could be degraded effectively below this concentration.The toxicity inhibition of phenol on strain WY-01 directly reflected in the length of adaptation period: the higher the concentration of phenol was,the longer the adaptation period was.Through SEM observation,it was testified that phenol did have toxic effects on strain WY-01.When severe,the bacteria cell would gather to mass and appear local shrinkage or depression on the surface.The phenol degradation pathway of strain WY-01 was synthetically investigated on the basis of enzyme activity,gene detection and metabolites analysis,and phenol was suggested to degrade through ortho-pathway to produce non-toxic intermediate compounds that entered the TCA cycle to produce CO₂ ultimately.Finally,the phenol removal performance of strain WY-01 was optimized using the single-factor experiments,and the optimal conditions for phenol degradation were 30–35°C,pH from 7 to 8,DO?4.79 mg/L and C/N=14.?6?By adding certain concentration of carbon source,the capability of strain WY-01 in simultaneous removal of phenol and ammonium was achieved,and the removal efficiency of ammonium was also significantly improved.However,the effect of external sodium acetate on phenol degradation was significantly varied with different concentration of phenol.When the concentration of phenol was 500 mg/L,exogenous carbon source could decrease the degradation rate of phenol.Once the phenol concentration was 800–1000 mg/L,adding sodium acetate could stimulate the growth of WY-01,which would shorten the adaptation period of WY-01 in high concentration of phenol and thus enhance the phenol degradation rate.Adding carbon source could promote co-metabolic degradation of ammonium and phenol,but this method still could not improve WY-01's maximum tolerable concentration of phenol.?7?Considering the adsorption characteristic of phenol on CAD-40 macroporous resins and the degradation performance of strain WY-01 that could utilize phenol to degrade ammonium,an adsorption–desorption–biodegradation system was built to simultaneously biodegrade phenol and ammonium in phenol-rich wastewater.In this system,up to 6000 mg/L phenol could be completely degraded by WY-01;meanwhile,99.03% of ammonium was removed from the initial concentration of 384 mg/L.Moreover,the resin was regenerated during the biodegradation process without any additional manipulations.All results indicated that the system had a good performance for removing ammonium and phenol,and also showed a good reusability.This system provided an efficient and economic method for simultaneous removal of ammonium and phenol in phenol-rich wastewater.Then the key enzyme genes involved in simultaneous biodegradation of phenol and ammonium were quantitatively analyzed by qRT–PCR,and both the phenol biodegradation genes,PH and CatA,and the denitrifying genes,nir K and nosZ were prominently expressed at transcription level.