Journal of Environmental Protec tion, 2013, 4, 74-79
doi:10.4236/jep.2013.41b014 Published Online January 2013 (http://www.SciRP.org/journal/jep)
Study on Characteristics in the Removal Pr oces s of
Ammonia Nitrogen and Nitra t e Nit roge n by a n Isolated
Heterotrophic Nitri f icat i on-Aerobic Denitrification Strain
Rhodococcus sp.
Weisi Li*
Department of chemi cal engin eer in g, China University of Petroleum, 266555, Qingdao, China.
Received 2013
ABSTRACT
Removal of ammonia nitrogen and nitrate nitrogen by an heterotrophic nitrification-aerobic denitrification strain is an
economical and effective method. In this article, a ki nd o f hetero tro phic nitrificatio n -aerob ic de nitrificatio n strai n which
has aero bic denitrification and heterotro phic nitrificat ion ability was selecte d, and t he n wa s identified as rhodococcus sp.
by 16S rRNA sequencing analysis and morphological ob se r vation. After that, carbon source utilization and nitrification-
denitrification activity of this strain in different C/N , initial nitrogen concentration were studied. In addition, the assi-
milation and denitrification activities of ammonia and nitrate were also researched under the condition of nitrate and
ammonia coexisted in the solution. T he res ults show t hat the s trai n can gr ow in sod ium acetate, gl ucose, sodium succi-
nate and sodium citr ate soluti ons, and it can no t survive i n sodium oxalate, sucrose and soluble starch solutions. Initial
concentration and C/N were important for nitrogen removal rate. This strain can completely remove nitrate/ammonia
when nitrate/ammonia concentration was lower than 15 mg·l–1/80 mg·l–1. the C/N of 10 and of 12 were the optimum
C/N ratio in the nitrate and ammonia removal process respectively. pH value rose up sharply in the de nitrificatio n
process and it increased relatively slowly in the nitrificatio n process, which sho ws that p H is one of the most i mportant
factor inhibiting the denitrification removal process. Nitrite concentration was much higher in denitrifica tio n process
than in nitrification process. I n addition, this strai n gave priority to utilizing ammonia as nitrogen sourc e when am moni a
and nitrate coexisted in the solution.
Keywords: Aerobic Denitrification; Heterotrophic Nitrification; Rhodococcus sp.; Nitroge n Removal
1. Introduction
Microbial nitrification and denitrification are totally dif-
ferent biochemical processes according to traditional
theory[1] . Nitrification hap pens only in aero bic conditio n
by two kinds of chemoautotroph bacteria. Ammonia is
oxidized to nitrite by nitrosobacteria and then nitrite is
oxidized to nitrate by nitrobacterium[2]. Denitrification
happens only in anaerobic or faculta tive aero bic condi-
tion. Nitrate and nitrite are reduced to N2 or nitrogen
oxides by heterotrophic denitrifying bacteria. However,
some bacterium which have heterotrophic nitrification
and aerobic denitrification abi lity were selected in recent
20 years [3-5]. T hese st rains can conduct nitrification a nd
denitrification in aerobic condition using or ga ni cs as
carbon source. Based on this principle, a new nitrogen
removal method called simultaneous nitrification and
denitrification (SND) is designed and applicated in
wastewater treatment process. Comparing with tradition-
al met hods, this method has t he follo win g adva ntages: 1)
Nitrification and denitrification can conduct in one reac-
tion cell, which saves floor space and money. 2) pH of
water would rise in denitrification process and OH- can
neutralize H+ generated in nitrification process. 3) Aero-
bic process is easily controlled and of simple operation.
However, these organisms are hard isolated from envi-
ronment. How to select the special strain is the first
problem in nitrogen removal experiments. This work
successfully selected a strain which can conduct hetero-
trophic nitrificat ion and aerobic denitrificatio n. In former
research, rhodococcus sp. wa s lesser studied on nitrogen
removal than on desulfurization[6]. In this work, the
changing characteristics of pH, nitrate, nitrite, ammonia
and growth increment in nitrification or denitrification
activity were researched in different C/N, initial nitrogen
*Corresponding author.

Study on Characteristics in the Removal Proces s of Ammonia Nitrogen and Nitrate Nitrogen by an Isolated
Heterotrophic Nitrification-Aerobic Denitrification Strain Rhodococcus sp.
75
concentration.
2. Method s
2.1. Microorganism
Substrate sludge was collected from a river which was
polluted by domestic sewage. 0.2 ml supernatant, which
was gradient diluted 103, 104…109 times by deionized
water, was coated BTB medium[7] after being fully sha-
ken up. Then incubate it at 30˚C for 3d. T he supernatant
quality condition was as follows (mg·l–1): COD, 57; TN,
6.33; NH3-N, 0.875; NO3-N, 4.53; DO, 6; TP, 0.134; Cl–,
187; pH, 8.6. The BTB medium contained the followin g
(g·l–1): bromthymol blue (BTB, dissolved by 0.5mL ethyl
alcohol), 0.01; agar, 20; NaNO3, 1; KH2PO4, 1; NaCl,
0.15; CH3COONa·3H2O, 8; pH, 7.0. Autoclave all BTB
mediums at 121˚C for 20 min.
Several bacterial strains were observed after three days
incubation, then select strains whose BTB medium had
changed from green to blue. After that, purify these
strains by successive streak transfer on BTB medium.
The denitrification ability/nitrification ability of these
strains was ide ntified by t he aid of LB/HB liq uid denitri-
fication medium. After two days growth in LB/HB me-
dium with temperature of 30˚C and rotating rate of
160r/min, some nitrite chromogenic reagent was added
into the solution. The strain is related to denitrification
and nitrification abilit y if its solution had reddened. The
nitrite chromogenic reagent contained the following:
4-aminobenzene sulfonamide, 20 g; N-1-naphthyl ethy-
lenediamine hydrochloride, 1 g; phosphoric acid 50 ml;
water, 250 ml, then dil ute them to 500 ml. The LB(HB)
liquid medium contained the following (g·l–1): NaNO3
(NH4Cl), 1; KH2PO4, 1; NaCl, 0.15; CH3COONa·3H2O,
8; pH, 7.0-7.3. Autoclave the medium at 121˚C for 20
min.
2.2. 16S rRNA Gene Sequences, Phylogenetic
Analysis and Morphological Observa tion
Genomic DNA of isolate HY-1 was extracted using a
SK1201-UNIQ-10 column type bacterial DNA Isolation
Kit (Sa n gon ，Shanghai，Chin a). The 16S rRNA was am-
plified by polymerase chain reaction(PCR Thermal Cycler,
BBI, Canada) using universal primers (7f, 5'-CAGAG T-
TT GATCCTGGCT-3' and 1540r(1522), 5'-AGGAGGT-
GATCCAGCCGCA-3'). The PCR reaction system con-
sisted of: 10pmol of template; 1ul of primer up (10 uM);
1 ul of pri mer down (10 uM); 1 ul of dNTP mix (10 Mm
each); 5 ul 10*Taq reaction buffer; 0.25 ul Taq(5 u/ul)
and added water to 50 ul. Genes were amplified by
pre-denaturation at 9 8˚C for 5min, followed by 35 cycles
of denaturation at 95 ˚C for 35 s, annealing at 55˚C for 35
s, and elongation at 72˚C for 90 s, then by a final of ex-
tension at 72˚C for 8 min. The PCR products were puri-
fied and sequencing by a DNA Sequence r (3730, ABI,
USA). The closet matching sequences in the GenBank
database were searched using the BLAST program and
the phylogenetic tree was established by the PHYLIP
software. Some fresh HY-1 cells were put on the micro-
slide and dyed by safranine for 1-2min, then observe it
by fluorescence convert microscope (LEICA, DMI3000B,
Germany). Gram stain method and colony characteristic
were also considered.
2.3. Carbon Source Utilization
Seven kinds of carbon sources, including sodium acetate,
sodium citrate, glucose, soluble starch, sodium oxalate,
sucrose and sodium succinate, were used as different
substra t es in experiments. The carbon source medium
contained the following (g·l–1): NaNO3, 1; KH2PO4, 1;
NaCl, 0.15; pH, 7.0-7.3; C/N (molar ratio), 12; water,
100 ml. Autoclave them at 121˚C for 20 min. Then
transfer some HY-1 cells from plate medium into 10 ml
aseptic water which was fully shaken up. After that, in-
oculate 0.5 ml cell suspension into every different carbon
source medium. Culture them in the shaking table with
stirring rate of 160 r/min and 30˚C for two days. Initial
values of medium were as follo ws: NO3-N, 221.3 mg·l–1;
pH, 7.3; NO2-N, 0 mg·l–1. Growth condition was ob-
served and nitrate removal rate, nitrite concentration,
OD600 (Spectrophotometer, 2100, Unico, USA) and pH
(pH Meter, pH221, HANNA, Italy) were analyzed.
2.4. Influence of Initial Ammonia and Nitrate
Concentration
The optimum carbon source was set as sodium acetate
and original mediu m of initial nitrate (a mmonia) concen-
tration contained the follo wing (mg·l–1): NO3-N (NH3-N),
150 (160.5) ; KH2PO4, 500; NaCl, 150; pH, 7.0-7.3; C/N,
36; water, 100 ml. Experiment mediums were obtained
by diluting the original medium to 1, 2, 4, 8, 16, 32 times
by deionized water. T hen autoclave them at 121˚C for 20
min. Inoculatio n and culture methods were the same as
section 2.3. Removal rates of nitrate (ammonia), nitrite
concentration, OD600 and pH were analyzed after the
mediums were centri fuged with 7000 r/min for 5 min
(High-Speed Freezing Centrifuge, Allegra 25R, BECK-
MAN , USA).
2.5. Effect of C/N
The C/N medium contained the following (g·l–1): KH2PO4,
0.5; NaCl, 0.15; pH, 7.0-7.3; water, 100 ml. Sodium ace-
tate was set as carbon source in the C/N experiment. C/N
was set to a series of 2, 4, 6, 8, 10, 12, 14, 16 and me-
diums were autoclaved at 121˚C for 20 min. Inoculation

Study on Characteristics in the Removal Proces s of Ammonia Nitrogen and Nitrate Nitrogen by an Isolated
Heterotrophic Nitrification-Aerobic Denitrification Strain Rhodococcus sp.
76
and culture methods were the same as section 2.3. Initial
nitrate (ammonia) concentration was 50.1 mg·l–1 (79.9
mg·l–1) and initial nitrite concentration was 0 mg·l–1. Fi-
nal nitrate (ammonia) concentration, nitrite concentration,
OD600 and pH were analyzed after the mediums were
centrifuged.
2.6. Ammonia and Nitrate removal Process of
the Strain in the Mixed Solution
To clarify the competitive utilization of nitrate and am-
monia by the strain, a time series experiment was carried
out when nitrate and ammonia coexisted in the same so-
lution. The experiment medium contained the following
(mg·l–1): NO3-N, 48.9; NH3-N, 77.8; KH2PO4, 500; NaCl,
150; pH, 7.0-7.3; C/N, 12; water, 100 ml. Autoclave
these mediums at 1 21˚C for 20 min . Inoculation and cul-
ture methods were the same as section 2.3. Take out o ne
erlenmeyer flask ever y 2.5 h and centrifuge the medium.
The n measure the nitrate, nitrite, ammonia concentratio n,
OD600 and pH of the centrifugal liquid.
3. Results and Discussion
3.1. Identification, Phylogenetic Analysis and
Morpho log ica l Charac ter of the St rain
HY-1 strai n bacterial colony wa s salmon p ink and moist.
Unde r microscope, cells of the strain were rod or coccus,
and they exhibited gram positive staining. Rhodococcus
sp. had these remark able charac teristics. Figure 1 showed
the neighbor joining phylogenetic tree using HY-1 gene
fragment and GenBank database sequences. HY-1 isolate
was most similar (99%) to Rhodococcus sp. from Gen-
Bank database and phylogenetic tr ee, which coincided with
the conclusion of morphological character of the strain.
So HY-1 was co nfirmed to b e the strai n Rhodococcus sp
3.2. Carbon Source Utilization by the Strain
Carbon source[8] and C/N[9-10] are key factors influen-
cing the aerobic denitrification process. The main rea-
son is that perip la smic reductase whose activity was in-
fluenced by carbon source was the most important re-
ductase for nitrate removal. In this experiment, the re-
sults show that sodium acetate, glucose, sodium succi-
nate, sodiu m citrate can be used by the stra i n, b ut s ucrose,
soluble starch and sodium oxalate can not be used (Table
1). It can be obtained from above result that the strain is
inclined to using ionic organic carbon source or small
organic molecules. Nitrate removal rate and cell growth
increment were higher in ionic organic carbon solution
than in glucose solution. Meanwhile, pH increased much
faster in ionic organic carbon solution than in glucose
solution. Obviously, low nitrate removal rate is due to
strong basicity because alkalinity inhibited the microor-
ganism growth and aerobic denitrification process. Ni-
trite did not accumulate in all solutions possibly for the
reason that nitrite reductase has higher activity than ni-
trate reductase of HY -1 s t rain.
Temperature and initial pH influence cell growth[11]
and nitrate removal rate[12]. This strain can survive be-
twee n 20˚C and 40˚C. 30˚C is the optimum temperature.
Initial pH of between 5 and 9 is suitable for the strain.
The re was no gro wth p heno me non when pH≤4 or pH≥10.
Further more, this strain can not grow in anaerobic con-
dition, and it is an absolutely aerobic bacteria.
3.3. Effect of Initial Nitrate and Ammonia
Concentration
The influence of initial c oncentration on nitrification and
denitrification are briefly discussed as the following as-
pects : 1) High initial nitrogen concentration or metabo-
lites harm the microbial growth or enzyme activity; 2)
Microorganism has the maximum cell density, and the
nitrification or denitrification do not occur any more till
cell density reaches the maximum. In this denitrification
Figure 1. Phy logenetic tree of the strai n.
Table 1. Carbo n source uti lization.
carbon source Nitrate removal rate Final pH OD600 Nitrite (mg l-1)
sodi um acetate 30.5% 9.57 ↑ 1.87 0.002
glucose 18. 7% 7.74 ↑ 1.44 0.016
sodium succinate 27.8% 9.54 ↑ 1.83 0.004
sodium citrate 29.3% 9.51 ↑ 1.86 0.008
↑ represented pH rising.

Study on Characteristics in the Removal Proces s of Ammonia Nitrogen and Nitrate Nitrogen by an Isolated
Heterotrophic Nitrification-Aerobic Denitrification Strain Rhodococcus sp.
77
experiment, pH reached to about 9.5 and nitrate was not
removed completely when nitrate concentration was
more than 15 mg·l–1. Obviously, OH- inhibited the deni-
trification process or enzyme activity. When nitrate con-
centration was lower than 15 mg·l–1, final pH did not
reach 9.5 and nitrate was completely removed. However,
it was surprise that OD600 was proportional to initial ni-
trate concentration, which illuminated that cells kept
growing even when pH reached 9.5. The possible reason
is that this strain had two nitrate metabolic mode. The
strain conduct s denitrification and it produces OH– when
pH is lower than 9.5. Nitrate is absorbed by the strain
on ly as nitroge n so urce , and pH keeps invariant when pH
is higher t han 9.5. Nitrite conc e ntra tion kept low bet ween
0.04 mg·l-1 and 0.17 mg·l–1 in all various initial nitrate
conc entration so luti on.
Compared with denitrificatio n, HY-1 s train had higher
ammonia re moval rate in nitrification. Ammonia removal
rate can reach 93% when initial ammonia concentration
was 80 mg·l–1. pH rose up relatively slo w in nitrification
process than in denitrification process, which was totally
different from H+ produced process of autotrophic nitri-
fication.
3.4. Effect of C/N
C/N is a key factor influencing on the form and amount
of metabolites. In a certain range, the higher the carbon
source concentration, the faster the denitrification rate
[9,13,14]. In addition, different C/N can result in differ-
ent biochemical process and metabolites in nitrification
and denitrification process, and then it influences on the
nitrate (ammonia) removal rate.
From Fig ure 3 (Left), pH of all C/N solutions rose
sharply from about 7.0 to about 9.5. It showed that the
denitrification of HY-1 strain is an alkali producing
process when carbon source is sodium acetate. Nitrate
removal rates rose up at beginning with C/N increasing,
and then it decreased when C/N>10. Nitrate removal rate
reached the maximum with removal rate of 62.5% when
C/N=10. Meanwhile, OD600 a lso got the max imum va lue
when C/N=10, which illustrated that the strain needed an
optimum substrate co ncentrat ion for gro wth and re moval
process. Nitr ite did not accumulate in most C/N solutions.
However,nitrite concentration reached 0.42 mg·l–1 when
C/N=10 and it was much higher than in other C/N solu-
tions.
Compared with denitrification (Fig ure 3, Right), pH
of all C/N solutions rose up relatively slow in nitrifica-
tion process and the highest pH was 9.47 with a C/N of
12. Definitely, final pH was influenced by C/N and car-
bon s ource wa s not suffic ient wh en C/N<1 2. In thi s situ-
ation, the higher the C/N, the higher the ammonia re-
moval rate. Ammonia removal rate exceeded 94% when
C/N≥12. Moreover, OD600 showed similar changes to pH
rising and nitrate removal rate . Nitrite concentr ation kep t
very low in al l C/N solut io ns.
Figure 2. Effect of initial nitrate/ammonia concentration on pH, nitr ite and nitrate/ammonia concentr a tion.
Figure 3. Effect of C/N on pH, nitrite and nitrate (ammonia) concentration.

Study on Characteristics in the Removal Proces s of Ammonia Nitrogen and Nitrate Nitrogen by an Isolated
Heterotrophic Nitrification-Aerobic Denitrification Strain Rhodococcus sp.
78
Figure 4. Ammonia and nitrate re moval process of the st rai n in the mixe d solution.
3.5. Ammonia and Nitrate Removal Process of
the Strain in the Mixed Solution
In this experiment (Figure 4), OD600 and ammonia re-
moval rate increased slowly during the beginning 20 h,
and then both sharply rose up after 22.5 h. Meantime,
ammonia removal rate and pH also rose up sharply. At
this time, the strain was possible in the logarithmic
gr owth phase. Ammonia removal rate increased from
73.6% of 20th hour to 95.8% of 22.5th hour. The pH,
whi ch increased from 8.88 to 9.34, kept moving on to
about 9.55. Nitrate concentration was almost unchanged
during beginning 20 h. After 20 h, nitrate concentration
decreased lightly, and then reduced from 48.9 mg·l–1 to
45.7 mg·l–1. So the nitrate removal rate was only 6.5%
after 30 h. Obviously, the strain conducted nitrification
prior to denitrification when ammonia and nitrate coex-
isted in the s ol ution. Therefore, ammonia was use d fir st ly
by the strain and nitrate was used only when ammonia
was completely removed. Nitrite concentration kept a
very low level(<0.01 mg·l–1) during the beginning 25 h,
and then increased slightl y to 0.06 mg·l–1 aft er 2 5 h. This
phenomenon also showed that denitrification started
conducting when ammonia was al most removed.
4. Conclusions
This work selected an aerobic heterotr ophic nitrification-
aerobic denitrificatio n strain which belongs to rhodococcus
sp.. This strain can use nitrate and ammonia as nitrogen
source, and it also can use sodium acetate, glucose, so-
dium succinate and sodium citrate as carbon source in
denitrificatio n. 15 mg·l–1 and 80 mg·l–1 were the best
initial ammonia and nitrate concentration for denitrific a-
tion and nitrification re spectively. A C/N of 10 and a C/N
of 12 were the best C/N ra tio in the nitrate and a mmonia
removal process respectively. pH value was the most
important factor inhibiting nitrate and ammonia removal
process because pH rose up to a very high value in both
processes. In addition, this strain gave priority to utilize
ammonia as nitrogen source to nitrate when ammonia
and nitrate coexisted in the solution.
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