Bioresource Technology 108 (2012) 35–44

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Bioresource Technology
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The characteristics of a novel heterotrophic nitrification–aerobic

denitrification 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 a b s t r a c t

Article history: Bacillus methylotrophicus strain L7, exhibited efficient heterotrophic nitrification–aerobic denitrification
Received 1 November 2011 ability, with maximum NHþ 
4 -N and NO2 -N removal rate of 51.58 mg/L/d and 5.81 mg/L/d, respectively.
Received in revised form 28 December 2011 Strain L7 showed different gaseous emitting patterns from those strains ever described. When 15NH4Cl,
Accepted 28 December 2011
or Na15NO2, or K15NO3 was used, results of GC–MS indicated that N2O was emitted as the intermediate
Available online 9 January 2012
of heterotrophic nitrification or aerobic denitrification, while GC–IRMS results showed that N2 was pro-
duced as end product when nitrite was used. Single factor experiments suggested that the optimal con-
Keywords:
ditions for heterotrophic nitrification were sodium succinate as carbon source, C/N 6, pH 7–8, 0 g/L NaCl,
Bacillus methylotrophicus L7
Heterotrophic nitrification–aerobic
37 °C and a wide range of NHþ 4 -N from 80 to 1000 mg/L. Orthogonal tests showed that the optimal

denitrification conditions for aerobic denitrification were C/N 20, pH 7–8, 10 g/L NaCl and DO 4.82 mg/L (shaking speed
Nitrite 50 r/min) when nitrite was served as substrate.
Nitrous oxide Ó 2012 Elsevier Ltd. All rights reserved.
Nitrogen removal

1. Introduction as Paracoccus denitrifican), capable of heterotrophic nitrification–

The removal of nitrogen from wastewater by nitrifiers and den-
itrifiers was the most efficient method in wastewater treatment.
Autotrophic nitrification and anoxic denitrification played impor-
tant roles in this process. Nitrifiers convert ammonia to nitrite,
followed by nitrate. Denitrifiers reduce nitrate to nitrite, finally
to N2, in which NO and N2O were the main intermediate products
(Joo et al., 2005). Due to the totally differences in physiology and
biochemistry, the nitrifiers and denitrifiers had some disadvan-
tages in the process of nitrogen removal treatment of wastewater.
Nitrifiers were sensitive to organic matter (Kulikowska et al.,
2010). However, organic compounds were necessary to the denit-
rifiers. In addition, the growth of nitrifiers relied on oxygen that
was toxic to denitrifiers (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 nitrifiers 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 nitrification
was first isolated from natural environment (Verstrae and
Alexande, 1972). In 1983, Thiosphaera pantotropha (now known
⇑ Corresponding author. Address: Institute of Microbiology, Chinese Academy of
Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China. Tel.:
+86 10 62653757; fax: +86 10 62538564.
E-mail address: liuzhp@sun.im.ac.cn (Z.-P. Liu).
aerobic denitrification, was isolated from activated sludge in the
wastewater treatment plant (Robertson and Kuenen, 1983). After-
wards, the study on heterotrophic nitrification and aerobic denitri-
fication had drawn more and more attentions. Compared with the
traditional removal of nitrogen, nitrogen removal by heterotrophic
nitrification and aerobic denitrification had several advantages.
Firstly, the utilization of organic substrates and the tolerance to
oxygen agreed with each other, achieving the vision of simulta-
neous nitrification and denitrification (SND) in one reactor (Third
et al., 2005). In addition, the process of denitrification could bal-
ance the change of pH in the reactor, avoiding the acidification
caused by nitrification. Furthermore, the diversity of substrates
and products of heterotrophic nitrification 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 nitrification–aerobic denitrification 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 nitrification–aerobic deni-
trification mainly focused on substrate removal and accumulation
of intermediate (Wan et al., 2011). The most common way to prove
the denitrification and the production of gaseous nitrogen com-
pounds from the denitrification 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

0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.12.139
36 Q.-L. Zhang et al. / Bioresource Technology 108 (2012) 35–44
8% of the input nitrogen was not recovered (Gonenc and Harrem-
oes, 1985). Besides, compared with nitrate, the research of the
characteristics of denitrification using nitrite, a link intermediate
between nitrification and denitrification, 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 nitrification–aerobic denitrification 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 specific inorganic nitrogen metabolism
pathway by strain L7 was proposed. Our results might provide an
alternate microbial resource for nitrogen removal treatment of
wastewater, and also might be benefit to the elucidation of the
mechanisms of heterotrophic nitrification–aerobic denitrification,
especially by Gram-positive bacteria.
2. Methods
2.1. Identification of strain L7
Strain L7 was isolated from wastewater sample. Physiological
and biochemical characteristics were tested using API 20 NE and
API ZYM strips (bioMérieux, French) following the manufacturer’s
instructions.
16S rRNA gene was PCR amplified using bacterial universal
primers 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 -GG
TTACCTTGTTACGACTT-30 ) and sequenced by Meiji Corp. (Beijing,
China). Sequence alignment was performed using Basic Local
Alignment Search Tool program (BLAST: http://www.ncbi.nlm.-
nih.gov/blast/Blast.cgi). A neighbor-joining tree was constructed
using MEGA 3.1 program (Kumar et al., 2004).
2.2. Medium
Heterotrophic nitrification medium (HNM, g/L of distilled
water): (NH4)2SO4 0.66, sodium succinate 4.72, KH2PO4 0.50, Na2H-
PO4 0.50, MgSO47H2O 0.20, NaCl 30.00, trace element solution
2.00 mL, pH 7.5.
Denitrification medium (DM, g/L of distilled water): KNO3 1.00,
sodium succinate 4.68, MgSO47H2O 0.20, CaCl2 0.01, EDTA 0.07,
KH2PO4 0.50, Na2HPO4 0.50, FeSO4 0.01, trace element solution
2.00 mL, pH 7.5.
Nitrite denitrification medium (NDM, g/L of distilled water):
NaNO2 0.28, sodium succinate 3.16, MgSO47H2O 0.20, CaCl2 0.01,
EDTA 0.07, KH2PO4 0.50, Na2HPO4 0.50, FeSO4 0.01, trace element
solution 2.00 mL, pH 7.5.
Hydroxylamine oxidation medium (HO, g/L of distilled water):
hydroxylamine 0.165, sodium succinate 2.36, KH2PO4 0.50, Na2H-
PO4 0.50, MgSO47H2O 0.20, NaCl 30.00, trace element solution
2.00 mL, pH 7.5.
Trace element solution (Joo et al., 2005) (g/L of distilled water):
EDTA2Na 57.10, ZnSO47H2O 3.90, CaCl22H2O 7.00, MnCl24H2O
1.00, FeSO47H2O 5.00, (NH4)6Mo7O244H2O 1.10, CuSO45H2O
1.60, CoCl26H2O 1.60, pH 6.0.
LB medium (g/L of distilled water): Tryptone 10, Yeast extract 5,
NaCl 10, pH 7.5. For preparation of LB plates, 1.5% (w/v) agar was
added.
2.3. The qualitative assay of heterotrophic nitrification and
denitrification of strain L7
One milliliter pre-culture of strain L7 in LB overnight was inoc-
ulated into 100 mL HNM and 100 mL DM, respectively. The cul-
tures were incubated for 5 days at 30 °C on a rotary shaker at
160 r/min and then centrifuged at 8000 r/min for 5 min. NO 2 and
NO3 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
600 nm (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 (Nbio) could be expressed as Nbio (mg/
L) = 9.026  OD600.
2.5. Detection of gaseous nitrogen compounds
For these experiments, strain L7 was incubated in 100 mL med-
ium (HNM, or DM, or NDM) containing 50% 15NH4Cl, or 50%
K15NO3, or 50% Na15NO2 (by atomic fraction, Spectra Corp., USA),
respectively, in 250 mL serum bottle sealed with a robber stopper
at 30 °C on a rotary shaker at 160 r/min. N2O or N2 from headspace
was detected after hermetic incubation for 10 days according to Ai
et al (2011). N2O was detected by GC–MS (Agilent, USA) with 50 lL
upper gas using 100 lL gastight syringe, and N2 was measured by
GC–IRMS (Thermo Fisher Scientific, USA) with 5 lL upper gas using
10 lL gastight syringe. Both gas detection devices were equipped
with GS–Carbon Plot (30 m  0.32 mm  3.0 lm, Agilent, USA).
2.6. Single-factor experiments to study the factors influencing the
nitrification performance of strain L7
Single-factor experiments were conducted for studying the het-
erotrophic nitrification characteristics of strain L7 under 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-
centration 140 mg/L, C/N 7, initial pH 7.5, NaCl 30 g/L, culturing tem-
perature 30 °C, shaking speed 160 r/min. In C/N ratio experiments,
the content of carbon source was changed in order to adjust C/N ratio
to 2, 4, 6, 8, 10, 15 and 20, respectively, by fixing nitrogen concentra-
tion at 140 mg/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 to 5, 6, 7, 8, 9 and 10 using 6 mol/L HCl or
10 mol/L NaOH. The salinity was set at 0, 10, 20, 30 and 40 g/L NaCl.
Culturing temperature was adjusted to 20, 25, 30 and 37 °C. Initial
 Q.-L. Zhang et al. / Bioresource Technology 108 (2012) 35–44 37

ammonium nitrogen concentration was adjusted to 80, 400, 99 Strain L7 (JN 635497)
1000 mg/L, representing low, mediate and high ammonium concen- 81 Bacillus methylotrophicus CBMB205T (EU 194897)
trations, respectively; and sodium succinate content varied accord- 80 Bacillus amyloliquefaciens ATCC 23350 T (X 60605)
ingly to keep C/N ratio at 7. All of the above experiments were Bacillus subtilis NCDO 1769T (X 60646)
99
conducted in triplicate with inoculation size of 2% (v/v), and non- 82 Bacillus mojavensis IFO 15718T (AB 021191)
seeded samples and seeded without nitrogen source samples were 72 Bacillus atrophaeus JCM 9070T (AB 021181)
also conducted as controls. Unless otherwise stated, all the hetero- Bacillus sonorensis NRRL B-23154 T (AF 302118)
trophic nitrification experiments were conducted at shaking speed 100 Bacillus aerius 24KT (AJ 831843)
160 r/min for 108 h. Then strain growth (OD600), and the contents Bacillus pumilus DSM 27T (AY 456263)

of ammonium nitrogen (NHþ 4 -N) and nitrite (NO2 -N) were 100 Bacillus safensis FO-36BT (AF 234854)
determined. Bacillus acidicola 105-2T (AF 547209)
100 Bacillus shackletonii LMG 18435T (AJ 250318)
2.7. Orthogonal test to study the factors influencing the denitrification
0.005
performance of strain L7
Fig. 1. Neighbor-joining tree based on 16S rRNA gene sequences showing the
Orthogonal tests were designed and analyzed with SPSS statis- phylogenetic position of strain L7 and representatives of some other related taxa.
tical software (SPSS 16.0 version for windows, SPSS, Inc., Chicago, Bootstrap values (expressed as percentages of 1000 replications) >50% are shown at
IL, USA) to illustrate the effect of different factors on the aerobic the branch points. Bar, 0.005 substitutions per nucleotide position.
denitrification performance of strain L7. The selected factors and
their levels were detailed in Table 1. Differences were considered
strain L7 was related to members of genus Bacillus, and showed
to be significant when P < 0.05. Cells of strain L7 grown in LB over-
highest sequence similarity (100%) to B. methylotrophicus
night were harvested by centrifugation at 8000 r/min for 5 min,
CBMB205T. Phylogenetic tree (Fig. 1) further indicated that strain
washed twice with sterile saline solution, and then re-suspended
L7 together with B. methylotrophicus CBMB205T formed a distinct
in sterile saline solution. 1 mL of cell suspension was inoculated
linkage in the tree with 99% bootstrap support. Combining above
into 100 mL NDM in 250 mL flask. All the experiments were done
results, strain L7 was identified as B. methylotrophicus L7. So far,
in triplicates.
there was no report about the heterotrophic nitrification–aerobic
denitrification of this species.
3. Results and discussion

3.2. The property of heterotrophic nitrification and aerobic

3.1. The identification of strain L7
denitrification of strain L7
Strain L7 was Gram-positive. Colonies were creamy white con-
The qualitative assay results (data not shown) indicated that
vex opaque with regular edges, sticky not conducive to be picked
strain L7 could utilize ammonium to produce nitrite whereas ni-
and 2–5 mm in diameter after incubation for 48 h at 30 °C on LB
trate was not detected, indicating that L7 was able to heterotrophic
plates. The results of API 20NE test indicated that strain L7 could
nitrify. Nitrite was the dominant product in the process of hetero-
utilize D-glucose, L-arabinose, D-mannose, D-mannitol, maltose,
trophic nitrification (Castignetti and Hollocher, 1984; Verstrae and
potassium gluconate and malic acid. Strain L7 was positive for
Alexande, 1972), while nitrate was produced mainly by fungi
methanol utilization and activities of catalase, oxidase, protease,
(Marshall and Alexander, 1962) and just a few bacteria (Joo et al.,
amylase, alkaline phosphatase, esterase (C4), esterase lipase (C8)
2005; Yang et al., 2011). When strain L7 was cultivated in HNM,
and naphthol-AS-BI-phosphohydrolase; and negative for urease
there was not significant increase in amount of hydroxylamine,
and b-galactosidase activity. All these properties were in agree-
an important intermediate in nitrification (Lees, 1952), possibly
ment with those described for B. methylotrophicus CBMB205T
due to the instability of hydroxylamine and fast transformation
(Madhaiyan et al., 2010), except of a-glucosidase activity.
to the downstream intermediates such as nitrite. When nitrate
Almost complete 16S rRNA gene (1422 nt) of strain L7 was
was used as sole nitrogen source (in DM), the production of nitrite
amplified and sequenced. It was deposited in GenBank under
indicated that strain L7 could denitrify nitrate to nitrite or gaseous
accession number of JN 635497. Homology searches revealed that
nitrogen compounds.

Table 1 3.3. Detection of gaseous nitrogen compounds

L16 (4)4 orthogonal test design for denitrification of strain L7.

Test Factor A (shaking Factor B (C/ Factor C Factor D GC–MS results showed that N2O was produced on both nitrate
no. speed, r/min) N ratio) (salinity, g/L) (initial pH) (Fig. 2A) and nitrite (Fig. 2B) served as substrate, respectively.
1 50 (A1) 5 (B1) 0 (C1) 6 (D1) The results also indicated that strain L7 emit N2O when 15NH4Cl
2 100 (A2) 10 (B2) 10 (C2) D1 (HNM, Fig. 2C) and 15NH4Cl plus hydroxylamine (HNM plus
3 150 (A3) 15 (B3) 20 (C3) D1
hydroxylamine, Fig. 2D) as nitrification substrates. N2O isotopic
4 200 (A4) 20 (B4) 30 (C4) D1
5 A4 B2 C1 7 (D2)
abundance ratios (Fig. 3) showed that the labeled 15,14N2O,
15,15
6 A3 B1 C2 D2 N2O did appear in the headspace gas of HNM sample, although
7 A2 B4 C3 D2 the abundance of N2O from NH4Cl was far less than that of nitrate,
8 A1 B3 C4 D2 nitrite and NH4Cl plus hydroxylamine (Fig. 2). These results sug-
9 A2 B3 C1 8 (D3)
gested that strain L7 was a heterotrophic nitrification–aerobic
10 A1 B4 C2 D3
11 A4 B1 C3 D3 denitrifier. It could not only aerobically denitrify nitrate or nitrite
12 A3 B2 C4 D3 to form N2O, but also nitrify ammonia to form N2O, and the later
13 A3 B4 C1 9 (D4) was rarely reported in other strains. Only a few strains were
14 A4 B3 C2 D4 described with nitrification of ammonia to form N2O, such as T.
15 A1 B2 C3 D4
16 A2 B1 C4 D4
pantotropha (Arts et al., 1995) and A. faecalis No.4 (Joo et al.,
2005), however, non of them was Gram-positive. Strain L7 was
38 Q.-L. Zhang et al. / Bioresource Technology 108 (2012) 35–44
Fig. 2. Detection of N2O from incubation samples, un-seed controls and normal air samples (curves from top down, respectively). The 15N-labeled substrates included K15NO3
(A), Na15NO2 (B), 15NH4Cl (C) and 15NH4Cl plus unlabeled hydroxylamine (D).
the first 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 denitrification. Much more
amounts of non-labeled N2O was detected when 5 mM hydroxyl-
amine was added into 15N–HNM than that from 4 mM nitrite as
substrate in DM (Fig. 2B, D).This data suggested that oxidation of
hydroxylamine and aerobic denitrification occurred simultaneous
in strain L7.
N2, the end product of denitrification, was also detected when
the labeled nitrite was used as substrate, for that the d15N/14N ratio
of the labeled sample reached 8.343, much higher than that of the
blank control with d15N/14N of 0.081. When d15N/14N > 1, it can be
concluded that the 15N2 was produced according to the detection
accuracy (Ai et al., 2011). However, 15N2 was not detected with la-
beled nitrate and ammonia as substrates.
The above gas-producing patterns of strain L7 were different
from those strains ever reported. For example, T. pantotropha could
 
utilize NHþ 4 , NO3 , NO2 , respectively, to produce more N2O than N2
(Arts et al., 1995). A. faecalis No.4 could convert NHþ4 -N to more N2
than N2O (Joo et al., 2005), whereas another A. faecalis strain was
able to emit equivalent amounts of N2 and 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 N2 in 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 N2 and denitrifying nitrite
and nitrate to N2O in aerobically growing culture has never been
reported in Gram-positive bacteria. Whether N2O or N2 or both
gases was produced during the denitrification process depended
largely on the oxygen tension (Lloyd et al., 1987). Paracoccus halo-
denitrificans produced N2 in the culture condition absence of oxy-
gen, while as the oxygen tension gradually increased, the
 Q.-L. Zhang et al. / Bioresource Technology 108 (2012) 35–44
Fig. 2 (continued)
denitrification 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-
cific inorganic nitrogen dissimilation pathway of heterotrophic
nitrification–aerobic denitrification by strain L7 was 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. Nitrification characteristics of strain L7 under various conditions
3.4.1. Effect of carbon source
Carbon source was considered to be an important factor
influencing heterotrophic nitrification ability. The results in
39
Table 2 showed that sodium succinate and glucose could well
support the growth of strain L7, OD600 reached 0.42 and 0.39,
respectively; meanwhile, strain L7 exhibited efficient nitrifying
abilities, the total NHþ4 -N removal percentage were 48.00% and
38.40%, respectively, despite there were about 3.79 mg/L
(2.70%) and 3.53 mg/L (2.50%) of NHþ 4 -N consumed as nitrogen
source for its growth, respectively. An accumulation of NO 2 -N
of 0.22 ± 0.02 mg/L was observed on sodium succinate, but not
on glucose, implying that there might be different mechanisms
for strain L7 to perform heterotrophic nitrification on different
carbon sources. These results were consistent with those re-
ported for strain Bacillus MS 30 (Mevel and Prieur, 2000). Strain
MS 30 grew quite well (97.60% of input nitrogen was converted
to biomass nitrogen), but showed a relatively poor nitrification
ability with nitrification rate of 0.03 lmol NO 2 mg
1
dry weight
when glucose was served as carbon source. The results in Table
2 also indicated that other carbon sources did not support the
40 Q.-L. Zhang et al. / Bioresource Technology 108 (2012) 35–44
Fig. 3. N2O isotopic abundance ratios in 50% 15NH4Cl medium, indicating the production of 14, 15N2O and 15, 15N2O. Blank control (A) and 50% 15NH4Cl experimental sample (B)
were analyzed.
Fig. 4. Proposed nitrification and denitrification pathway of strain L7.
growth of strain L7, and worse nitrification performances were
also observed. Accordingly, sodium succinate was employed in
the following experiments.
3.4.2. Effect of C/N ratio
The NHþ 4 -N removal percentage was not significantly different
among C/N 2–20 as 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 L7 still exhibited satisfying nitrification ability with a NHþ4-
N removal percentage of 44.80%. The tolerance of strain L7 to a
wide C/N range expanded its application scope including the pig-
gery waste with C/N of 4–7 and the municipal landfill leachate
with high C/N (Kim et al., 2006). Limited formation of NO 2 -N
(<0.15 mg/L) at different C/N ratios in strain L7 would 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-
fication of strain L7. Take cost effectiveness into consideration, C/N
6 was 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 NHþ 4 -N removal percentage
increased from 6.10% at 20 °C to 78.40% at 37 °C. This might be due
 Q.-L. Zhang et al. / Bioresource Technology 108 (2012) 35–44 41

Table 2
Nitrification performance of strain L7 on different carbon source.

Carbon source OD600 NO

2 -N (mg/L) NHþ
4 -N

Initial Final Removal

(mg/L) (mg/L) percentage
(%)
Sodium 0.421 ± 0.025 0.218 ± 0.022 142.16 73.92 48.00
succinate
Sodium 0.141 ± 0.008 0.141 ± 0.031 141.58 131.95 6.80
pyruvate
Sodium 0.033 ± 0.006 0 142.07 137.67 3.10
acetate
Sodium 0.089 ± 0.012 0 142.47 132.35 7.10
citrate
Potassium 0.044 ± 0.005 0 141.96 140.11 1.30
sodium
tartrate
Glucose 0.388 ± 0.021 0 142.59 87.84 38.40

Values are means ± SD for triplicates.

to the increase of both the enzyme activity of heterotrophic nitri-

fication and the concentration of free ammonia, the substrate of
ammonia monooxygenase (AMO), at high temperatures (Kim
et al., 2006).

3.4.4. Effect of salinity

It could be concluded from Fig. 5C that HNM containing no NaCl
was optimal for strain L7 with NHþ 4 -N removal percentage of
83.40%. The more NaCl in the medium, the lower ammonium re-
moval percentage, 58.70% at 30 g/L NaCl, and 39.50% at 40 g/L NaCl,
was achieved, respectively. Salinity was an important parameter
affecting nitrification, even those strains isolated from marine
environment were inhibited by high salinity (Finstein and Bitzky,
1972). A thermophilic strain, Bacillus MS 30 (Mevel and Prieur,
2000) can be taken as an example, whose optimal growth salinity
was 16 g/L NaCl versus optimal nitrification salinity was 9.60 g/L
NaCl, and it could not grow when the salinity was up to 28.50 g/
L NaCl. Strain L7 could be defined as a halotolerant bacterium since
more than 58.70% of ammonium was removed within 0–30 g/L
NaCl. This characteristic expanded its application scope, regardless
of the treatment of municipal water with low salinity or the aqua-
culture wastewater containing high salinity.

3.4.5. Effect of pH
Strain L7 performed efficient nitrification ability at initial pH of
7–8, namely neutral or slightly alkaline environment, with the
NHþ 4 -N removal percentage of 58.40%, 55.00%, respectively, as
shown in Fig. 5D. Acidic (pH 5–6) or alkaline (pH 9–10) condition
was harmful to the growth of strain L7. Slightly alkaline environ-
ment was conducive to heterotrophic nitrification because more
free ammonia (NH3) contained in medium according to the theory
that the substrate of taken advantage by AMO was NH3 other than
NHþ 4 (Mevel and Prieur, 2000). A thermophilic strain, Bacillus MS 30
was reported to exhibit excellent nitrification ability at pH 7.5–8,
also a slightly alkaline condition, while the best growth was
achieved at pH 6.0–6.5 (Mevel and Prieur, 2000).

3.4.6. Effect of ammonia concentration

The higher initial ammonia concentration in the medium, the
better bacterial growth and ammonia removal were obtained, with
removal efficiency of 11.08, 41.22, 90.95 mg/d, at low (78.75 mg/L),
intermediate (427.44 mg/L) and high (1121.24 mg/L) initial NHþ 4 -N
concentration, respectively, as shown in Table 3. The removal effi-
Fig. 5. Effect of factors on the growth and nitrification ability of strain L7. C/N ratio
ciency was increased with the increase of initial NHþ
4 -N concentra- (A), temperature (B), salinity (C) and initial pH (D). N, strain growth; j,
tion, suggesting that high ammonia content in the wastewater accumulation of NO2; , removal percentage of NHþ 4 -N. Values are means ± SD
would not inhibit the heterotrophic nitrification activity of (error bars) for three replicates.
42 Q.-L. Zhang et al. / Bioresource Technology 108 (2012) 35–44

Table 3 strain L7. The tolerance to high ammonia load made this strain a
Nitrification performance of strain L7 cultivated in HNM with different ammonia
promising candidate in treatment of municipal landfill leachate
concentrations.
with about 1000 mg/L NHþ 4 -N (Kim et al., 2006). This tolerance to
Initial OD600 NO2 -N (mg/ Final NHþ
4 -N
Removal ammonia was much higher than that of another heterotrophic
NHþ 4 -N
L) (mg/L) efficiency
nitrifier, Providencia rettger YL, which was reported to maximum
(mg/L) (mg/d)
tolerance to 300 mg/L NHþ 4 -N (Taylor et al., 2009). In traditional
78.750 0.256 ± 0.009 0.108 ± 0.005 28.901 ± 1.047 11.08 high ammonium load wastewater treatment, the nitrite accumula-
427.444 0.360 ± 0.064 0.179 ± 0.021 229.110 ± 25.783 41.22
tion was inevitable during to the high free ammonia which would
1121.241 0.644 ± 0.052 0.154 ± 0.017 711.988 ± 29.466 90.95
inhibit the activity of nitrite oxidation microbes (Yun and Kim,
Values are means ± SD for triplicates. 2003). In this study, the amount of accumulated nitrite was
0.15 ± 0.02 mg/L when 1121.21 mg/L NHþ 4 -N was supplied.

3.5. Nitrifying performance of strain L7 under the optimal conditions

The NHþ 4 -N content gradually reduced from initial 146.71–

38.29 mg/L after incubation for 9 days, and the maximum NHþ 4 -N
removal rate of 51.58 mg/L/d was achieved, as shown in Fig. 6A.
The accumulation of nitrite was maintained at low level
(<0.1 mg/L). The growth of strain L7 kept at a stable level (OD600
ca. 1.30) from day 4.
Take the practical application in marine aquaculture into con-
sideration, 30 g/L NaCl was added into the medium. Then a differ-
ent pattern of strain growth and NHþ 4 -N removal was observed, as
shown in Fig. 6B. The highest growth was achieved at 54 h (OD600
ca. 1.20) with NHþ 4 -N removal percentage of 51.03%, followed by a
decline growth phase. The accumulation of nitrite began at 36 h
and maintained a sustained increase to a maximum amount of
0.11 mg/L. The calculated maximum NHþ 4 -N removal rate was
3.08 mg/L/h during the incubation. Compared with that in optimal
medium, the higher removal rate in salinity medium made strain
L7 suitable for the marine aquaculture wastewater treatment.
However, the maximum NHþ 4 -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 L7 to 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 denitrification performance of

strain L7

Fig. 6. Nitrification performance of strain L7 under optimal culture conditions (A) The results of orthogonal tests were tabulated in Table 4 and 5.
and 30 g/L NaCl (B). N, strain growth; j, NO þ
2 ; , NH4 -N. The maximum NO 2 -N removal rate was 5.81 mg/L/d calculated
from Table 4.

Table 4
Results of orthogonal tests for aerobic denitrification performance of strain L7 from day 4–7.

Day 4 Day 5 Day 6 Day 7

OD600 NO
2 -N (mg/L) OD600 NO
2 -N (mg/L) OD600 NO
2 -N (mg/L) OD600 NO
2 -N (mg/L)

1 0.03 66.12 ± 0.65 0.02 63.63 ± 0.00 0.02 65.29 ± 0.52 0.02 66.22 ± 0.78
2 0.01 66.86 ± 0.13 0.01 66.03 ± 0.52 0.01 67.14 ± 1.05 0.01 67.05 ± 0.39
3 0.02 61.41 ± 4.97 0.02 65.29 ± 0.78 0.02 65.20 ± 0.39 0.02 65.20 ± 0.13
4 0.04 65.20 ± 0.65 0.03 65.20 ± 0.65 0.03 65.48 ± 0.52 0.03 64.74 ± 1.05
5 0.01 66.95 ± 0.26 0.01 67.42 ± 1.44 0.01 67.79 ± 1.18 0.01 68.43 ± 0.52
6 0.10 65.94 ± 0.92 0.14 64.64 ± 3.00 0.24 57.81 ± 2.22 0.15 57.44 ± 0.13
7 0.07 66.86 ± 0.92 0.09 65.66 ± 1.05 0.24 59.38 ± 2.61 0.34 51.89 ± 0.65
8 0.06 66.31 ± 0.13 0.08 66.22 ± 0.00 0.15 61.87 ± 0.39 0.16 58.92 ± 0.13
9 0.02 65.48 ± 0.26 0.05 64.92 ± 0.52 0.07 64.92 ± 0.78 0.07 67.14 ± 0.26
10 0.26 58.55 ± 1.70 0.30 53.45 ± 0.65 0.39 47.64 ± 2.22 0.40 44.87 ± 2.48
11 0.06 66.68 ± 0.39 0.05 66.77 ± 0.78 0.06 66.40 ± 0.00 0.10 66.40 ± 0.26
12 0.05 65.48 ± 1.05 0.04 66.22 ± 0.52 0.05 65.38 ± 0.39 0.052 61.60 ± 5.75
13 0.04 67.05 ± 0.92 0.11 63.54 ± 4.84 0.14 54.39 ± 4.70 0.089 51.34 ± 0.92
14 0.09 67.05 ± 0.13 0.10 65.48 ± 1.31 0.15 63.17 ± 1.44 0.227 60.02 ± 0.13
15 0.05 67.79 ± 0.39 0.05 67.23 ± 0.13 0.08 64.92 ± 0.26 0.161 61.41 ± 1.57
16 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.

 Q.-L. Zhang et al. / Bioresource Technology 108 (2012) 35–44 43

Table 5 4. Conclusion
Analysis results of orthogonal tests with software SPSS 16.0.

A. Tests of between-subjects effects In this study, Bacillus methylotrophicus L7 was first reported
Dependent variable: NITRITE Gram-positive bacterial strain to denitrify nitrite to N2 and denitri-
fying nitrite and nitrate to N2O in aerobic condition. Strain L7
Source Type III sum of df Mean F Sig.
squares square exhibited efficient heterotrophic nitrifying–aerobic denitrifying
ability with maximum NHþ 4 -N removal rate of 51.58 mg/L/d and
Corrected 732.26a 12 61.02 14.63 0.019
model maximum NO 2 -N removal rate of 5.81 mg/L/d. Besides, more than
Intercept 60224.84 1 60224.84 1.44  104 0.000 90 mg/d ammonia removal efficiency was obtained even in the ex-
A (shaking 122.71 3 40.91 9.81 0.046 tremely high ammonia load (>1000 mg/L). Therefore, L7 is a prom-
speed) ising candidate in the extensive application of various pollution
B (C/N ratio) 404.46 3 134.82 32.33 0.009
C (salinity) 115.20 3 38.40 9.21 0.051
control system including municipal wastewater, aquaculture
D (pH) 89.89 3 29.96 7.18 0.070 industry, etc.
Error 12.51 3 4.17
Total 60969.61 16
Corrected 744.77 15
Acknowledgement
total
B. Estimated marginal means
This work was supported by grants from the Knowledge Inno-
Level A B C D vation Program of the Chinese Academy of Sciences (No. KJCX2-
YW-L08).
1 57.575 63.720 63.440 65.385
2 62.888 65.938 56.883 59.423
3 60.070 62.890 61.640 60.668 References
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