at

, Warbinhina

gjian

rophic n was: Nbon cou

te reductase were detectable. The putative nitrogen removal process by the s: NH4+?NH2OH?NO2?NO3, then NO3?NO2?N2. Biological activated

concentratio n in surface water in China increased gradually during

PR China, http://datacenter .mep.gov.cn/ ). To guarantee safe drink- ing water, the Ministry of Health of the PR China requires anammonium concentration of lower than 0.5 mg/L accordin g tothe Standards for Drinking Water Quality (GB5749-2006). Tradi-

sirable chlorinated by-product. before chlorina- me et al.,two-step p

4 O2-N anNO2-N into NO3-N occurs. However , low temperature s gedrastical ly affect nitrifying bacterial process rate. The optimperature for nitrication in pure culture ranges from 25 to 35 C.Below 15 C, the nitrication rate drops sharply, which could result in nitrite accumulation (Andersson et al., 2001; Groenew eg et al.,1994; Kors et al., 1998 ). During winter, the average temperature in Northern China is always below 10 C, so nitrifying bacteria cant play an effective role in nitrication process.

Ammonium removal by heterotrophic microorgan isms has usu- ally been reported to oxidize NH4+-N to NO2-N or NO3-N and

Corresponding author. Address: Box No. 2602, School of Municipal and Environmental Engineering, 73 Huanghe Road, Harbin Institute of Technology,Harbin 150090, PR China. Tel.: +86 13904512510; fax: +86 451 86283003.

Bioresource Technology 137 (2013) 147152

Contents lists available at

Biore source T

elsE-mail addresses: weiguanglihit@hotmail.com, hitlwg@126.com (W. Li).the past ten years. For example, annual average ammonium con- centration of the Songhua River was 0.51 mg/L in 2004, but it has increased to 0.85 mg/L by 2011 (The data were calculated accord- ing to a report of the Ministry of Environmental Protection of the

The removal of ammonium by biodegradat iontion decrease short-term chlorine demand (HolAmmonium removal by nitrifying bacteria is ain which sequential oxidation of NH +-N into N0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.03.0941999 ).rocess

d then nerally al tem- AmmoniumDrinking water Low temperature Microbacterium sp.

carbon attached with the strain SFA13 could effectively remove ammonium in surface water with the rate of 2.68 0.273.16 0.25 mg NH4

+-N/L/h at C/N 210, temperature 10 C, and DO > 5.2 mg/L. 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Ammonium in raw surface water used for drinking water pro- duction is undesirable, because it might cause taste and odor prob- lem, as well as decrease disinfecti on efciency. While ammonium

tional drinking water treatment process used in China, such ascoagulation , sedimentati on and ltration, could not effectively re- move ammoniu m in raw water. Break-point chlorination is a clas- sical process used for ammonium removal. However, the increase of chlorine content in water could stimulate the formation of unde- Keywords:Heterotrophic nitrication

nitrite reductase and nitrastrain SFA13 was as follow Microbacterium sp. SFA13 with heterot The putative nitrogen removal process Strain SFA13 attached on activated car

a r t i c l e i n f o

Article history:Received 22 January 2013 Received in revised form 9 March 2013 Accepted 13 March 2013 Available online 21 March 2013 itrifying ability at 5 C.H4+?NH2OH?NO2?NO3, then NO3?NO2?N2.ld effectively remove ammonium in surface water at C/N 210, temperature 10 C, and DO > 5.2 mg/L.

a b s t r a c t

The strain SFA13, isolated from Songhua River, demonstrates ability to convert ammonium to nitrogen under aerobic conditions at low temperature. On the basis of 16S rRNA gene sequence, the strain SFA13 was a species in genera Microbacterium. The isolate showed unusual ability of autotrophic nitri-cation with the ratio of 0.11 mg NH4+-N/L/h at 5 C. Ammonium was consumed by the strain SFA13 with the biodegrad ation of organic carbon and without nitrite or nitrate accumulation. NO3-N or NO2-N was reduced by the strain SFA13. The denitrication ratio was 0.24 mg NO3-N/L/h. Hydroxylamine oxidase,h i g h l i g h t sRemoval of ammonium in surface water isolated Microbacterium sp. strain SFA13

Duoying Zhang a,c, Weiguang Li a,b,, Xiaofei Huang aa School of Municipal and Environmental Engineering, Harbin Institute of Technology, Hb State Key Laboratory of Urban Water Resource and Environment, Harbin 150090, PR Cc Department of Food Science and Environmental Engineering, East University of Heilon

journal homepage: www.low temperature by a newly

en Qin a, Miao Liu a

150090, PR China g, Harbin 150086, PR China

SciVerse ScienceDi rect

echnology

evier .com/locate /bior tech

echsimultaneou sly covert NO2-N or NO3-N to N2O and/or N2 (Chenet al., 2012; Khardenavi s et al., 2007; Robertso n et al., 1988 ). Pre- vious studies reported that heterotrophic bacteria could remove ammonium at low temperature (Zhang et al., 2011c ). Because het- erotrophic microorganism s often require high concentratio ns ofammonium and organic carbon (ammonium above 50 mg/L and C/N above 8), they are generally used in wastewater treatment (Joo et al., 2005,2006; Kim et al., 2005; Taylor et al., 2009 ). In the present study, a heterotrophi c strain, designated SFA13, was iso- lated from Songhua River which is one of main drinking water re- sources for Harbin citizens. The purpose of the study is todetermine the bacterial ability to remove ammonium under low ammonium and temperat ure condition.

2. Methods

2.1. Isolation of heterotrophic nitrifying bacteria

One liter raw water taken from Songhua River was prepared asenrichment medium (pH 7.2) by adding 5 g tryptone, 5 g yeast ex- tract and 8 g NaCl and incubated at 30 C for 3 days. Then, serial dilutions were prepared and spread on solidied enrichme nt med- ium containing 2% (w/v) agar and incubated at 30 C until visible colonies had formed. Separate colonies were chosen and incubated in inorganic medium solution (1.0 g/L Na2HPO4, 2.0 g/L KH2PO4,0.01 g/L MgSO 47H2O, 0.005 g/L CaCl 22H2O, 1.0 g/L NH4Cl, pH7.2) for 3 days at 30 C. The growing bacteria were gradually culti- vated at descending temperature s (30, 20, 15, 10, 8, 6, 5 C). Bacte- ria grew at 5 C were chosen and individually tested for ammonium removal efciency.

The isolated bacterium was inoculated into the basic medium (contain 0.5 g/L NH4Cl, 1.0 g/L CH3CH2ONa, 0.05 g/L MgSO 47H2O,0.2 g/L K2HPO4, 0.12 g/L NaCl, 0.01 g/L MnSO 44H2O, 0.01 g/L FeSO 4,pH7.0) and cultured at 5 C for 34 days when the population amount reached to above 107 cells/mL. One liter liquid culture was collected and centrifuged at 6000 rpm for 5 min. The sedimen- tation were collected and washed with sterile pure water for 3times. Then the collected bacteria were inoculated to triplicate 50 mL sterile ammoniu m sulfate solution (about 5 mg/L NH4+-N).The same sterile ammonium sulfate solution without inoculate was used as control. After shaking at 5 C, 140 rpm/min for 0.5, 2,4, 6, 8, 10, 15, 20, 25 and 30 h, the samples were collected respec- tively to detect the remaining ammonium concentration for assessing the ammonium removal efciency.

2.2. Identication of the selected bacteria

Initial identication schemes were performed with biochemical tests as suggested by the Bergeys Manual of Systematic Bacteriol- ogy and Systematic Determinat ive Manual of General Bacteria (Garrity et al., 2004 ). The sequence s of the 16S rDNA fragments were extracted and sequenced by TAKARA biotechnolo gy Dalian Co., Ltd. (Dalian, China). The 16S rDNA sequences were compared with that of other microorgan isms by way of BLAST (http://blas-t.ncbi.nlm.n ih.gov/Blast.cg i).

2.3. Assessment of nitrifying characteri stics

The isolated bacterium was inoculated into the basic medium and cultured at 5 C for 34 days when the population amount reached to more than 107 cells/mL. One liter liquid culture was col- lected and centrifuged at 6000 rpm for 5 min. The sedimentation were collected and washed with sterile pure water for 3 times.

148 D. Zhang et al. / Bioresource TThen the collected bacteria were inoculated to triplicate 50 mLsterile ammonium sulfate solution (about 5 mg/L NH4+-N). Car- bon/Nitrogen (C/N) was adjusted to 4 with sodium acetate as car- bon source. The same sterile solution without inoculate was used as control. The medium and headspace were subsequently evacu- ated and aerated with pure oxygen at constant pressure (0.3 MPa) for 810 min and aerated with O2He (95:5) gas. After shaking at 10 C, 140 rpm/min for 0.5, 2, 4, 6, 8, 10, 15, 20, 25and 30 h, the liquid samples were collected respectively to de- tected NH4+-N, NH2OH, NO2-N, NO3-N and TOC concentratio n.The bacterial contents were determined by measuring OD600. Atthe end of experiment, the gas samples were collected to detect N2 and N2O production.

2.4. Assessme nt of nitrite and nitrate removal

Nitrate and nitrite were used to elucidate the denitricationprocess of the isolate. Based on the Standards for Drinking Water Quality (GB5749-2006), NO2-N and NO3-N were adjusted to10 mg/L respectively. Carbon/Ni trogen (C/N) was adjusted to 4with sodium acetate as carbon source. After cultured in basic med- ium (the population amount reached to more than 107 cells/mL),the cells were collected and inoculated intro triplicate 50 mL ni- trate or nitrite solution with sodium acetate. After shaking at10 C, 140 rpm/min for 0.5, 2, 4, 6, 8, 10, 15, 20, 25 and 30 h, the samples were collected respectively to detected NO2-N, NO3-Nand TOC concentratio n. The bacterial contents were determined by measuring OD600.

2.5. Assessme nt of enzyme activity

The isolate was prepared as previously described for detecting hydroxylam ine oxidase (HAO) activity (Wehrfrit z et al., 1993 ).The HAO activity was determined by the reduction of potassium ferricyani de at 400 nm (Otte et al., 1999 ). The activity of nitrite reductas e (NiR) and nitrate reductas e (NR) were analyzed as previ- ously described (Alefounder and Ferguson, 1980; Bell et al., 1990 ).

2.6. Biological activated carbon experiment of the strain SFA13

Approxim ately 109 cells of the strain SFA13 were collected and immobilized on 5.00 g sterile granular activated carbon (GAC) par- ticles to form biologica l activated carbon (BAC) (Pesce and Wun- derlin, 1997; Zhang et al., 2011a ). The GAC particles were purchase d from Tangshan Co., China (http://www.jianxincar- bon.com). 5.00 g BAC samples were immersed in 50.00 mL sterile ammoniu m solution (5 mg/L NH4+-N) for 3 days to adapt to oligo- trophic condition. Then the solutions were discharged, and BAC samples were collected to degrade 5 mg/L ammonium solution (50.00 mL). The contact time was adjusted to 30 min (Sere-dyn ska-Sobe cka et al., 2006 ). For evaluating the effect of carbon re- source on the ammonium removal, glucose, sodium citrate, sodium acetate, glycerol and calcium carbonat e were added into the ammoniu m solution. Four-times the quantity of C (20 mg/L) was provided in the solution. The parameters were chosen based onthe worst water quality condition in Northern China. The optimal carbon source was selected to study the effect of Carbon/Ni trogen (C/N) ratio on ammonium removal. The Carbon/Nitr ogen (C/N) ra- tios were adjusted to 0.2, 0.5, 1, 1.5, 2, 4 and 10, and xed the ammoniu m concentr ation at 5 mg/L, temperature at 10 C with 140 rpm/min shaking. For investigatin g the inuence of tempera- ture on ammonium removal, the temperat ures were controlled at5, 6, 8, 10, 15 and 20 C, and xed the ammonium concentration at 5 mg/L, temperature at 10 C with 140 rpm/min shaking. For testing the inuence of dissolved oxygen on ammoniu m removal,the dissolved oxygen was adjusted by controlling rotating speed

nology 137 (2013) 147152of shaker to values of 0 rpm/min (DO2.87 mg/L), 100 rpm/min (DO3.54 mg/L), 120 rpm/min (DO4.33 mg/L), 140 rpm/min (DO5.21 mg/L), 160 rpm/min (DO5.82 mg/L) and 180 rpm/min

(DO6.05 mg/L), and xed the ammoniu m concentratio n at 5 mg/ L, temperature at 10 C with 140 rpm/min shaking.

2.7. Analytical methods

The growth of isolates was tested by spectrophotom etry at awavelength of 600 nm. The ammonium concentratio n was deter- mined colorimetric ally according to Water quality-Deter mination of ammonium -Nesslers reagent colorimetric method (GB7479-87). NH2OH was analyzed by indirect spectrophotom etry (Mingand Fengke, 1999 ). The nitrate and nitrite were analyzed using N-(1-naphthalene)-diaminoethane photometry and phenol disul- phonic acid methods according to the State Environm ental Protec- tion Administr ation of China (Ministry of Environm ental Protection of the Peoples Republic of China, 2002 ). TOC were analyzed by

SFA13 had unusual ability of autotrophic nitrication. Yang et al.(2011) also reported the autotrophic nitrication ability of Bacillussubtilis strain A1 with broad range of ammonium load (from 105.58 to 1014.17 mg/L).

It has been reported that the nitrication ratio of Pseudomona sstutzeri YZN-001 was approximat ely 0.3 mg NH4+-N/L/h at 4 C(Zhang et al., 2011c ), which was a little higher than the strain SFA13. However, the initial ammoniu m concentratio n was 106.3 mg/L, which was much higher than that of present study.In this research, the bacterial growth might be restricted in the low nutrient condition, which resulted in low ammonium removal rate. The previous studies also showed that heterotrophi c nitrify- ing activity was affected by nitrogen and carbon components (Bri-erley and Wood, 2001; Joo et al., 2005; Kim et al., 2005 ).

3.2. Identication and nitrication characters of the strain SFA13

0.70 Nitrate concentration TOC40

D. Zhang et al. / Bioresource Technology 137 (2013) 147152 149using an Aurora Combusti on Total Organic Carbon Analyzer 1030C (OI, America). Tests to detect the changes in N2 and O2respectively were conducted using gas chromatography (GC-14B,Shimadzu, Japan). Gas samples were extracted using a 100 lLair-tight glass syringe. The carrier gas Ar had a ow rate of20 mL/min. Column, injector and detector temperat ures were 80,100 and 110 C, respectively .

2.8. Statistical analysis

The ammonium removal rate formula is (C0-Cn)/h. C0 is initial concentratio n of NH4+-N (NO2-N or NO3-N). Cn is the nal con- centration of NH4+-N (NO2-N or NO3-N) at n hour. h is the time of SFA13 treatment.

3. Results and discussion

3.1. Ammonium removal by isolate strain

In the study, the strain SFA13 was found to remove ammonium at 5 C. The ammonium removal process of the strain SFA13 isshowed in Fig. 1. The initial ammoniu m concentratio n was 5.09 0.05 mg/L. After 30 h, 1.85 0.04 mg NH4+-N/L remained inthe solution. The average nitrication ratio at 5 C was 0.11 mgNH4+-N/L/h. High ammonium removal rate appeared in the initial 4 h, the average ammonium removal rate was 0.46 mg NH4+-N/L/h. In the initial 30 min, 11.39% NH4+-N was removed, and the re- moval rate was 1.16 mg NH4+-N/L/h. Then with the prolonging bio- degradation time, the removal rate decrease d. The maximum NH4+-N removal rate was 63.65%. Exceeding 30 h, ammonium con- centration in the solution was relatively stable with little change (the data were not shown). The results showed that the strain

0 5 10 15 20 25 301.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

Time (hours)

Amm

oniu

m c

once

ntra

tion

(mg/

L)Fig. 1. Ammonium removal at 5 C by the strain SFA13. Error bars: mean S.D. ofthree replicates.0 5 10 15 20 25 300.50

0.52

0.54

0.56

0.58

0.60

0.62

0.64

0.66

0.68

Bacterial content (OD

600 )

Time (hours)

0

5

10

15

20

25

30

35

Con

cent

ratio

n (m

g/L)The strain SFA13 was selected based on ammonium removal at5 C. The cells are short rods, Gram-positive , catalase- positive, oxi- dase-neg ative, non-motile and non-spore-form ing. The 16S rDNA sequence of the strain SFA13 was 1422 bp and submitted to NCBI with accession No. HM486420. Bootstrapped neighbor- joining relationship was estimate d with MEGA version 4.1 (Molecular Evo- lutionary Genetics Analysis [http://www.m egasoftware .net ]). The strain SFA13 was putatively identied as Microbacterium sp. Micro-bacterium species have been reported to reduce heavy metal inwater (Humphries et al., 2005; Mokashi and Paknikar, 2002 ) and remove organic chemical s (Sheng et al., 2009 ). However , their role in heterotrop hic nitrication was rarely reported. Hence, it is nec- essary to study the ammoniu m removal characterist ic of Microbac-terium sp. SFA13.

Microbacter ium sp. SFA13 exhibited ammonium removal ability in low NH4+-N concentratio n at 5 C. But NH4+-N could not be de- graded to zero after 30 h. For improving the ammonium removal efciency and evaluating the nitrication characterist ics of Micro-bacterium sp. SFA13, sodium acetate was added as carbon source (C/N = 4) and the temperature was enhanced to 10 C. The produc- tion of intermediates was tested, such as NH2OH-N, NO3-N and NO2-N. The results are shown in Fig. 2. The NH4+-N was consumed to 0.26 0.12 mg/L with the biodegradation of TOC after 30 h.Compare d with sole NH4+-N degradat ion (showed in Fig. 1), the average ammonium removal rate increased from 0.11 mg NH4+-N/L/h to 0.16 mg NH4+-N/L/h. Especially in the initial 30 min, the ammoniu m removal rate increased from 1.16 mg NH4+-N/L/h to

Bacteria content Nitrite concentrationFig. 2. Characteristics of nitrication by the strain SFA13 with the initial ammo- nium level of 5 mg/L at 10 C. Error bars: mean S.D. of three replicates.

0.66

ech0.64

0.66

0.68

0.70

Bact

Bacteria content Nitrite concentration Nitrate concentration TOC

30

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/L)

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600 )

B0.68

0.70 Bacterial content Nitrite concentration Nitrate concentration TOC

40

45A

150 D. Zhang et al. / Bioresource T1.64 mg NH4+-N/L/h. The results showed that the carbon source were important for heterotrophi c nitrication by the strain SFA13. With the decrease of ammonium and TOC, the OD600 in-creased slightly, indicating that low substrate was not enough for rapid growth of the strain SFA13. The ammoniu m removal in this study was mainly attributed to high population cells (OD600 higherthan 0.5) added at the beginning of experiment.

NH2OH was produced at a little higher concentratio n (average0.29 0.09 mg/L) than NO3-N and NO2-N. It is proposed that ammonium is initially oxidized to NH2OH. It has been reported that NH2OH production was directly related to changes in ammo- nium concentratio ns (Taylor et al., 2009 ). There was little NO3-Nand NO2-N accumulati on, which indicated that Microbacter ium sp.SFA13 has the capability of nitrogen removal. At the end of exper- iments, N2 and N2O production was tested using gas chromatogra- phy. N2O production was not detected. However, 9.23% 0.6 N2was found in the gas. The results indicated that the strain SFA13 had the ability of simultaneou s heterotrophi c nitrication and aer- obic denitrication. The denitrify ing product is N2.

3.3. Utilization of nitrite and nitrate by SFA13 under aerobic conditions

For evaluating denitrication of Microbacter ium sp. SFA13,NO3-N and NO2-N were used as sole nitrogen source for the strain SFA13 respectively , and sodium acetate was used as carbon source (C/N = 4). The nitrogen concentratio n decreased no matter

0 5 10 15 20 25 300.50

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0.60

0.62erial content (O

D600 )

Time (hours)

0

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cent

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n (m

g

Fig. 3. Changes in NO3, NO2, TOC and OD600 by Microbacterium sp. SFA13 at 10 C.Nitrate (A) and nitrite (B) were used as sole nitrogen source respectively. Error bars:mean S.D. of two replicates.whether using NO3-N or NO2-N as sole nitrogen source (Fig. 3).The results showed that either NO3-N or NO2-N could be re- duced by Microbacter ium sp. SFA13 under aerobic culture condi- tions. Approximately 70% of the nitrate was removed in 30 h. The denitrication ratio was 0.24 mg NO3-N/L/h. The capability of aer- obic denitrication by Rhodococcus sp. CPZ24 and Pseudomona sstutzeri YZN-001 were demonstrated by Chen et al. (2012) andZhang et al. (2011c). However, Alcaligen es faecalis NR and Acineto-bacter calcoaceticus HNR could not denitrify NO3 or NO2 (Zhaoet al., 2012, 2010a ), but removed NH4+-N in the formatio n of N2or N2O. In the study, when nitrate was added, there was noNO2-N accumulation (Fig. 3A). However, when nitrite was added,1.67 mg/L NO3-N was observed at the beginning of the nitrite re- moval process (Fig. 3B). The phenomenon was in accordance with the report of Zhang et al. (2011c). This observation caused by oxi- dation originating from the brief exposure to air.

3.4. Enzyme assay

For understand ing the pathway involved in the ammonium re- moval process by the strain SFA13, a preliminary study on the en- zyme activities of HAO, NiR and NR was conducte d under aerobic condition . 0.15 lmol/min HAO activity was detected. NiR and NRactivity were 0.21 and 0.44 lmol/min, respectivel y. In the study,NH2OH is the substrate of HAO (Otte et al., 1999 ), it was oxidized to NO2 and NO3, and subsequently reduced to N2 by NiR and NR.Although the nitrite and nitrate was too low to detect, it was not uncommon because the transfer of nitrite and nitrate may have been too rapid to allow for adequate detection (Taylor et al.,2009). It can be conrmed that the N2 production by the strain SFA13 was via nitrate and nitrite. This was quite different from the mechanis m of denitrication process of Alcaligen es faecalis No. 4, Acinetobacte r calcoaceticus HNR and Alcaligenes faecalis NR(NH4+?NH2OH?N2O/N2) (Joo et al., 2005; Zhao et al., 2012,2010a). The pathway is also different from the report of Richardsonet al. (1998) (NH4+?NH2OH?NO2?N2O?N2). It has been re- ported that Providencia rettgeri YL heterotrop hic nitrication pro- cess was as follows: NH4+?NH2OH?NO2?NO3 and the denitrication process was: NO3?NO2?N2 (Taylor et al.,2009). The nitrogen transformat ion process was similar to that ofMicrobacter ium sp. SFA13. This ammonium removal pathway testi- ed that different nitrogen removal pathways existed in the het- erotroph ic microorgan isms.

3.5. The effect of carbon source, temperature and dissolved oxygen onthe ammoniu m removal by the strain SFA13

Biologica l activated carbon (BAC) is effective in bio-adsorpt ion,biodegra dation and bio-regener ation (Gao et al., 2010; Zhang et al.,2011b). Thus it is a widely used drinking water treatment process.In the present study, Microbacter ium sp. SFA13 was attached onGAC particles to form BAC that was applied to study the effect ofcarbon source, temperature and dissolved oxygen on the ammo- nium removal by the strain SFA13. The experimental time was ad- justed to 30 min according to empty bed contact time (EBCT) ofBAC lter.

Glucose was generally used as carbon source for heterotrop hic nitrifying bacteria (Kim et al., 2005; Zhao et al., 2010b ). Acetate and citrate have been reported to make the medium more alkaline during nitrication, which was better for nitrication process (Bri-erley and Wood, 2001 ). According to the previous reports, glucose,sodium citrate, sodium acetate, glycerol and calcium carbonate were selected as carbon source for evaluating the optimal carbon

nology 137 (2013) 147152source for Microbacterium sp. SFA13. The results showed that the ammoniu m removal reached to the highest (2.07 mg NH4+-N/L/h)in acetate medium solution (Table 1). So sodium acetate was se-

cit

0.15

+-N (mg/L) Final NH4 -N (mg/L) Ammonium removal rate (mg NH4 -N/L/h)

6 4.01 0.15 1.80 0.33 1 4 2 9 5 3 3 1 6 5 9 9 5 7 1 0 1 6

Techlected as carbon source to detect the inuence of pH, C/N and DOon ammonium removal efciency.

Table 1Nitrication of ammonium in 30 min at 5 C under different carbon sources.

Glucose Sodium

Ammonium removal rate (mg NH4+-N/L/h) 1.43 0.23 0.95

Data are means S.D. of three replications.

Table 2Ammonium removal rate by Microbac terium sp. SFA13 under different condition.

No. C/N ratio Shaking speed (rpm) Temperature (C) Initial NH4

1 4 140 5 4.91 0.012 4 140 6 5.03 0.033 4 140 8 5.15 0.054 4 140 10 5.19 0.045 4 140 15 4.82 0.036 4 140 20 4.99 0.047 0.2 140 10 5.12 0.068 0.5 140 10 4.99 0.059 1 140 10 5.17 0.0210 2 140 10 5.28 0.0211 4 140 10 5.24 0.0412 10 140 10 4.88 0.0413 20 140 10 5.21 0.0414 2 0 10 4.97 0.0415 2 100 10 5.24 0.0316 2 120 10 5.09 0.0417 2 140 10 4.87 0.0518 2 160 10 5.07 0.0419 2 180 10 5.11 0.05

Data are means S.D. of three replications.

D. Zhang et al. / Bioresource The ammonium removal by Microbacter ium sp. SFA13 at differ- ent temperatures was investigated in ammoniu m solution (about5 mg/L) at C/N 4. The results are showed in Table 2. The ammonium removal rates at 58 C were almost the same (No. 13 in Table 2).But above 10 C, the ammoniu m removal rate increased obviously (from 1.98 mg NH4+-N/L/h to 3.16 mg NH4+-N/L/h). At 1020 C,the ammonium removal rates slightly changed.

The effect of C/N on ammonium removal was also investiga ted.The results showed that under low carbon content (C/N 0.22), the ammonium removal rate increased rapidly from 0.82 mg NH4+-N/L/h to 2.68 mg NH4+-N/L/h with the enhancem ent of C/N ratio.However, it increased slightly when C/N ratio rise from 2 to 10.At C/N 20, ammonium removal rate decreased. The results signi-cantly differ from previous studies. Zhao et al. (2010b) reportedthat C/N = 15 was the optimal condition for Bacillus sp. LY to re- move nitrogen when initial ammonium was 41.1 mg/L. Tayloret al. (2009) reported that low carbon supply for Providenc ia rettge- ri resulted in the reduce NH4+-N removal ability and the most proper C/N was 10 when the initial ammonium was 120 mg/L.NH4+-N removal efciency by Bacillus strains (Bacillus cereus , Bacil-lus subtilis and Bacillus licheniform is) was the maximum at C/N = 8when the initial ammoniu m was 1.05 g/L (Kim et al., 2005 ). The re- sults of these studies showed that the heterotrophic nitricationneed high C/N ratio. In the present study, at C/N 10, ammoniu m re- moval rate was the highest. But at C/N 2, ammoniu m removal rate was 91.8% of that at C/N 10. The results showed that Microbacter i-um sp. SFA13 could effectively remove ammonium at low C/N.Microbacter ium sp. SFA13 was isolated from surface water contain- ing low nutrient, so it could adapt oligotrophic condition .

There has been reported that oxygen affected nitrication and denitrication of Thiosphaera pantotropha (Robertson et al.,1988). It has been accepted that the level in dissolved oxygen con- centration in any solution is strongly affected by variation in shak- ing speed (Taylor et al., 2009 ). Joo et al. reported that nitricationratio of Alcaligenes faecalis No. 4 was signicantly lower at 90 rpm

3.92 0.10 2.22 0.19 4.16 0.11 1.98 0.28 3.61 0.11 3.16 0.25 3.35 0.14 2.94 0.21 3.38 0.14 3.22 0.33 4.71 0.12 0.82 0.30 4.54 0.10 0.90 0.30 4.46 0.13 1.42 0.30 3.94 0.11 2.68 0.27 3.85 0.13 2.78 0.34 3.42 0.10 2.92 0.22 3.89 0.14 2.64 0.20 4.87 0.11 0.21 0.17 4.55 0.11 1.39 0.29 3.86 0.12 2.46 0.29 3.45 0.12 2.85 0.18 3.56 0.09 3.03 0.27 3.57 0.12 3.09 0.36 rate Sodium acetate Glycerol Calcium carbonate

2.07 0.27 0.34 0.22 0.72 0.18

+ +

nology 137 (2013) 147152 151(3.39 mg N/L/h) than at 120 rpm and 150 rpm (23.5 mg N/L/h and 25.3 mg N/L/h). Ammonium removal by Providencia rettgeri YL was also reported to be strongly affected by oxygen availability and depende nt on aerobic conditions. The results obtained in this study were in accordance with the previous research. The ammonium re- moval rate improved with the increase of shaking speed. However,the ammonium removal rate increased slightly when the shaking speed exceeded 140 rpm (DO over 5.21 mg/L).

Above all, when the initial ammoniu m was about 5 mg/L, the ammoniu m removal rate was 2.68 0.273.16 0.25 mg NH4+-N/L/h at C/N 210, 10 C and DO > 5.21 mg/L, which was higher than the rate of previous experiment (1.64 mg NH4+-N/L/h, showed inFig. 2). GAC with large surface area and rough surface texture (Liet al., 2012 ) act as carrier and protect the bacteria attached on itfrom washing out. It provided a good survival condition for bacte- ria and promoted the bio-remova l efciency. The previous study also found that the immobilized bacteria could enhance the ammo- nium removal rate (Strotmann and Windecker, 1997 ).

4. Conclusion

The strain SFA13 was isolated form Songhua River and identi- ed as Microbacterium sp. 5.13 0.16 mg/L NH4+-N was removed after 30 h without NO3-N and NO2-N accumulation. The conver- sion product was nitrogen gas. Trace NH2OH was detected during NH4+-N degradation . NO3-N and NO2-N could be utilized as sole nitrogen source. HAO, NiR and NR were detectable under aerobic condition . At C/N 210, 10 C and DO > 5.21 mg/L, the ammonium removal rate of the strain SFA13 attached on GAC was 2.68 0.273.16 0.25 mg NH4+-N/L/h. The strain SFA13 could beused for removing ammonium in surface water at low temperat ure.

Acknowled gements

This study is supported by grants from National Natural Science Foundation of China (Grant No. 51078106), Project of Outstanding Academic Youth of Heilongjian g Province (Grant No. 1251G049)and Heilongjian g Provincial Science Foundation for Distinguished Youth Scholar (Grant No. JC200708).

We are greatly indebted to our colleagues in this program that have made signicant contributions to the collection, analysis and/ or interpretation of samples and results. We are grateful for the useful and constructive comments of reviewer s.

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Removal of ammonium in surface water at low temperature by a newly isolated Microbacterium sp. strain SFA131 Introduction2 Methods2.1 Isolation of heterotrophic nitrifying bacteria2.2 Identification of the selected bacteria2.3 Assessment of nitrifying characteristics2.4 Assessment of nitrite and nitrate removal2.5 Assessment of enzyme activity2.6 Biological activated carbon experiment of the strain SFA132.7 Analytical methods2.8 Statistical analysis

3 Results and discussion3.1 Ammonium removal by isolate strain3.2 Identification and nitrification characters of the strain SFA133.3 Utilization of nitrite and nitrate by SFA13 under aerobic conditions3.4 Enzyme assay3.5 The effect of carbon source, temperature and dissolved oxygen on the ammonium removal by the strain SFA13

4 ConclusionAcknowledgementsReferences