Nitrogen removal characteristics of heterotrophic nitrification-aerobic denitrification by Alcaligenes faecalis C16☆

Yuxiang Liu *,Yao Wang Yi Li ,Hua An Yongkang Lv

1 College of Environmental Science and Engineering,Taiyuan University of Technology,Taiyuan 030024,China

2 National Major Science and Technology Program Management Of fi ce for Water Pollution Control and Treatment,Beijing 100029,China

3 Shanxi Key Lab of Coal Science and Technology,Taiyuan University of Technology,Taiyuan 030024,China

Keywords:Heterotrophic nitrification–aerobic denitrification Alcaligenes faecalis Hydroxylamine oxidase Nitrate reductase Nitrite reductase

A B S T R A C T Alcaligenes faecalis C16 was found to have the ability to heterotrophically nitrify and aerobically denitrify.In order to further understand its nitrogen removal ability and mechanism,the growth and ammonium removal response were investigated at different C/N ratios and ammonium concentrations in the medium with citrate and acetate as carbon source separately.Furthermore,experiments of nitrogen sources,production of nitrogen gas and enzyme assay were conducted.Results show that the bacterium converts NH4+-N and produces NH2OH during the growing phase and nitrite accumulation is its distinct metabolic feature.A.faecalis C16 is able to tolerate not only high ammonium concentration but also high C/N ratio,and the ammonium tolerance is associated with carbon source and C/N ratio.The nitrogen balance under different conditions shows that approximately 28%–45%of the initial ammonium is assimilated into the cells,44%–60%is denitrified and several percent is converted to nitrification products.A.faecalis C16 cannot utilize hydroxylamine,nitrite or nitrate as the sole nitrogen source for growth.How ever,nitrate can be used when ammonium is simultaneously present in the medium.A possible pathway for nitrogen removal by C16 is suggested.The preliminary enzyme assay provides more evidence for this nitrogen removal pathway.

1.Introduction

Conventional ammonium removal in wastewater treatment systems involves aerobic nitrification by autotrophs and anaerobic denitrification by heterotrophs.There are some problems in this system,for example,it requires separate treatment processes and strict condition control,and autotrophic nitrifiers are vulnerable to high concentrations of organic matter and ammonium[1–4].Thus the operating cost is prohibitively high,and conventional nitrification can be carried out only after reducing the C/N ratio or diluting the wastewater[5–7].Recently,bacteria that are capable of performing heterotrophic nitrification have received more attention because of their contribution to the nitrogen cycle and the possibility of application for w astewater treatment[8].Although heterotrophic nitrification activities are generally low,the sum of their activities in the field may be comparable to those of autotrophs because the growth rate of heterotrophs is much faster than that of autotrophs[9].Research has concluded that heterotrophic nitrogen removal may be particularly relevant for the treatment of wastewater containing high C/N ratios,because heterotrophic microorganisms often require high concentrations of organic carbon[3,10].Furthermore,most heterotrophic nitrifying bacteria are capable of aerobic denitrification.Under aerobic conditions,ammonium compounds can be converted to gaseous products in a single aeration phase by these heterotrophic microorganisms,and carbon can be removed simultaneously[11].Therefore,these microorganisms may be used to overcome problems inherent in the existing wastewater treatment method[12–14].

To date,many heterotrophic nitrifying–aerobic denitrifying microorganisms have been reported.How ever,most bacterial isolates accumulate only low amounts of nitrification products.Therefore,little is known about the physiology of heterotrophic nitrifying bacteria and the conditions forming nitrification products.A heterotrophic nitrifier,A.faecalis C16,has been isolated from the activated sludge of a coking wastewater treatment plant,presenting high-strength ammonium removal ability with high nitrite accumulation.The factors affecting ammonium removal and nitrite accumulation by A.faecalis C16 have been reported[15].The aim of this study is to further investigate the nitrogen removal ability by strain C16 at different conditions.In addition,the distribution of removed ammonium is clarified by nitrogen balance.And enzyme assay is conducted to confirm its possible metabolic pathway of nitrogen removal.

2.Materials and Methods

2.1.Microorganisms and culture conditions

A.faecalis C16 was isolated from the aeration tank of the wastewater treatment plant of a coking factory located in Shanxi Province,China.The medium was prepared by dissolving the following in 1 L of distilled water:(NH4)2SO40.472 g,MgSO4·7H2O 0.05 g,K2HPO40.2 g,NaCl 0.12 g,Mn SO4·4H2O 0.01 g,and FeSO40.01 g.Sodium acetate and trisodium citrate were used as carbon source separately in the medium,abbreviated as acetate medium and citrate medium,respectively.The initial pH value of medium was adjusted to pH 7.0.

Experiments with growing organisms were performed in shaken Erlenmeyer flasks(250 ml)containing 100 ml of medium and 1% preculture of A.faecalis C16 at 30°C on a rotary shaker at 120 r·min?1.

2.2.Shaking culture experiment

In order to elucidate the influence of carbon to nitrogen molar ratio(C/N)on the nitrifying capacity,the amount of carbon was changed to adjust the C/N ratio to 7,14 and 28 while fixing the amount of ammonium sulfate as the nitrogen source at approximately 100 mg·L?1.The effect of ammonium concentration was investigated in the acetate medium and citrate medium separately.In the acetate medium,the initial ammonium concentration was adjusted to approximately 100,200,400 and 800 mg·L?1on the basis of N content at the fixed ratio of C/N 7 and 14.And in the citrate medium,the initial ammonium concentration was adjusted to approximately 100,200,400,800 and 1200 mg·L?1at the fixed ratio of 14.Samples were taken periodically to examine changes in growth,concentrations of ammonium and intermediates.The culture conditions were as described above.

2.3.Consumption of nitrogen sources

Three nitrogen compounds,hydroxylamine,nitrite,and nitrate,found in nitrification,were used instead of ammonium in the citrate medium to elucidate the utilization of nitrogen sources of C16.NH2OH,NO2?,and NO3?were adjusted to about 100 mg·L?1on the basis of N content separately.In addition,20 mg·L?1NO3?-N was added to the citrate medium to clarify the denitrification by C16.For each compound,the amount of citrate as the carbon source was changed to adjust the C/N ratio to 14.The culture conditions were as described above.

2.4.Gas detection experiment

The citrate medium at C/N of 14 was inoculated with the bacterium and sealed in 100 ml serum bottle.The medium and head space were subsequently evacuated and aerated with pure oxygen at constant pressure(0.6 MPa)for 5–6 min.Then the serum bottle was cultivated at 30°C and 120 r·min?1.The gas samples were collected periodically by a gas tight syringe to detect the changes of N2using gas chromatography(SP-2100,Beifen-Ruili,China).

2.5.Enzyme assay

The bacteria for preparation of extracts were harvested from the citrate medium at C/Nof 14 after 48 h cultivation at 30°Cand 120 r·min?1.The cells were centrifuged(4°C,15 min,10000 r·min?1)and then suspended in a 0.01 mol·L?1potassium phosphate buffer(pH=7.4).The cells were disrupted by ultrasonication.The cell debris was removed by centrifugation at 15000 r·min?1and 4°C for 30 min.The supernatant fraction was immediately used for enzyme assay.

Hydroxylamine oxidoreductase(HAO)activity was determined by the reduction of potassium ferricyanide as described by Otte et al.[13].The activities of nitrate reductase(NR)and nitrite reductase(NiR)were determined by the reduction of NADH(nicotinamide adenine diuncleotide hydrogen)according to Zhao et al.[16].Protein concentration in crude extract was determined by the Bradford Reagent Kit(Sangon,Shanghai).One unit of enzyme activity(U)is defined as the amount of enzyme that catalyzes the transformation of 1 μmol of substrate per minute.The specific activity(U·mg?1)is defined as the amount of enzyme units divided by the concentration of protein.

2.6.Analytical methods

Grow th of bacteria was monitored by measuring the optical density at 600 nm(OD600nm)of the culture broth using a spectrophotometer.Culture samples were centrifuged at 12,000 r·min?1and filtered through a membrane filter,and the filtrate was used for chemical analysis.The concentration of ammonium was analyzed by Nessler's reagent photometry.The nitrite concentration was determined by N-(1-naphthalene)-diaminoethane photometry method.The nitrate concentration was measured by ion chromatography(DIONEX ICS-90).Hydroxylamine was determined colourimetrically by the method of Frear and Burrell[17].Total nitrogen(TN)was analyzed using a TOC/TN analyzer(SHIMADZU TNM-1).Intracellular nitrogen was calculated from the relationship between OD600nmand nitrogen content in dry biomass obtained using an element analyzer(EuroEA 3000).The dry biomass was prepared by drying the bacteria at 105°C overnight.The denitrification ratio was calculated according to the method of Joo et al.[3].All tests were conducted in duplicate or triplicate,with the exception of the growth experiment.

3.Results

3.1.Growth and heterotrophic nitrification of C16 at different C/N ratios

3.1.1.Growth characteristics

Fig.1 shows the growth profiles of C16 at different C/N ratios in the citrate and acetate media in shaking culture.Grow th curves are established by measurement of the OD value at 600 nm.The growth of the isolate at different C/N ratios all results in sigmoid curves,with three distinguished phases.In the citrate medium,three growth curves of C16 at different C/N ratios were similar.The bacterium showed the lag phase of 6–8 h,and then it entered the exponential phase which lasted for about 20 h.In the acetate medium under similar conditions,the growth patterns of C16 were different from those in the citrate medium.The lag phases of three growth curves were all longer than that in the citrate medium,especially at C/N ratio of 28.At C/N ratio of 7,the exponential phase was shorter than those at C/N of 14 and 28.How ever,at C/N of 14,the changes in growth were minimal after 48 h while at C/N of 28 the optical density readings continued to increase gradually.At C/N of 7 in both media,the stationary phase was not apparent mainly due to the exhaustion of the carbon source.

3.1.2.Heterotrophic nitrification

In order to identify possible correlations among growth,ammonium removal,and release of products during heterotrophic nitrification,C16 was inoculated into citrate and acetate media under different C/Nratios.The cultures were sampled each day for analyses.Fig.2 show s the concentrations of cell(a,d),NH4+-N(b,e)and TN(c,f).Fig.3 shows the changes in nitrification products(hydroxylamine,nitrite,nitrate)during a 96-h growth period.

Fig.1.Growth of C16 at different C/N ratios in the citrate medium(a)and acetate medium(b).Square:C/N 7;circle:C/N 14;triangle:C/N 28.

In the citrate medium,the consumption rates of NH4+-N and TN did not vary distinctly at three C/N ratios.In 96 h,the average ammonium removal ratios were 92.5%,93.2%and 91.2%at C/N of 7,14 and 28,respectively.In the acetate medium,the consumption rates of ammonium and TN were slightly different at three C/N ratios.At C/N of 7,the growth patterns of C16 and the consumption of NH4+-N were similar to those in the citrate medium,the average ammonium removal ratio was 90.5%in 48 h.However,72 h was needed for almost similar removal ratio at C/N of 14 and 28 because of a long transient time.The maximum growth and TN removal were observed at C/N of 14 followed by C/N of 28 and 7[Fig.2(d,e,f)].The results indicate that the effect of C/N ratio on the removal rate of ammonium and TN is much lower in the citrate medium than in the acetate medium.

Hydroxylamine,nitrite,and nitrate were formed by the bacterium during the heterotrophic nitrogen removal process in both media.In Fig.3(a),the hydroxylamine concentration in the citrate medium increased immediately,reached a maximum in 24 h(during exponential growth phase)at three C/N ratios,and then decreased.The amount of nitrite accumulation at C/N of 14 was higher than that at C/N of 7 and 28.In Fig.3(b),the hydroxylamine concentration reached maximum within 24 h at C/N of 7 in the acetate medium,while 48 h was needed at C/N of 14 and 28.Especially at C/N of 28,the hydroxylamine concentration increased dramatically after 24 h,that is,just after the lag phase.The maximum nitrite accumulation by C16 in the acetate medium occurred at C/N of 7.

The nitrogen balance of C16 in the citrate and acetate mediaat different C/N ratios is shown in Table 1.At C/N of 7,14,and 28 in the citrate medium,on average,28.26%,38.01%and 38.53%of there moved ammonium were converted to cell mass,12.23%,17.77%and 7.74%were converted to nitrification products,so 59.50%,44.22%and 53.72%were removed by aerobic denitrification,respectively.In the acetate medium,on average 28.15%,44.58%and 39.91%of the removed ammonium were assimilated by the bacterium,19.76%,11.11%and 5.66%were converted to nitrification products,so 52.09%,44.31%and 54.43% were removed by aerobic denitrification,respectively.

3.2.Heterotrophic nitrification at different ammonia concentrations

The influence of ammonium concentration on growth,ammonium removal and nitrification product was investigated in the acetate medium.In Fig.4(a),at ratio of C/N 7,the growth was recorded at all concentrations,indicating that C16 is able to tolerate a broad range of ammonium concentrations.And higher NH4+-N concentrations resulted in higher optical densities.In addition,at 800 mg·L?1NH4+-N,the growth exhibited longer lag time than the other concentrations.As shown in Fig.4(b),on average,NH4+-N was removed 89.7%,80.4%and 52.8% within 144 h at 200,400 and 800 mg·L?1,respectively,while at 100 mg·L?1NH4+-N,the average removal ratio was 94.1%in 72 h.

As described above,the hydroxylamine formation was associated with the cell growth.The hydroxylamine concentration in the medium increased immediately after the lag phase,reached the maximum during the exponential growth phase,and then decreased after the stationary phase[Fig.4(c)].The hydroxylamine maintained at higher level before the ammonium was almost consumed[compare Fig.4(b)with(c)].Higher concentration of ammonium led to slower degradation of hydroxylamine.The concentrations of nitrite began to increase after the start of hydroxylamine production and reached the maximum when the hydroxylamine was almost exhausted.Significant increase in nitrite concentration was always accompanied by a marked decrease in the amount of NH2OH[compare Fig.4(c)with(d)].In therange of-N 100–400 mg·L?1,the nitrite accumulation increased with the increase of-N.It is clear that there is a high correlation between ammonia oxidation and nitrite accumulation.Nitrate accumulation remained very low.

Table 1Nitrogen balance of ammonium removal by C16 at different C/N ratios in the citrate medium and acetate medium

Fig.2.Characteristics of ammonium removal at different C/N ratios in the citrate medium(a,b,c)and acetate medium(d,e,f).Square:C/N 7;circle:C/N 14;triangle:C/N 28;error bar:mean±SD for duplicate.

Fig.3.Nitrification products at different C/N ratios in the citrate medium(a)and acetate medium(b).Square:C/N 7;circle:C/N 14;triangle:C/N 28;closed symbol:NO2?-N;open symbol:NO3?-N;dash line:NH2OH-N;error bar:mean±SD for duplicate.

As shown in Fig.5(a),at C/N ratio of 14 in the acetate medium,the growth was inhibited by 400 and 800 mg·L?1ammonium concentrations.And at the initial NH4+-N concentration of 200 mg·L?1,the lag time lasted up to about 24 h.And the NH4+-N removal process was slower both at 100 and 200 mg·L?1than that at the same ammonium concentrations at C/N of 7[Fig.5(b)].However,at the same C/N ratio in the citrate medium,the growth was recorded at all concentrations from NH4+-N 100–1200 mg·L?1and NH4+-N was removed efficiently at all concentrations(Fig.6).This tolerance to ammonia is much higher than that of Providencia rettger YL[18].

Fig.4.Characteristics of ammonium removal at different ammonia concentrations in the acetate medium at C/N 7.NH4+-N concentration/mg·L?1:square—100;circle—200;triangle—400;diamond—800;error bar:mean±SD for triplicate.

Fig.5.Characteristics of growth and ammonium removal by A.faecalis C16 at different ammonia concentrations in the acetate medium at C/N 14.NH4+-Nconcentration/mg·L?1:square—100,circle—200;triangle—400;diamond—800;error bar:mean±SD for triplicate.

3.3.Consumption of nitrogen sources

When hydroxylamine,nitrite and nitrate were used as the nitrogen sources instead of ammonium in the citrate medium,no growth of C16 was observed.This indicates that C16 cannot utilize them as the nitrogen source for growth.How ever,as shown in Fig.7,with 20 mg·L?1NO3?-N added to the citrate medium,the ammonium removal is not affected by the nitrate,with an average of 91.3%of NO3?-N consumed within 72 h.

3.4.Detection of nitrogen gas

The nitrogen balance of C16 suggested possible occurrence of NH4+-N conversion to N2by the process of aerobic denitrification.To verify this possibility,gas chromatographic tests were conducted to identify the production of N2by C16.Fig.8 shows that from an original concentration of 100 mg·L?1NH4+-N,N2level increased to an average of 47.39% with in 48 h.

3.5.Enzyme assay

To further verify the heterotrophic nitrogen removal capability,we examined the activities of enzymes involved in nitrification and denitrification.The results show the detected activities of HAO,NR and NiR in crude extract prepared from the cells incubated at 30°C and 120 r·min?1for 48 h.The specific activities of HAO,NR and NiR are(0.042± 0.003),(0.019± 0.001),(0.021± 0.005)U·mg?1protein,respectively.The results suggest that C16 contains a conventional nitrifying and denitrifying system common to bacteria.

4.Discussion

Our previous study has showed that citrate and acetate are suitable organic substrates for heterotrophic nitrification by A.faecalis C16[15].This result is consistent with that of A.faecalis no.4[3].In this study,we investigate the growth profiles of C16 at different C/N ratios in the citrate and acetate media.The results indicate that C16 in acetate medium exhibits longer lag time,especially at C/N of 28,but the influence of C/N ratio on the growth of C16 in the citrate medium is not significant.Therefore,in terms of the cell growth,citrate is a more suitable carbon source for C16 compared to acetate,which maybe because citrate could involve in citric acid cycle directly.The final cell concentration of C16 is also related to the C/N ratio and 14 is the optimum for the cell growth in both media.Similarly,the effect of C/N ratio on the removal rate of ammonium and TN is much lower in the citrate medium than in the acetate medium.Carbon-dependency of heterotrophic nitrification has been reported.And the carbon sources favoring heterotrophic nitrification are not unique among different organisms.A possible reason is that ammonium is oxidized by a sequence involved in some way with the metabolism of a special carbon source,or aspecific carbon source could provide the acceptor molecules for the synthesis of intermediate organic nitrogen compounds[19].Furthermore,the results suggest that C/N ratio does not play an important role in the process of heterotrophic nitrification of C16,especially in the citrate medium.Even if the C/N ratio is as high as 28,which is too high for the growth of autotrophic nitrifying bacteria,C16 still exhibits satisfactory nitrification ability.This ability expands its application scope in wastewater treatment.

At three C/N ratios in both media,hydroxylamine,nitrite and nitrate are formed by the bacterium during the heterotrophic nitrogen removal process.It can be seen from the relationship between cell growth and hydroxylamine formation that hydroxylamine concentration in the media increases after the lag phase,reaches the maximum during the exponential growth phase,and then decreases after the stationary phase.Accumulation of hydroxylamine accompanies the decrease of NH4+-N and the hydroxylamine decreases to the lowest level when the ammonium is almost consumed(compare Fig.3 with Fig.2).The experimental results at different ammonia concentrations also show that NH2OH-N concentrations maintain at higher levels before NH4+-N is nearly exhausted[compare Fig.4(b)with(c)].These results indicate that NH4+-N is the source of NH2OH-N.

The experimental results at different C/N ratios and ammonia concentrations show that the concentrations of nitrite and nitrate increase after the start of hydroxylamine production.Nitrite concentration increases dramatically with the decrease of hydroxylamine concentration,and nitrite accumulation increases with the initial ammonia concentration,indicating that hydroxylamine is the source of nitrite formation.The nitrite accumulation is much higher than that of nitrate.In contrast to autotrophic nitrifiers,most heterotrophs seem to excrete nitriteor nitrate only after the active growing phase[20–22]and C16 is no exception.This is the reason why several researchers have suggested that heterotrophic nitrification might be associated with autolytic processes[23]or secondary metabolism[24].How ever,the present study indicates that heterotrophic nitrite/nitrate production is linked to growth,which supports the notion that heterotrophic nitrification can take place during the entire growing phase[25,26].Like C16,nitrite is the dominant product in the process of heterotrophic nitrification by some other bacteria,such as Bacillus methylotrophicus strain L7[27].How ever,for A.faecalis strain NR[28],Achromobacter sp.GAD3 and Comamonas sp.GAD4[29],nitrate is the dominant product in the process of heterotrophic nitrification.

Fig.6.Characteristics of growth and ammonium removal by A.faecalis C16 at different ammonia concentrations in the citrate medium at C/N 14.NH4+-Nconcentration/mg·L?1:square—100;circle—200;triangle—400;diamond—800;star—1200;error bar:mean±SD for triplicate.

Fig.7.Concentrations of ammonium,hydroxylamine,nitrite and nitrate with 20 mg·L?1-N added to the citrate medium.Square:-N;circle:NH2OH-N;uptriangle:-N;downtriangle:-N;error bar:mean±SD for triplicate.

Fig.8.Nitrogen gas production by C16 grown in the citrate medium at C/N 14.Error bar:mean±SD for triplicate.

It can be seen from the nitrogen balance of ammonium removal by C16 that approximately 28%–45%of the initial ammonium is assimilated into the cells with 44%–60%of ammonium denitrified.This efficiency is similar with A.faecalis No.4,with 40%–50%of ammonium denitrified[3].Also,several percent is converted to nitrification products that remain in the medium.This indicates that NH4+-Nremoved by C16 distributes mainly to denitrification and assimilation,not to nitrification.Furthermore,the amounts of ammonium used for cell synthesis at C/N of 14 and 28 are larger than those at C/N of 7 in both media,while the denitrification ratios at C/N of 7 and 28 are larger than those at C/N of 14 due to the low intracellular nitrogen content or nitrification products.This phenomenon is different from A.faecalis No.4,in which denitrification seems to preferentially proceed at lower C/N ratio[3].The result indicates that at the initial concentration of 100 mg·L?1NH4+-N,C/N 28 is the optimal for achieving cost-effective denitrification with lower accumulation of nitrite and C16 is appropriate for the treatment of wastewater with high organic substance content.

Grow th of organism is considered to be directly affected by the availability of carbon and nitrogen sources.The experimental results show that the final cell concentration is in proportion to ammonia concentration.Higher-N concentrations result in higher optical densities.This result is consistent with that of Taylor et al.[18]working with other genus.Although the removal process is slower at higher concentrations,the heterotrophic nitrification is not inhibited by higher ammonium concentrations.This is a beneficial feature for applications in ammonium removal from wastewater with high ammonium concentrations.Considering the experimental results at different ammonia concentrations,for C16,the tolerance to ammonia is associated with carbon source and C/N ratio,and citrate is a more suitable carbon for high ammonia concentration and high C/N ratio.

C16 cannot utilize hydroxylamine,nitrite or nitrate asthe sole nitrogen source for growth.How ever,nitrate can be used when ammonium simultaneously exists in the medium.This is different from A.faecalis strain NR[28],in which nitrate could not be utilized by strain NR under aerobic conditions when ammonium is present.Generally,there are 3 nitrate reductions in bacteria:nitrate assimilation,nitrate respiration and nitrate dissimilation.Nitrate assimilation,catalyzed by cytoplasmic assimilatory nitrate reductase,can convert nitrate to ammonium,which can be used as a nitrogen source for growth.Nitrate dissimilation,catalyzed by dissimilatory nitrate reductase,can reduce nitrate to nitrite under both aerobic and anaerobic conditions[30,31].The result suggests that the nitrate consumption by C16 is likely to be a result of their dissimilatory nitrate reductase activity and C16 should have the capability of aerobic denitrification.How ever,C16 could not use nitrate as the nitrogen source for growth,probably due to the absence of assimilatory nitrate reductase or repressed activity.

Based on the production of intermediates and the detection of nitrogen gas,a possible pathway for ammonium removal by C16 is suggested:ammonium is initially oxidized to hydroxylamine then to nitrite followed by nitrate,which is then reduced to nitrite and eventually to nitrogen gas.This is consistent with a previous report by Taylor et al.[18].

The preliminary enzyme assay provides more evidence for the nitrogen removal pathway of C16.The result shows that HAO,NR and NiR activities are detected in crude extract prepared from the cells incubated at 30°C and 120 r·min?1for 48 h.The specific activity of HAO is 0.042 U·mg?1protein.This is in the same order of magnitude as the HAO specific activity of Acinetobacter calcoaceticus HNR[16]and A.faecalis TUD[13],while in heterotrophic nitrifiers of Agrobacterium sp.LAD9,Achromobacter sp.GAD3 and Comamonas sp.GAD4,the specific activities of HAO are far less than the activity of C16[29].The specific activities of NR and NiR are 0.019,0.021 U·mg?1protein,respectively,which is in the same order of magnitude as the result in Providencia rettgeri strain YL[32].Neither NR nor NiR activity is detectable in Acinetobacter calcoaceticus HNR[16]and other A.faecalis strains such as A.faecalis no.4[3],strain NR[28],etc.Therefore,denitrification in these bacteria is considered via hydroxylamine rather than nitrite or nitrate.

Ammonia oxidation is often reported to be the rate-limiting step in the nitrification process[3].And hydroxylamine oxidation may be the rate-limiting step in the heterotrophic nitrification–aerobic denitrification process[29].In this study,apparent hydroxylamine production,found in nitrification process of C16,confirms this possibility.Apparent accumulation of nitrite instead of nitrate is found in C16,which may be due to the reduced activity of denitrification enzymes.Further study on the mechanism of heterotrophic nitrogen removal by C16 is necessary.

5.Conclusions

A.faecalis C16 is capable of heterotrophic nitrification and aerobic denitrification.The removal ability is not inhibited by higher ammonium concentrations.During the growing phase,C16 converts NH4+-N,produces NH2OH and accumulates nitrite.From the nitrogen balance,28%–45%of the initial ammonium was assimilated into the cells and 44%–60%of ammonium was denitrified.A possible pathway for ammonium removal by C16 is suggested:ammonium is initially oxidized to hydroxylamine then to nitrite,follow ed by nitrate,which is then reduced to nitrite and eventually to nitrogen gas.The preliminary enzyme assay provides more evidence for the removal pathway.