Removal of ammonium in surface water at low temperature by a newly

isolated Microbacterium sp. strain SFA13

Duoying Zhang
a,c
, Weiguang Li
a,b,
, Xiaofei Huang
a
, Wen Qin
a
, Miao Liu
a
a
School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, PR China
b
State Key Laboratory of Urban Water Resource and Environment, Harbin 150090, PR China
c
Department of Food Science and Environmental Engineering, East University of Heilongjiang, Harbin 150086, PR China
h i g h l i g h t s
Microbacterium sp. SFA13 with heterotrophic nitrifying ability at 5 C.
The putative nitrogen removal process was: NH
4
+
?NH
2
OH?NO
2

?NO
3

, then NO
3

?NO
2

?N
2
.
Strain SFA13 attached on activated carbon could effectively remove ammonium in surface water at C/N 210, temperature 10 C, and DO > 5.2 mg/L.
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
Keywords:
Heterotrophic nitrication
Ammonium
Drinking water
Low temperature
Microbacterium sp.
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 NH
4
+
-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. NO
3

-N or NO
2

-N was
reduced by the strain SFA13. The denitrication ratio was 0.24 mg NO
3

-N/L/h. Hydroxylamine oxidase,

nitrite reductase and nitrate reductase were detectable. The putative nitrogen removal process by the
strain SFA13 was as follows: NH
4
+
?NH
2
OH?NO
2

?NO
3

, then NO
3

?NO
2

?N
2
. Biological activated
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 NH
4
+
-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
concentratio n in surface water in China increased gradually during
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
PR China, http://datacenter .mep.gov.cn/ ). To guarantee safe drink-
ing water, the Ministry of Health of the PR China requires an
ammonium concentration of lower than 0.5 mg/L accordin g to
the Standards for Drinking Water Quality (GB5749-2006). Tradi-
tional drinking water treatment process used in China, such as
coagulation , 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-
sirable chlorinated by-product.
The removal of ammonium by biodegradat ion before chlorina-
tion decrease short-term chlorine demand (Holme et al., 1999 ).
Ammonium removal by nitrifying bacteria is a two-step process
in which sequential oxidation of NH
4
+
-N into NO
2

-N and then
NO
2

-N into NO
3

-N occurs. However , low temperature s generally

drastical ly affect nitrifying bacterial process rate. The optimal tem-
perature 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 NH
4
+
-N to NO
2

-N or NO
3

-N and
0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.03.094

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.
E-mail addresses: weiguanglihit@hotmail.com, hitlwg@126.com (W. Li).
Bioresource Technology 137 (2013) 147152
Contents lists available at SciVerse ScienceDi rect
Biore source Technology
j our nal homepage: www. el sevi er . com/ l ocat e/ bi or t ech
simultaneou sly covert NO
2

-N or NO
3

-N to N
2
O and/or N
2
(Chen
et 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 of
ammonium 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 to
determine 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 as
enrichment 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 Na
2
HPO
4
, 2.0 g/L KH
2
PO
4
,
0.01 g/L MgSO
4
7H
2
O, 0.005 g/L CaCl
2
2H
2
O, 1.0 g/L NH
4
Cl, pH
7.2) 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 NH
4
Cl, 1.0 g/L CH
3
CH
2
ONa, 0.05 g/L MgSO
4
7H
2
O,
0.2 g/L K
2
HPO
4
, 0.12 g/L NaCl, 0.01 g/L MnSO
4
4H
2
O, 0.01 g/L FeSO
4
,
pH7.0) and cultured at 5 C for 34 days when the population
amount reached to above 10
7
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 3
times. Then the collected bacteria were inoculated to triplicate
50 mL sterile ammoniu m sulfate solution (about 5 mg/L NH
4
+
-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 10
7
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.
Then the collected bacteria were inoculated to triplicate 50 mL
sterile ammonium sulfate solution (about 5 mg/L NH
4
+
-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 O
2
He (95:5) gas. After
shaking at 10 C, 140 rpm/min for 0.5, 2, 4, 6, 8, 10, 15, 20, 25
and 30 h, the liquid samples were collected respectively to de-
tected NH
4
+
-N, NH
2
OH, NO
2

-N, NO
3

-N and TOC concentratio n.

The bacterial contents were determined by measuring OD
600
. At
the end of experiment, the gas samples were collected to detect
N
2
and N
2
O production.
2.4. Assessme nt of nitrite and nitrate removal
Nitrate and nitrite were used to elucidate the denitrication
process of the isolate. Based on the Standards for Drinking Water
Quality (GB5749-2006), NO
2

-N and NO
3

-N were adjusted to
10 mg/L respectively. Carbon/Ni trogen (C/N) was adjusted to 4
with sodium acetate as carbon source. After cultured in basic med-
ium (the population amount reached to more than 10
7
cells/mL),
the cells were collected and inoculated intro triplicate 50 mL ni-
trate or nitrite solution with sodium acetate. After shaking at
10 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 NO
2

-N, NO
3

-N
and TOC concentratio n. The bacterial contents were determined
by measuring OD
600
.
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 10
9
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 NH
4
+
-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 on
the 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 at
5, 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
of 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
148 D. Zhang et al. / Bioresource Technology 137 (2013) 147152
(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 a
wavelength 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). NH
2
OH was analyzed by indirect spectrophotom etry (Ming
and 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
using an Aurora Combusti on Total Organic Carbon Analyzer
1030C (OI, America). Tests to detect the changes in N
2
and O
2
respectively were conducted using gas chromatography (GC-14B,
Shimadzu, Japan). Gas samples were extracted using a 100 lL
air-tight glass syringe. The carrier gas Ar had a ow rate of
20 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 (C
0
-C
n
)/h. C
0
is initial
concentratio n of NH
4
+
-N (NO
2

-N or NO
3

-N). C
n
is the nal con-
centration of NH
4
+
-N (NO
2

-N or NO
3

-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 is
showed in Fig. 1. The initial ammoniu m concentratio n was
5.09 0.05 mg/L. After 30 h, 1.85 0.04 mg NH
4
+
-N/L remained in
the solution. The average nitrication ratio at 5 C was 0.11 mg
NH
4
+
-N/L/h. High ammonium removal rate appeared in the initial
4 h, the average ammonium removal rate was 0.46 mg NH
4
+
-N/L/
h. In the initial 30 min, 11.39% NH
4
+
-N was removed, and the re-
moval rate was 1.16 mg NH
4
+
-N/L/h. Then with the prolonging bio-
degradation time, the removal rate decrease d. The maximum
NH
4
+
-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
SFA13 had unusual ability of autotrophic nitrication. Yang et al.
(2011) also reported the autotrophic nitrication ability of Bacillus
subtilis strain A1 with broad range of ammonium load (from105.58
to 1014.17 mg/L).
It has been reported that the nitrication ratio of Pseudomona s
stutzeri YZN-001 was approximat ely 0.3 mg NH
4
+
-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
The strain SFA13 was selected based on ammonium removal at
5 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 Microbacteriumsp. Micro-
bacterium species have been reported to reduce heavy metal in
water (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 mremoval characterist ic of Microbac-
terium sp. SFA13.
Microbacter ium sp. SFA13 exhibited ammonium removal ability
in low NH
4
+
-N concentratio n at 5 C. But NH
4
+
-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 NH
2
OH-N, NO
3

-N and
NO
2

-N. The results are shown in Fig. 2. The NH

4
+
-N was consumed
to 0.26 0.12 mg/L with the biodegradation of TOC after 30 h.
Compare d with sole NH
4
+
-N degradat ion (showed in Fig. 1), the
average ammonium removal rate increased from 0.11 mg NH
4
+
-
N/L/h to 0.16 mg NH
4
+
-N/L/h. Especially in the initial 30 min, the
ammoniu m removal rate increased from 1.16 mg NH
4
+
-N/L/h to
0 5 10 15 20 25 30
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Time (hours)
A
m
m
o
n
i
u
m

c
o
n
c
e
n
t
r
a
t
i
o
n

(
m
g
/
L
)
Fig. 1. Ammonium removal at 5 C by the strain SFA13. Error bars: mean S.D. of
three replicates.
0 5 10 15 20 25 30
0.50
0.52
0.54
0.56
0.58
0.60
0.62
0.64
0.66
0.68
0.70

B
a
c
t
e
r
i
a
l

c
o
n
t
e
n
t

(
O
D
6
0
0
)
Bacteria content Nitrite concentration
Nitrate concentration TOC
Time (hours)
0
5
10
15
20
25
30
35
40

C
o
n
c
e
n
t
r
a
t
i
o
n

(
m
g
/
L
)
Fig. 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.
D. Zhang et al. / Bioresource Technology 137 (2013) 147152 149
1.64 mg NH
4
+
-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 OD
600
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 (OD
600
higher
than 0.5) added at the beginning of experiment.
NH
2
OH was produced at a little higher concentratio n (average
0.29 0.09 mg/L) than NO
3

-N and NO
2

-N. It is proposed that

ammonium is initially oxidized to NH
2
OH. It has been reported
that NH
2
OH production was directly related to changes in ammo-
nium concentratio ns (Taylor et al., 2009 ). There was little NO
3

-N
and NO
2

-N accumulati on, which indicated that Microbacter ium sp.

SFA13 has the capability of nitrogen removal. At the end of exper-
iments, N
2
and N
2
O production was tested using gas chromatogra-
phy. N
2
O production was not detected. However, 9.23% 0.6 N
2
was 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 N
2
.
3.3. Utilization of nitrite and nitrate by SFA13 under aerobic conditions
For evaluating denitrication of Microbacter ium sp. SFA13,
NO
3

-N and NO
2

-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
whether using NO
3

-N or NO
2

-N as sole nitrogen source (Fig. 3).

The results showed that either NO
3

-N or NO
2

-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 NO
3

-N/L/h. The capability of aer-

obic denitrication by Rhodococcus sp. CPZ24 and Pseudomona s
stutzeri YZN-001 were demonstrated by Chen et al. (2012) and
Zhang et al. (2011c). However, Alcaligen es faecalis NR and Acineto-
bacter calcoaceticus HNR could not denitrify NO
3

or NO
2

(Zhao
et al., 2012, 2010a ), but removed NH
4
+
-N in the formatio n of N
2
or N
2
O. In the study, when nitrate was added, there was no
NO
2

-N accumulation (Fig. 3A). However, when nitrite was added,

1.67 mg/L NO
3

-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 NR
activity were 0.21 and 0.44 lmol/min, respectivel y. In the study,
NH
2
OH is the substrate of HAO (Otte et al., 1999 ), it was oxidized
to NO
2

and NO
3

, and subsequently reduced to N

2
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 N
2
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
(NH
4
+
?NH
2
OH?N
2
O/N
2
) (Joo et al., 2005; Zhao et al., 2012,
2010a). The pathway is also different from the report of Richardson
et al. (1998) (NH
4
+
?NH
2
OH?NO
2

?N
2
O?N
2
). It has been re-
ported that Providencia rettgeri YL heterotrop hic nitrication pro-
cess was as follows: NH
4
+
?NH
2
OH?NO
2

?NO
3

and the
denitrication process was: NO
3

?NO
2

?N
2
(Taylor et al.,
2009). The nitrogen transformat ion process was similar to that of
Microbacter 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 on
the 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 on
GAC particles to form BAC that was applied to study the effect of
carbon 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) of
BAC 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
source for Microbacterium sp. SFA13. The results showed that the
ammoniu m removal reached to the highest (2.07 mg NH
4
+
-N/L/h)
in acetate medium solution (Table 1). So sodium acetate was se-
0 5 10 15 20 25 30
0.50
0.52
0.54
0.56
0.58
0.60
0.62
0.64
0.66
0.68
0.70

B
a
c
t
e
r
i
a
l

c
o
n
t
e
n
t

(
O
D
6
0
0
)
Bacteria content Nitrite concentration
Nitrate concentration TOC
Time (hours)
0
5
10
15
20
25
30
35
40

C
o
n
c
e
n
t
r
a
t
i
o
n

(
m
g
/
L
)
0 5 10 15 20 25 30
0.50
0.52
0.54
0.56
0.58
0.60
0.62
0.64
0.66
0.68
0.70
Bacterial content Nitrite concentration
Nitrate concentration TOC
Time (hours)
0
5
10
15
20
25
30
35
40
45

C
o
n
c
e
n
t
r
a
t
i
o
n

(
m
g
/
L
)
B
a
c
t
e
r
i
a
l

c
o
n
t
e
n

(
O
D
6
0
0
)
A
B
Fig. 3. Changes in NO
3

, NO
2

, TOC and OD
600
by Microbacteriumsp. SFA13 at 10 C.
Nitrate (A) and nitrite (B) were used as sole nitrogen source respectively. Error bars:
mean S.D. of two replicates.
150 D. Zhang et al. / Bioresource Technology 137 (2013) 147152
lected as carbon source to detect the inuence of pH, C/N and DO
on ammonium removal efciency.
The ammonium removal by Microbacter ium sp. SFA13 at differ-
ent temperatures was investigated in ammoniu m solution (about
5 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 NH
4
+
-N/L/h to 3.16 mg NH
4
+
-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 NH
4
+
-N/
L/h to 2.68 mg NH
4
+
-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) reported
that C/N = 15 was the optimal condition for Bacillus sp. LY to re-
move nitrogen when initial ammonium was 41.1 mg/L. Taylor
et al. (2009) reported that low carbon supply for Providenc ia rettge-
ri resulted in the reduce NH
4
+
-N removal ability and the most
proper C/N was 10 when the initial ammonium was 120 mg/L.
NH
4
+
-N removal efciency by Bacillus strains (Bacillus cereus , Bacil-
lus subtilis and Bacillus licheniform is) was the maximum at C/N = 8
when the initial ammoniu mwas 1.05 g/L (Kim et al., 2005 ). The re-
sults of these studies showed that the heterotrophic nitrication
need high C/N ratio. In the present study, at C/N 10, ammoniu mre-
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 nitrication
ratio of Alcaligenes faecalis No. 4 was signicantly lower at 90 rpm
(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 NH
4
+
-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 NH
4
+
-N/L/h, showed in
Fig. 2). GAC with large surface area and rough surface texture (Li
et al., 2012 ) act as carrier and protect the bacteria attached on it
from 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 NH
4
+
-N was removed
after 30 h without NO
3

-N and NO
2

-N accumulation. The conver-

sion product was nitrogen gas. Trace NH
2
OH was detected during
NH
4
+
-N degradation . NO
3

-N and NO
2

-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 NH
4
+
-N/L/h. The strain SFA13 could be
used for removing ammonium in surface water at low
temperat ure.
Table 1
Nitrication of ammonium in 30 min at 5 C under different carbon sources.
Glucose Sodium citrate Sodium acetate Glycerol Calcium carbonate
Ammonium removal rate (mg NH
4
+
-N/L/h) 1.43 0.23 0.95 0.15 2.07 0.27 0.34 0.22 0.72 0.18
Data are means S.D. of three replications.
Table 2
Ammonium removal rate by Microbac terium sp. SFA13 under different condition.
No. C/N ratio Shaking speed (rpm) Temperature (C) Initial NH
4
+
-N (mg/L) Final NH
4
+
-N (mg/L) Ammonium removal rate (mg NH
4
+
-N/L/h)
1 4 140 5 4.91 0.016 4.01 0.15 1.80 0.33
2 4 140 6 5.03 0.031 3.92 0.10 2.22 0.19
3 4 140 8 5.15 0.054 4.16 0.11 1.98 0.28
4 4 140 10 5.19 0.042 3.61 0.11 3.16 0.25
5 4 140 15 4.82 0.039 3.35 0.14 2.94 0.21
6 4 140 20 4.99 0.045 3.38 0.14 3.22 0.33
7 0.2 140 10 5.12 0.063 4.71 0.12 0.82 0.30
8 0.5 140 10 4.99 0.053 4.54 0.10 0.90 0.30
9 1 140 10 5.17 0.021 4.46 0.13 1.42 0.30
10 2 140 10 5.28 0.026 3.94 0.11 2.68 0.27
11 4 140 10 5.24 0.045 3.85 0.13 2.78 0.34
12 10 140 10 4.88 0.049 3.42 0.10 2.92 0.22
13 20 140 10 5.21 0.049 3.89 0.14 2.64 0.20
14 2 0 10 4.97 0.045 4.87 0.11 0.21 0.17
15 2 100 10 5.24 0.037 4.55 0.11 1.39 0.29
16 2 120 10 5.09 0.041 3.86 0.12 2.46 0.29
17 2 140 10 4.87 0.050 3.45 0.12 2.85 0.18
18 2 160 10 5.07 0.041 3.56 0.09 3.03 0.27
19 2 180 10 5.11 0.056 3.57 0.12 3.09 0.36
Data are means S.D. of three replications.
D. Zhang et al. / Bioresource Technology 137 (2013) 147152 151
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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