The characteristics of a novel heterotrophic nitrification–aerobic

The characteristics of a novel heterotrophic nitri?cation–aerobic denitri?cation bacterium,Bacillus methylotrophicus strain L7

Qing-Ling Zhang,Ying Liu,Guo-Min Ai,Li-Li Miao,Hai-Yan Zheng,Zhi-Pei Liu ?

State Key Laboratory of Microbial Resources,Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China

a r t i c l e i n f o Article history:

Received 1November 2011

Received in revised form 28December 2011Accepted 28December 2011Available online 9January 2012Keywords:

Bacillus methylotrophicus L7

Heterotrophic nitri?cation–aerobic denitri?cation Nitrite

Nitrous oxide Nitrogen removal

a b s t r a c t

Bacillus methylotrophicus strain L7,exhibited ef?cient heterotrophic nitri?cation–aerobic denitri?cation

ability,with maximum NH t4-N and NO à

2-N removal rate of 51.58mg/L/d and 5.81mg/L/d,respectively.Strain L7showed different gaseous emitting patterns from those strains ever described.When 15NH 4Cl,or Na 15NO 2,or K 15NO 3was used,results of GC–MS indicated that N 2O was emitted as the intermediate of heterotrophic nitri?cation or aerobic denitri?cation,while GC–IRMS results showed that N 2was pro-duced as end product when nitrite was used.Single factor experiments suggested that the optimal con-ditions for heterotrophic nitri?cation were sodium succinate as carbon source,C/N 6,pH 7–8,0g/L NaCl,37°C and a wide range of NH t4-N from 80to 1000mg/L.Orthogonal tests showed that the optimal conditions for aerobic denitri?cation were C/N 20,pH 7–8,10g/L NaCl and DO 4.82mg/L (shaking speed 50r/min)when nitrite was served as substrate.

1.Introduction

The removal of nitrogen from wastewater by nitri?ers and den-itri?ers was the most ef?cient method in wastewater treatment.Autotrophic nitri?cation and anoxic denitri?cation played impor-tant roles in this process.Nitri?ers convert ammonia to nitrite,followed by nitrate.Denitri?ers reduce nitrate to nitrite,?nally to N 2,in which NO and N 2O were the main intermediate products (Joo et al.,2005).Due to the totally differences in physiology and biochemistry,the nitri?ers and denitri?ers had some disadvan-tages in the process of nitrogen removal treatment of wastewater.Nitri?ers were sensitive to organic matter (Kulikowska et al.,2010).However,organic compounds were necessary to the denit-ri?ers.In addition,the growth of nitri?ers relied on oxygen that was toxic to denitri?ers (Lloyd et al.,1987).Because of the different tolerance to organic matter and oxygen,they had to be separated in the wastewater treatment system.Meanwhile,the slow growth of autotrophic nitri?ers made the wastewater treatment process not only time-consuming but also large system,thus increasing the cost of wastewater treatment (Khin and Annachhatre,2004).In 1972,Arthrobacter sp.capable of heterotrophic nitri?cation was ?rst isolated from natural environment (Verstrae and Alexande,1972).In 1983,Thiosphaera pantotropha (now known as Paracoccus denitri?can ),capable of heterotrophic nitri?cation–aerobic denitri?cation,was isolated from activated sludge in the wastewater treatment plant (Robertson and Kuenen,1983).After-wards,the study on heterotrophic nitri?cation and aerobic denitri-?cation had drawn more and more https://www.360docs.net/doc/d516024526.html,pared with the traditional removal of nitrogen,nitrogen removal by heterotrophic nitri?cation and aerobic denitri?cation had several advantages.Firstly,the utilization of organic substrates and the tolerance to oxygen agreed with each other,achieving the vision of simulta-neous nitri?cation and denitri?cation (SND)in one reactor (Third et al.,2005).In addition,the process of denitri?cation could bal-ance the change of pH in the reactor,avoiding the acidi?cation caused by nitri?cation.Furthermore,the diversity of substrates and products of heterotrophic nitri?cation realized the mixed cul-ture with other strains and expanded the application scope (Mar-azioti et al.,2003).

With the advancement of research,more and more heterotro-phic nitri?cation–aerobic denitri?cation strains were isolated and characterized,such as Alcaligenes faecalis No.4(Joo et al.,2005),Bacillus sp.strains (Yang et al.,2011)and Pseudomonas sp.(Wan et al.,2011).

Current studies about heterotrophic nitri?cation–aerobic deni-tri?cation mainly focused on substrate removal and accumulation of intermediate (Wan et al.,2011).The most common way to prove the denitri?cation and the production of gaseous nitrogen com-pounds from the denitri?cation was by nitrogen balance calcula-tion (Joo et al.,2005;Yang et al.,2011).However,there was an error existed in the calculation of nitrogen balance,since about

?Corresponding author.Address:Institute of Microbiology,Chinese Academy of Sciences,No.1West Beichen Road,Chaoyang District,Beijing 100101,China.Tel.:+861062653757;fax:+861062538564.

E-mail address:liuzhp@https://www.360docs.net/doc/d516024526.html, (Z.-P.Liu).

8%of the input nitrogen was not recovered(Gonenc and Harrem-oes,1985).Besides,compared with nitrate,the research of the characteristics of denitri?cation using nitrite,a link intermediate between nitri?cation and denitri?cation,was rarely reported (Wan et al.,2011).Furthermore,nitrite accumulation was a critical issue inherent in the aquaculture industry because of its toxicity to aquatic animals.Therefore,study on nitrite as denitrifying sub-strate was urgent and important not only in theoretic research but also application research to better understand the mechanism of heterotrophic nitri?cation–aerobic denitri?cation and control nitrite pollutions.

In this study,a Gram-positive bacterial strain,Bacillus methylot-rophicus strain L7,was characterized for its heterotrophic nitrify-ing–aerobic denitrifying performance using ammonia,nitrite and nitrate as substrate.In addition,highly precise analyzing methods including gas chromatography–mass spectrometry(GC–MS)and gas chromatography–isotope ratio mass spectrometry(GC–IRMS) were employed to determine the gaseous nitrogen compounds. Based on these results,a speci?c inorganic nitrogen metabolism pathway by strain L7was proposed.Our results might provide an alternate microbial resource for nitrogen removal treatment of wastewater,and also might be bene?t to the elucidation of the mechanisms of heterotrophic nitri?cation–aerobic denitri?cation, especially by Gram-positive bacteria.

2.Methods

2.1.Identi?cation of strain L7

Strain L7was isolated from wastewater sample.Physiological and biochemical characteristics were tested using API20NE and API ZYM strips(bioMérieux,French)following the manufacturer’s instructions.

16S rRNA gene was PCR ampli?ed using bacterial universal primers27F(50-AGAGTTTGATCCTGGCTCAG-30)and1492R(50-GG TTACCTTGTTACGACTT-30)and sequenced by Meiji Corp.(Beijing, China).Sequence alignment was performed using Basic Local Alignment Search Tool program(BLAST:https://www.360docs.net/doc/d516024526.html,/blast/Blast.cgi).A neighbor-joining tree was constructed using MEGA3.1program(Kumar et al.,2004).

2.2.Medium

Heterotrophic nitri?cation medium(HNM,g/L of distilled water):(NH4)2SO40.66,sodium succinate4.72,KH2PO40.50,Na2H-PO40.50,MgSO4á7H2O0.20,NaCl30.00,trace element solution 2.00mL,pH7.5.

Denitri?cation medium(DM,g/L of distilled water):KNO31.00, sodium succinate4.68,MgSO4á7H2O0.20,CaCl20.01,EDTA0.07, KH2PO40.50,Na2HPO40.50,FeSO40.01,trace element solution 2.00mL,pH7.5.

Nitrite denitri?cation medium(NDM,g/L of distilled water): NaNO20.28,sodium succinate3.16,MgSO4á7H2O0.20,CaCl20.01, EDTA0.07,KH2PO40.50,Na2HPO40.50,FeSO40.01,trace element solution2.00mL,pH7.5.

Hydroxylamine oxidation medium(HO,g/L of distilled water): hydroxylamine0.165,sodium succinate2.36,KH2PO40.50,Na2H-PO40.50,MgSO4á7H2O0.20,NaCl30.00,trace element solution 2.00mL,pH7.5.

Trace element solution(Joo et al.,2005)(g/L of distilled water): EDTAá2Na57.10,ZnSO4á7H2O3.90,CaCl2á2H2O7.00,MnCl2á4H2O 1.00,FeSO4á7H2O 5.00,(NH4)6Mo7O24á4H2O 1.10,CuSO4á5H2O 1.60,CoCl2á6H2O1.60,pH6.0.

LB medium(g/L of distilled water):Tryptone10,Yeast extract5,

NaCl10,pH7.5.For preparation of LB plates,1.5%(w/v)agar was added.

2.3.The qualitative assay of heterotrophic nitri?cation and

denitri?cation of strain L7

One milliliter pre-culture of strain L7in LB overnight was inoc-ulated into100mL HNM and100mL DM,respectively.The cul-tures were incubated for5days at30°C on a rotary shaker at 160r/min and then centrifuged at8000r/min for5min.NOà

and

NOà

in supernatants were qualitative detected by Griess–Romijn reagent and diphenylamine reagent(Xu and Zheng,1986), respectively.

2.4.Analytical methods

Nitrite was determined using N-(1-naphthyl)-1,2-diaminoe-thane dihydrochloride spectrophotometry(Mahmood et al.,2009). Ammonium was determined by the method of Nessler’s reagent spectrophotometry(Zhang,2009).Hydroxylamine was measured colorimetrically according to Frear and Burrell(1955).Bacterial growth was determined by monitoring the optical density at 600nm(OD600)using a spectrophotometer(UV-7200,UNICO, Shanghai).Biomass nitrogen was measured by Center for Environ-mental Quality Test Center(Tsinghua University).For strain L7,the obtained results indicated that the relationship between OD600 and biomass nitrogen(N bio)could be expressed as N bio(mg/ L)=9.026?OD600.

2.5.Detection of gaseous nitrogen compounds

For these experiments,strain L7was incubated in100mL med-ium(HNM,or DM,or NDM)containing50%15NH4Cl,or50% K15NO3,or50%Na15NO2(by atomic fraction,Spectra Corp.,USA), respectively,in250mL serum bottle sealed with a robber stopper at30°C on a rotary shaker at160r/min.N2O or N2from headspace was detected after hermetic incubation for10days according to Ai et al(2011).N2O was detected by GC–MS(Agilent,USA)with50l L upper gas using100l L gastight syringe,and N2was measured by GC–IRMS(Thermo Fisher Scienti?c,USA)with5l L upper gas using 10l L gastight syringe.Both gas detection devices were equipped with GS–Carbon Plot(30m?0.32mm?3.0l m,Agilent,USA).

2.6.Single-factor experiments to study the factors in?uencing the nitri?cation performance of strain L7

Single-factor experiments were conducted for studying the het-erotrophic nitri?cation characteristics of strain L7under different culturing conditions,including carbon source,C/N ratio,pH,tem-perature,salinity and ammonia concentration.

In carbon source experiments,sodium succinate,sodium acetate, sodium citrate,sodium pyruvate,potassium sodium tartrate and glucose were employed as sole carbon source,respectively.The other experiment conditions were as follows:initial nitrogen con-centration140mg/L,C/N7,initial pH7.5,NaCl30g/L,culturing tem-perature30°C,shaking speed160r/min.In C/N ratio experiments, the content of carbon source was changed in order to adjust C/N ratio to2,4,6,8,10,15and20,respectively,by?xing nitrogen concentra-tion at140mg/L.The other culturing conditions were the same as the carbon source experiments.The effects of initial pH,salinity and temperature on the ammonium removal were also investigated in the optimum medium from carbon source test and C/N test.The initial pH was adjusted to5,6,7,8,9and10using6mol/L HCl or 10mol/L NaOH.The salinity was set at0,10,20,30and40g/L NaCl. Culturing temperature was adjusted to20,25,30and37°C.Initial

36Q.-L.Zhang et al./Bioresource Technology108(2012)35–44

ammonium nitrogen concentration was adjusted to80,400, 1000mg/L,representing low,mediate and high ammonium concen-trations,respectively;and sodium succinate content varied accord-ingly to keep C/N ratio at7.All of the above experiments were conducted in triplicate with inoculation size of2%(v/v),and non-seeded samples and seeded without nitrogen source samples were also conducted as controls.Unless otherwise stated,all the hetero-trophic nitri?cation experiments were conducted at shaking speed 160r/min for108h.Then strain growth(OD600),and the contents

of ammonium nitrogen(NHt

4-N)and nitrite(NOà

-N)were

determined.

2.7.Orthogonal test to study the factors in?uencing the denitri?cation performance of strain L7

Orthogonal tests were designed and analyzed with SPSS statis-tical software(SPSS16.0version for windows,SPSS,Inc.,Chicago, IL,USA)to illustrate the effect of different factors on the aerobic denitri?cation performance of strain L7.The selected factors and

their levels were detailed in Table1.Differences were considered to be signi?cant when P<0.05.Cells of strain L7grown in LB over-night were harvested by centrifugation at8000r/min for5min, washed twice with sterile saline solution,and then re-suspended in sterile saline solution.1mL of cell suspension was inoculated into100mL NDM in250mL?ask.All the experiments were done in triplicates.

3.Results and discussion

3.1.The identi?cation of strain L7

Strain L7was Gram-positive.Colonies were creamy white con-vex opaque with regular edges,sticky not conducive to be picked and2–5mm in diameter after incubation for48h at30°C on LB plates.The results of API20NE test indicated that strain L7could utilize D-glucose,L-arabinose,D-mannose,D-mannitol,maltose, potassium gluconate and malic acid.Strain L7was positive for methanol utilization and activities of catalase,oxidase,protease, amylase,alkaline phosphatase,esterase(C4),esterase lipase(C8) and naphthol-AS-BI-phosphohydrolase;and negative for urease and b-galactosidase activity.All these properties were in agree-ment with those described for B.methylotrophicus CBMB205T (Madhaiyan et al.,2010),except of a-glucosidase activity.

Almost complete16S rRNA gene(1422nt)of strain L7was ampli?ed and sequenced.It was deposited in GenBank under accession number of JN635497.Homology searches revealed that strain L7was related to members of genus Bacillus,and showed highest sequence similarity(100%)to B.methylotrophicus CBMB205T.Phylogenetic tree(Fig.1)further indicated that strain L7together with B.methylotrophicus CBMB205T formed a distinct linkage in the tree with99%bootstrap https://www.360docs.net/doc/d516024526.html,bining above results,strain L7was identi?ed as B.methylotrophicus L7.So far, there was no report about the heterotrophic nitri?cation–aerobic denitri?cation of this species.

3.2.The property of heterotrophic nitri?cation and aerobic

denitri?cation of strain L7

The qualitative assay results(data not shown)indicated that strain L7could utilize ammonium to produce nitrite whereas ni-trate was not detected,indicating that L7was able to heterotrophic nitrify.Nitrite was the dominant product in the process of hetero-trophic nitri?cation(Castignetti and Hollocher,1984;Verstrae and Alexande,1972),while nitrate was produced mainly by fungi (Marshall and Alexander,1962)and just a few bacteria(Joo et al., 2005;Yang et al.,2011).When strain L7was cultivated in HNM, there was not signi?cant increase in amount of hydroxylamine, an important intermediate in nitri?cation(Lees,1952),possibly due to the instability of hydroxylamine and fast transformation to the downstream intermediates such as nitrite.When nitrate was used as sole nitrogen source(in DM),the production of nitrite indicated that strain L7could denitrify nitrate to nitrite or gaseous nitrogen compounds.

3.3.Detection of gaseous nitrogen compounds

GC–MS results showed that N2O was produced on both nitrate (Fig.2A)and nitrite(Fig.2B)served as substrate,respectively. The results also indicated that strain L7emit N2O when15NH4Cl (HNM,Fig.2C)and15NH4Cl plus hydroxylamine(HNM plus hydroxylamine,Fig.2D)as nitri?cation substrates.N2O isotopic abundance ratios(Fig.3)showed that the labeled15,14N2O, 15,15N

O did appear in the headspace gas of HNM sample,although the abundance of N2O from NH4Cl was far less than that of nitrate, nitrite and NH4Cl plus hydroxylamine(Fig.2).These results sug-gested that strain L7was a heterotrophic nitri?cation–aerobic denitri?er.It could not only aerobically denitrify nitrate or nitrite to form N2O,but also nitrify ammonia to form N2O,and the later was rarely reported in other strains.Only a few strains were described with nitri?cation of ammonia to form N2O,such as T. pantotropha(Arts et al.,1995)and A.faecalis No.4(Joo et al., 2005),however,non of them was Gram-positive.Strain L7was

Table1

L16(4)4orthogonal test design for denitri?cation of strain L7.

Test no.Factor A(shaking

speed,r/min)

Factor B(C/

N ratio)

Factor C

(salinity,g/L)

Factor D

(initial pH)

150(A1)5(B1)0(C1)6(D1) 2100(A2)10(B2)10(C2)D1 3150(A3)15(B3)20(C3)D1 4200(A4)20(B4)30(C4)D1

5A4B2C17(D2) 6A3B1C2D2

7A2B4C3D2

8A1B3C4D2

9A2B3C18(D3) 10A1B4C2D3

11A4B1C3D3

12A3B2C4D3

13A3B4C19(D4) 14A4B3C2D4

15A1B2C3D4

16A2B1C4D4

Q.-L.Zhang et al./Bioresource Technology108(2012)35–4437

the?rst reported Gram-positive bacterium possessing this function.

The N2O might derive from two pathways.First,N2O was emit-ted as the byproduct in the process of oxidation of hydroxylamine into nitrite(Otte et al.,1999).Second,it was produced from the ni-trite through the pathway of aerobic denitri?cation.Much more amounts of non-labeled N2O was detected when5mM hydroxyl-amine was added into15N–HNM than that from4mM nitrite as substrate in DM(Fig.2B,D).This data suggested that oxidation of hydroxylamine and aerobic denitri?cation occurred simultaneous in strain L7.

N2,the end product of denitri?cation,was also detected when the labeled nitrite was used as substrate,for that the d15N/14N ratio of the labeled sample reached8.343,much higher than that of the blank control with d15N/14N of0.081.When d15N/14N>1,it can be concluded that the15N2was produced according to the detection accuracy(Ai et al.,2011).However,15N2was not detected with la-beled nitrate and ammonia as substrates.

The above gas-producing patterns of strain L7were different

from those strains ever reported.For example,T.pantotropha could utilize NHt

,NOà

,respectively,to produce more N2O than N2

(Arts et al.,1995).A.faecalis No.4could convert NHt

-N to more N2 than N2O(Joo et al.,2005),whereas another A.faecalis strain was able to emit equivalent amounts of N2and N2O(Robertson et al., 1995).Moreover,when nitrate or nitrite was employed as sole nitrogen source,A.faecalis strain TUD could denitrify nitrite,but not nitrate,to N2in anaerobic conditions,while N2O was not de-tected under anaerobic conditions(Vanniel et al.,1992).All of these strains were Gram-negative.Up to date,to our knowledge, the capability of denitrifying nitrite to N2and denitrifying nitrite and nitrate to N2O in aerobically growing culture has never been reported in Gram-positive bacteria.Whether N2O or N2or both gases was produced during the denitri?cation process depended largely on the oxygen tension(Lloyd et al.,1987).Paracoccus halo-denitri?cans produced N2in the culture condition absence of oxy-gen,while as the oxygen tension gradually increased,the

incubation samples,un-seed controls and normal air samples(curves from top down,respectively).The15N-labeled 15NH

Cl plus unlabeled hydroxylamine(D).

denitri?cation gaseous product converted to N2O because of the inhibition of oxygen to nitrous oxide reductase(Hochstein et al., 1984).Whether this theory could explain the phenomenon ob-served in this study or not,needed to be further studied.

Based on the detection of intermediate and end products,a spe-ci?c inorganic nitrogen dissimilation pathway of heterotrophic nitri?cation–aerobic denitri?cation by strain L7was proposed,as shown in Fig.4,which was largely consistent with that assumed by Richardson et al.(1998)except the mutual conversion between nitrate and nitrite and the production of NO in the process of ni-trite conversion to N2O.

3.4.Nitri?cation characteristics of strain L7under various conditions 3.4.1.Effect of carbon source

Carbon source was considered to be an important factor in?uencing heterotrophic nitri?cation ability.The results in Table2showed that sodium succinate and glucose could well

support the growth of strain L7,OD600reached0.42and0.39, respectively;meanwhile,strain L7exhibited ef?cient nitrifying abilities,the total NHt

-N removal percentage were48.00%and 38.40%,respectively,despite there were about 3.79mg/L

(2.70%)and3.53mg/L(2.50%)of NHt

-N consumed as nitrogen

source for its growth,respectively.An accumulation of NOà

-N of0.22±0.02mg/L was observed on sodium succinate,but not on glucose,implying that there might be different mechanisms for strain L7to perform heterotrophic nitri?cation on different carbon sources.These results were consistent with those re-ported for strain Bacillus MS30(Mevel and Prieur,2000).Strain MS30grew quite well(97.60%of input nitrogen was converted to biomass nitrogen),but showed a relatively poor nitri?cation ability with nitri?cation rate of0.03l mol NOà2ámgà1dry weight when glucose was served as carbon source.The results in Table 2also indicated that other carbon sources did not support

the Fig.2(continued)

growth of strain L7,and worse nitri?cation performances were also observed.Accordingly,sodium succinate was employed in the following experiments.

3.4.2.Effect of C/N ratio The NH t4-N removal percentage was not signi?cantly different among C/N 2–20as shown in Fig.5A,all of which can reach a level of 50%in C/N ratio 4–15,with the highest removal percentage of 58.00%at C/N ratio 6.Even if the C/N ratio was as high as 20,which

was too high for the growth of autotrophic nitrifying bacteria,strain L7still exhibited satisfying nitri?cation ability with a NH t4-N removal percentage of 44.80%.The tolerance of strain L7to a wide C/N range expanded its application scope including the pig-gery waste with C/N of 4–7and the municipal land?ll leachate with high C/N (Kim et al.,2006).Limited formation of NO à2-N (<0.15mg/L)at different C/N ratios in strain L7would satisfy the well-functioning SND system in which little nitrite or nitrate accu-mulated (Third et al.,2005).These results suggested that C/N ratio did not play an important role in the process of heterotrophic nitri-?cation of strain L7.Take cost effectiveness into consideration,C/N 6was used in following experiments.

3.4.3.Effect of temperature

Fig.5B showed that the ammonium removal ability was in-creased as the temperature rising.The NH t4-N removal percentage increased from 6.10%at 20°C to 78.40%at 37°C.This might be

due

ratios in 50%15NH 4Cl medium,indicating the production of 14,15N 2O and 15,15N 2O.Blank control (A)and 50%nitri?cation and denitri?cation pathway of strain L7.

Fig.5.Effect of factors on the growth and nitri?cation ability of strain L7.C/N ratio (A),temperature(B),salinity(C)and initial pH(D).N,strain growth; accumulation of NO2;?,removal percentage of NHt

-N.Values are means±SD

(error bars)for three replicates.

strain L7.The tolerance to high ammonia load made this strain a promising candidate in treatment of municipal land?ll leachate with about 1000mg/L NH t4-N (Kim et al.,2006).This tolerance to ammonia was much higher than that of another heterotrophic nitri?er,Providencia rettger YL,which was reported to maximum tolerance to 300mg/L NH t4-N (Taylor et al.,2009).In traditional high ammonium load wastewater treatment,the nitrite accumula-tion was inevitable during to the high free ammonia which would inhibit the activity of nitrite oxidation microbes (Yun and Kim,2003).In this study,the amount of accumulated nitrite was 0.15±0.02mg/L when 1121.21mg/L NH t4-N was supplied.3.5.Nitrifying performance of strain L7under the optimal conditions The NH t4-N content gradually reduced from initial 146.71–38.29mg/L after incubation for 9days,and the maximum NH t4-N removal rate of 51.58mg/L/d was achieved,as shown in Fig.6A.The accumulation of nitrite was maintained at low level (<0.1mg/L).The growth of strain L7kept at a stable level (OD 600ca.1.30)from day 4.

Take the practical application in marine aquaculture into con-sideration,30g/L NaCl was added into the medium.Then a differ-ent pattern of strain growth and NH t4-N removal was observed,as shown in Fig.6B.The highest growth was achieved at 54h (OD 600ca.1.20)with NH t4-N removal percentage of 51.03%,followed by a decline growth phase.The accumulation of nitrite began at 36h and maintained a sustained increase to a maximum amount of 0.11mg/L.The calculated maximum NH t4-N removal rate was 3.08mg/L/h during the https://www.360docs.net/doc/d516024526.html,pared with that in optimal medium,the higher removal rate in salinity medium made strain L7suitable for the marine aquaculture wastewater treatment.However,the maximum NH t4-N removal percentage was lower in salinity medium than that in optimal medium.The reason for these changes might be that the sensitivity of strain L7to the external high osmotic pressure environment caused by the high salinity at the stage of stationary phase and decline phase,as described pre-viously by Rysgaard et al.(1999).

3.6.Orthogonal Test for the aerobic denitri?cation performance of strain L7

The results of orthogonal tests were tabulated in Table 4and 5.

The maximum NO à2-N removal rate was 5.81mg/L/d calculated from Table 4.

Fig.6.Nitri?cation performance of strain L7under optimal culture conditions (A)

and 30g/L NaCl (B).N ,strain growth;j ,NO à2;?,NH t

4-N.

Table 4

Results of orthogonal tests for aerobic denitri?cation performance of strain L7from day 4–7.

Day 4Day 5Day 6Day 7OD 600

NO à2-N (mg/L)OD 600NO à2-N (mg/L)OD 600NO à2-N (mg/L)OD 600NO à2-N (mg/L)10.0366.12±0.650.0263.63±0.000.0265.29±0.520.0266.22±0.7820.0166.86±0.130.0166.03±0.520.0167.14±1.050.0167.05±0.3930.0261.41±4.970.0265.29±0.780.0265.20±0.390.0265.20±0.1340.0465.20±0.650.0365.20±0.650.0365.48±0.520.0364.74±1.0550.0166.95±0.260.0167.42±1.440.0167.79±1.18à0.0168.43±0.5260.1065.94±0.920.1464.64±3.000.2457.81±2.220.1557.44±0.1370.0766.86±0.920.0965.66±1.050.2459.38±2.610.3451.89±0.6580.0666.31±0.130.0866.22±0.000.1561.87±0.390.1658.92±0.1390.0265.48±0.260.0564.92±0.520.0764.92±0.780.0767.14±0.26100.2658.55±1.700.3053.45±0.650.3947.64±2.220.4044.87±2.48110.0666.68±0.390.0566.77±0.780.0666.40±0.000.1066.40±0.26120.0565.48±1.050.0466.22±0.520.0565.38±0.390.05261.60±5.75130.0467.05±0.920.1163.54±4.840.1454.39±4.700.08951.34±0.92140.0967.05±0.130.1065.48±1.310.1563.17±1.440.22760.02±0.13150.0567.79±0.390.0567.23±0.130.0864.92±0.260.16161.41±1.5716

0.04

67.23±1.70

0.04

67.51±0.52

0.04

65.57±1.18

0.052

65.11±0.00

Values are means ±SD (error bars)for triplicates.

42Q.-L.Zhang et al./Bioresource Technology 108(2012)35–44

Variance analysis(Table5A)indicated that the signi?cant val-ues of factor A(shaking speed),B(C/N ratio),C(salinity),D(pH) were0.046,0.009,0.051and0.070,respectively.Four factors had impact on the nitrite removal in the order of B>A>C>D,while factor A and B,namely dissolved oxygen(DO)and C/N ratio,had signi?cant difference with the value of P<0.05.The optimal condi-tions for nitrite removal according to the results of Estimated Mar-ginal Means(Table5B)were shaking speed50r/min,C/N20, salinity was10g/L NaCl and initial pH7–8.

C/N and DO were the most important factors in?uencing the performance of aerobic denitri?cation(Huang and Tseng,2001). Carbon source were indispensable for the growth of microorgan-isms and energy-producing,and at a certain carbon source concen-tration range,the more carbon source,the faster bacterial growth and the higher denitri?cation rate(Patureau et al.,2000).The essential difference between the emerging aerobic denitri?cation and the traditional one was whether the denitrifying microbes were vulnerable to oxygen.The aerobic denitri?er still exhibited satisfying denitri?cation activity even in the condition that the oxygen concentration approaching or exceeding the air saturation (Lloyd et al.,1987).Microvirgula aerodenitri?cans could denitri?ed at100%air saturation(7mg/L DO)with a critical DO point of 4.50mg/L,i.e.the deniti?cation ability gradually increased with the reduction of DO below the critical point while the denitri?ca-tion activity was not affected by the change of DO above the critical point(Patureau et al.,2000).The optimal DO for strain L7was 4.82mg/L;lower or higher than this,the denitri?cation activity was inhibited.The relationship between DO and the shaking speed was tabulated in Table6.4.Conclusion

In this study,Bacillus methylotrophicus L7was?rst reported

Gram-positive bacterial strain to denitrify nitrite to N2and denitri-fying nitrite and nitrate to N2O in aerobic condition.Strain L7 exhibited ef?cient heterotrophic nitrifying–aerobic denitrifying ability with maximum NHt

-N removal rate of51.58mg/L/d and

maximum NOà

-N removal rate of5.81mg/L/d.Besides,more than 90mg/d ammonia removal ef?ciency was obtained even in the ex-tremely high ammonia load(>1000mg/L).Therefore,L7is a prom-ising candidate in the extensive application of various pollution control system including municipal wastewater,aquaculture industry,etc.

Acknowledgement

This work was supported by grants from the Knowledge Inno-vation Program of the Chinese Academy of Sciences(No.KJCX2-YW-L08).

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Table5

Analysis results of orthogonal tests with software SPSS16.0.

A.Tests of between-subjects effects

Dependent variable:NITRITE

Source Type III sum of

squares d f Mean

square

F Sig.

Corrected

model

732.26a1261.0214.630.019

Intercept60224.84160224.84 1.44?1040.000

A(shaking

speed)

122.71340.919.810.046

B(C/N ratio)404.463134.8232.330.009

C(salinity)115.20338.409.210.051

D(pH)89.89329.967.180.070

Error12.513 4.17

Total60969.6116

Corrected

total

744.7715

B.Estimated marginal means

Level A B C D

157.57563.72063.44065.385

262.88865.93856.88359.423

360.07062.89061.64060.668

464.87552.86063.44559.933

Optimal level50r/min C/N2010g/L NaCl pH7

Dependent variable:Nitrite.Std.Error:1.021.Con?dence interval:95%.

a R2=0.98(adjusted R2=0.92)

Table6

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NaCl(g/L)DO AS DO AS DO AS DO AS

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20 4.6062.8 6.1784.3 6.7692.4 6.8193.2

30 4.1957.3 6.1583.8 6.7191.6 6.7592.2

DO:dissolved oxygen(mg/L).AS:air saturation(%).

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