Aerobic nitrification–denitrification by heterotrophic Bacillus strains

Bioresource Technology 96 (2005) 1897–1906

Aerobic nitrification–denitrification by heterotrophic Bacillus strains

Joong Kyun Kim a,*, Kyoung Joo Park a, Kyoung Sook Cho a, Soo-Wan Nam b,Tae-Joo Park c, Rakesh Bajpai d

a Division of Food Science and Biotechnology, Pukyong National University, Pusan 608-737, Republic of Koreab Department of Biotechnology and Bioengineering, Dong-Eui University, Pusan 614-714, Republic of Koreac Department of Environmental Engineering, Pusan National University, Pusan 609-735, Republic of Korea

d Department of Chemical Engineering, University of Missouri-Columbia, Columbia, MO, USA

Received in revised form 3 January 2005; accepted 28 January 2005

Available online 29 March 2005

Abstract

Twenty-four Bacillus strains predominantly outgrown in a night soil treatment system were isolated and characterized. Under

various culture conditions, cell interactions took place among them and cell population changed. Maximum removal of NHþ4 -N

and cell production by the isolates occurred under the conditions of 30% DO and C/N ratio of 8. Five dominant isolates were iden-

tified to be species of Bacillus cereus, Bacillus subtilis and Bacillus licheniformis with similarities of 78–94%. Additions of 0.8% pep-

tone and 0.3% yeast extract to a basal medium influenced the growth of isolates and the removal of NHþ4 -N in flask culture. Metal

ions such as Ca2+, Fe2+ and Mg2+ had a similar effect. The specific growth rates of the five isolates were found to be in a range of

0.43–0.55 h�1. During the flask experiment of nitrogen removal under aerobic growth conditions, active nitrification by the isolates

occurred largely in 1 h with a decrease of COD and alkalinity reduced to only 74.6% of theoretical value. From the nitrogen balance,

the percentage of nitrogen lost in the flask culture was estimated to be 33.0%, which was presumed to convert to N2 gas. This con-

version of ammonia to N2 without formation of nitrous oxide under aerobic growth conditions was confirmed by GC analysis.

From all the results, it has been found that the Bacillus strains were able to occur simultaneously aerobic nitrification/denitrification

and the B3 process using the Bacillus strains seemed to possess some economic advantages.

� 2005 Elsevier Ltd. All rights reserved.

Keywords: Aerobic nitrification–denitrification; Bacillus strains; Nitrogen removal; Cell-population balance

1. Introduction

The presence of nitrogenous substances in wastewater

discharges has attracted attention because of the role of

nitrogen in eutrophication of receiving waters. Nitrogenremoval is an important aspect of present day wastewater

treatment processes, and biological nitrification–denitri-

fication is one of the most economical processes for nitro-

gen removal from municipal wastewaters (Gupta and

Gupta, 2001). The nitrogenous substances in municipal

0960-8524/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2005.01.040

* Corresponding author. Tel.: +82 51 620 6186; fax: +82 51 620 6180.

E-mail address: [email protected] (J.K. Kim).

wastewater is mostly in the form of ammonium ion

requiring a treatment process involving the biological

oxidation of NHþ4 into NO

�3 (nitrification) followed by

the biological reduction of NO�3 into N2 (denitrification).

It has commonly been accepted that nitrification anddenitrification require aerobic and anoxic conditions,

respectively. However, there have been periodic reports

of anaerobic ammonium oxidation (anammox) (Fux

et al., 2002; Schmidt et al., 2003) and aerobic denitrifica-

tion (Meiberg et al., 1980; Robertson and Kuenen, 1983;

Su et al., 2001b; van Niel et al., 1992). The nitrification–

denitrification process has been also challenged by a one-

step process in which ammonium is oxidized directly to

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1898 J.K. Kim et al. / Bioresource Technology 96 (2005) 1897–1906

N2 (Astrid et al., 1995; Mulder et al., 1995). Recently, re-

searches focus on nitrite nitrification, which might be a

short cut process for savings in oxygen for nitrification

and carbon requirements for denitrification (Eum and

Choi, 2002; Rahman et al., 1995).

In Korea, �B3 (Bio Best Bacillus) process� (KoreanPatent No. 151928) has been known as an advanced

wastewater treatment system in which Bacillus strains

have predominated. It has been reported that the process

is able to remove nitrogen and phosphorus as well as or-

ganic matter efficiently (Choi et al., 2000). Various

microorganisms participate in the nitrogen cycle in the

natural ecosystem. Among them, the nitrification process

has been thought to be carried out mainly by ammonia-and nitrite-oxidizing bacteria that are obligately aerobic

and chemoautotrophic. However, a number of hetero-

trophic microorganisms have been reported to nitrify

many types of nitrogen compounds (Focht and Verstra-

ete, 1977). Despite the diversity of the reactions, their

activities have known to be generally lower than those

of autotrophs and to be not essential for their growth.

Hence, their contribution to the nitrification processhas remained unclear in spite of the studies on heterotro-

phic nitrification (Papen et al., 1989; Rho, 1986; Robert-

son and Kuenen, 1990; Robertson et al., 1988).

Bacillus strains have known to be involved in hetero-

trophic nitrification (Mevel and Prieur, 2000), but other

characteristics of the Bacillus strains have not been stud-

ied in detail so far. Understanding the characteristics of

microorganisms used in the biological nitrification–deni-trification process is very important in order to remain

high efficiency of treatment at all times, and the possible

microbiological nitrogen conversions should be pro-

vided. The purpose of this study was to understand

Bacillus strains predominantly occurring in a night soil

treatment system by isolating pure cultures and charac-

terizing these isolates.

2. Methods

2.1. Isolation of Bacillus strains

The Bacillus sludge and cell suspension were obtained

from five different sites of night soil treatment systems in

which Bacillus strains were predominantly occurringand stable growth was maintained. The samples were

first agitated to obtain homogeneous suspensions in

sterile 0.2% NaCl. One milliliter of the suspended liquid

was pipetted into a 10 mL tube that contained a 0.8%

nutrient broth medium. After two days of incubation

at 30 �C and 150 rpm, cells were spread with a platinum

loop on 1.5% nutrient agar plates. Purified isolates of

Bacillus cells were obtained by repeated streaking onfresh agar plates. Each isolate was maintained on a

1.5% nutrient agar plate.

2.2. Characterization of isolates and their taxonomic

identification

To observe the change of cell population among

Bacillus strains under various culture conditions, a 5-L

continuous stirred reactor was used with the workingvolume of 3.5 L. The pH and temperature in the reactor

were controlled at 7 and 30 �C, respectively. The agita-tion speed was 300 rpm, and the concentration of dis-

solved oxygen (DO) in the reactor was adjusted by

both air and argon gas at the same time. The same

amount of isolates was cultivated in flasks, and 10% of

those mixed cells were used as an inoculum. The compo-

sition of a medium used in this experiment was (g L�1):glucose, 1.05; NH4Cl, 0.382; KH2PO4, 0.131; peptone,

0.05; yeast extract, 0.05; and 1 mL-mineral solution.

The mineral solution contained (g L�1): FeSO4 Æ7H2O,

3; H3BO3, 0.01; Na2MoO4 Æ2H2O, 0.01; MnSO4 ÆH2O,

0.02; CuSO4 Æ5H2O, 0.01; ZnSO4, 0.01; and ethylenedia-

mine tetraacetic acid (EDTA), 0.5. Glucose or NH4Cl

was used to provide for various C (total organic carbon,

TOC)/N (NHþ4 -N) ratios. All media used were sterilized

by autoclaving at 121 �C for 15 min. The experiment

was conducted as a repeated batch. After environmental

conditions (DO and C/N ratio) were changed, first two

batches were wasted not to obtain transient data. Sam-

ples were taken and analyzed from the third and fourth

batches. The experiment was initiated again when more

than 30% of spores were found in the mixed culture after

changing culture conditions. Each batch was executedfor 8 h and measurements were performed in triplicates.

In flask experiments to study effects of growth factors

on growth of isolates, a basal synthetic medium was

used, which contained (g L�1): KNO3, 1; K2HPO4, 1;

EDTA, 3.5 · 10�3; ZnSO4 Æ7H2O, 2 · 10�3; FeSO4 Æ7H2O, 10 · 10�3; MnSO4 Æ7H2O, 2 · 10�3; CuSO4Æ5H2O, 1 · 10�3; Co(NO3)2 Æ6H2O, 0.2 · 10�3; and

H3BO3, 1 · 10�3. Vitamin solution contained (g L�1):nicotinic acid, 0.2; thiamine-HCl, 0.4; nicotinamide,

0.2; and biotin, 0.008. The vitamin solution was added

to the flask after autoclaving. After growth factors were

determined, a synthetic medium containing additional

0.8% peptone and 0.3% yeast extract in the basal med-

ium was used for cultures of pure and mixed cells in later

experiments. A bacterial suspension (10% inoculum) of

the isolates was inoculated into duplicate flasks. Themedium pH was maintained at 7, and flask cultures were

incubated at 150 rpm and 30 �C for 14 h.

Capacity of isolates for aerobic denitrification was

tested in a tightly sealed 1 L-branched flask in duplicate.

The flask was evacuated and pure oxygen was fully pres-

surized into the flask before autoclaving (initial DO was

approximately 70% saturated). A septum was equipped

on the mouth of the branched flask for gas analysis.The night soil was used as a main substrate, and the sub-

strate composition was adjusted by glucose, NH4Cl and

J.K. Kim et al. / Bioresource Technology 96 (2005) 1897–1906 1899

KH2PO4 to simulate a typical composition of municipal

wastewater in Korea. The final composition of the sub-

strate was (mg L�1): chemical oxygen demand (COD),

250; NHþ4 -N, 35; NO

�2 -N, 0; NO

�3 -N, 0; total nitrogen

(TN), 50; and total phosphorus (TP), 7. The isolates

were cultivated very actively under the environmentalcondition of 30% DO, and 10% of those cells were

inoculated into the flask. The flask was incubated at

150 rpm and 30 �C. Samples were taken from the flasks

periodically for measurements of optical density (OD),

dry cell weight (DCW), NHþ4 -N, NO

�2 -N, NO

�3 -N,

TN, TP, COD, alkalinity, oxygen and nitrogen gas

concentrations.

To obtain microscopic features of isolates, the Gramstaining and agar-stab culture were made. Cell size,

motility and morphology of isolates were also deter-

mined microscopically (Axiostar plus microscope, Carl

Zeiss, German) (1000·). For taxonomic identification,API 50CH identification system (Version 6.0) was used.

2.3. Analyses

Growth of isolates was determined spectrophotomet-

rically at a 510-nm wavelength (OPRON-3000, Hanson

Technology Co., Ltd., Korea). The DCW was deter-

mined by weighing the cell pellet after being dried in

an oven at 100 �C for 12 h. The cell pellet was prepared

by centrifuging a 5 mL sample of broth culture at

5000 rpm for 10 min and then by decanting the superna-

tant after washing twice with distilled water.The concentrations of NHþ

4 -N, COD of medium were

determined by procedures given in the Standard Meth-

ods (APHA, 1992). Estimation of NHþ4 -N was made

by a phenate method that monitored the absorbency

at 635 nm. COD was measured by a closed reflux color-

imetric method at 600 nm. Nitrite and nitrate were esti-

mated by ion chromatography (Metrohm 792 Basic IC,

Switzerland). TN and TP were determined by ultravioletadsorption method and ascorbic acid reduction method,

respectively (APHA, 1992). Alkalinity was determined

by the titration method.

For oxygen and nitrogen determination, 20 lL sam-

ples (injection volume) were taken by a press-lock syr-

inge for GC/TCD (Perkin Elmer Instruments, USA)

analysis. The carrier gas was helium at a flow rate of

20 mL min�1. The column used was a �molecular sieve5A� (stainless steel, mesh 80/100, 6 ft · 1/8 in). The col-umn and detector temperatures were 70 and 120 �C,respectively. The amounts of both oxygen and nitrogen

were calculated by applying the ideal gas law.

2.4. Statistical analyses

Statistical analyses were done with measurements ob-tained from this study. Since the sample observations

were not arranged in a frequency distribution, the stan-

dard deviations were calculated by the following proce-

dures: each deviation was squared, the sum of the

squares was divided by (n � 1), one less than the sample

size (n), (this resulted in the sample variance) and finally

extraction of the square root recovered the original scale

of measurement. Comparisons of means were performedby the Tukey method (Neter et al., 1985) using the SAS

program, since all sample sizes were equal. Differences

were considered significant at P < 0.05.

3. Results and discussion

3.1. Isolation of Bacillus strains

Bacillus strains were isolated by streaking on agar

plates. Various distinct colonies developed on the agar

plates after two days incubation. The microscopic fea-

tures of the 24 isolates are tabulated in Table 1.

Although some isolates exhibited similar characteristics,

they had different colony types in elevation and surface

on agar. Only few cells were isolated identically fromfive different sites of night soil treatment systems. This

fact suggested that dominant Bacillus strains in a main

process of wastewater treatment system could be chan-

ged significantly by different environmental conditions

in which cell interactions took place among microbial

population (Purtschert and Gujer, 1999). Only one

(PK22) out of 24 colonies was found to be Gram nega-

tive cell. It is, thus, not a Bacillus strain and it might bePseudomonas or Zoogloea that is generally present in

activated sludge.

Hence, a detailed experiment was performed in a 5-L

bioreactor in order to investigate the change of cell pop-

ulation under different culture conditions.

3.2. Change of cell population among isolates

To investigate the change of cell population among

Bacillus strains, all the isolates were combined in a batch

reactor incubated for 8 h under various culture condi-

tions. The result is tabulated in Table 2. For determina-

tion of each cell�s population in the mixed culture,

samples were diluted such that 25–50 colonies were

formed on an agar plate, and the identification of each

species was based upon information in Table 1. The col-onies that were not clearly identifiable were classified as

remaining Bacillus strains. As mentioned in Section 3.1,

cell interactions among the isolates were observed

distinctly.

When the reactor was first operated under conditions

of 30% DO and a C/N ratio of 4 (400 ppm TOC/

100 ppm NHþ4 -N), the most dominating species was

found to be PK15, followed by PK11, PK8, PK5,PK16 and the other species. This reflected consider-

able microbial interactions among isolates during

Table 1

The microscopic features of 24 isolates

Isolates Gram strain Sizea (lm) Colony pigmentb Chain forming Motilityc Agar-stab cultured

PK1 + L: 2.5–4, W: 0.8 I None ++ F.A. (s.g.)

PK2 + L: 3–4, W: 0.7–1 L.B. Pair ++ F.A. (s.g.)

PK3 + L: 4.5–6, W: 1 I Pair ++ F.A. (d.g.)

PK4 + L: 4–5, W: 0.5–0.8 I Pair + F.A. (d.g.)

PK5 + L: 4–5, W: 1.2 I 2–3 +++ F.A. (d.g.)

PK6 + L: 2–3, W: 0.8 I Pair ++ F.A. (d.g.)

PK7 + L: 7–9, W: 1.2–1.5 I 2–3 + F.A. (s.g.)

PK8 + L: 3–4, W: 0.8–1 I None ++ Aerobic

PK9 + L: 4–5, W: 1 S.T. None + F.A. (s.g.)

PK10 + L: 3–4, W: 1–1.2 I Pair + F.A. (s.g.)

PK11 + L: 3–4, W: 1.2 I Pair ++ Aerobic

PK12 + L: 4–5, W: 1–1.2 I None ++ F.A. (s.g.)

PK13 + L: 4–5, W: 1 I 2–4 + F.A. (s.g.)

PK14 + L: 6–7, W: 1.5 I 2–4 + F.A. (s.g.)

PK15 + L: 2.5–3, W: 0.8 S.T. None +++ Aerobic

PK16 + L: 1.5–2, W: 0.7 I None +++ F.A. (d.g.)

PK17 + L: 4–5, W: 1–1.2 I None ++ F.A. (s.g.)

PK18 + L: 2.5–3, W: 0.8–1 L.Y. None ++ F.A. (s.g.)

PK19 + L: 1.5–2, W: 0.7 I None ++ F.A. (d.g.)

PK20 + L: 3–4, W: 1.2 I Several ++ F.A. (d.g.)

PK21 + L: 4–5, W: 1.2–1.5 I 2–4 ++ F.A. (d.g.)

PK22 � L: 1–1.2, W: 1 S.T. None ++ F.A. (d.g.)

PK23 + L: 3.5–5, W: 1 I 2–3 ++ F.A. (d.g.)

PK24 + L: 1–1.2, W: 0.8–1 L.Y. None ++ F.A. (d.g.)

a L means length and W means width.b Symbols mean as follows: I, ivory; L.B., light brown; S.T., semi-translucence; and L.Y., light yellow.c + means the degree of motility: +, weak; ++, moderate; and +++, strong.d F.A. means facultatively anaerobic; s.g. shallow growth and d.g. deep growth.

Table 2

Change of cell-population balance for 8 h under various culture conditionsa

Culture conditions Removal

of NHþ4 -N

(mg L�1)

Cell

production

(g L�1)

Population balance

30% DO and C/N = 4 (400 ppm TOC/100 ppm NHþ4 -N) 30 ± 2e 0.25 ± 0.01e 73 ± 7%ab PK15, 8 ± 4% PK11, 8 ± 4% PK8,

4 ± 2%b PK5, 4 ± 2%a PK16, 5 ± 4% others

30% DO and C/N = 8 (800 ppm TOC/100 ppm NHþ4 -N) 118 ± 4c 0.60 ± 0.03d 83 ± 6%ab PK15, 6 ± 4%b PK5, 6 ± 4%a PK16,

7 ± 3% others

30% DO and C/N = 4 (800 ppm TOC/200 ppm NHþ4 -N) 163 ± 4b 0.86 ± 0.02b 87 ± 6%a PK15, 4 ± 2%b PK5, 4 ± 2%a PK16,

4 ± 2% others

30% DO and C/N = 8 (1600 ppm TOC/200 ppm NHþ4 -N) 226 ± 5a 1.10 ± 0.02a 90 ± 6%a PK15, 4 ± 2%b PK5, 4 ± 2%a PK16,

2 ± 2% others

15% DO and C/N = 8 (800 ppm TOC/100 ppm NHþ4 -N) 116 ± 3c 0.68 ± 0.03cd 85 ± 5%ab PK15, 4 ± 2%b PK5, 7 ± 4%a PK16,

4 ± 3% others

5% DO and C/N = 8 (800 ppm TOC/100 ppm NHþ4 -N) 63 ± 3d 0.40 ± 0.04e 52 ± 8%b PK15, 28 ± 4%a PK5, 19 ± 4%a PK16,

2 ± 2% others

30% DO and C/N = 8 (800 ppm TOC/100 ppm NHþ4 -N) 120 ± 4c 0.76 ± 0.03bc 90 ± 4%a PK15, 5 ± 2%b PK5, 5 ± 2%a PK16,

2 ± 2% others

a Means in the same column with different superscript are significantly different (P < 0.05). Values represent mean ± S.D. of two replicates.


heterotrophic nitrification (Rho, 1986). The domination

of PK15 increased with the increase of C/N ratio, while

the populations of PK8 and PK11 reduced significantly.

Even at the same C/N ratio, the PK15 was predominant

in the medium containing high concentrations of TOC

and NHþ4 -N. The results showed that carbon and nitro-

gen components in the medium affected the nitrifying

activity of the isolates. When the DO concentration de-

creased from 30% to 15%, the removal amount of

NHþ4 -N reduced to half with minimal changes of cell-

population balance. At 5% DO and a C/N ratio of 8,

the populations of PK5 and PK16 increased markedly,


while that of PK15 decreased significantly. From the

above results, it was found that PK15 grew very well

under aerobic conditions (more than 15% DO) and

PK5 and PK16 did under the condition of 5% DO. This

phenomenon could be explained by the results in Table

1. Maximum removal of NHþ4 -N and cell production oc-

curred under the conditions of 30% DO and C/N ratio

of 8. This fact suggested that PK15 was involved in

active nitrification (to remove ammonia) while PK5

and PK16 were involved in denitrification (during con-

version of nitrate to N2). Bacillus licheniformis (like

PK16) has been reported to possibly occur aerobic de-

nitrification (Brycki et al., 2000).

The effect of DO concentration on the removal effi-ciency of NHþ

4 -N and cell production was plotted in

Fig. 1. When DO concentration was maintained in a

range of 15% to 30%, DO did not affect the removal

efficiency of NHþ4 -N significantly, but the efficiency

was almost halved under the condition of 5% DO. This

trend could also be seen in cell production. Thus, this

indicates that cell activity for nitrification under an aer-

obic condition is possibly related to the efficient re-moval of ammonia and cell production as well. In

Fig. 2, the effect of substrate composition on the re-

moval efficiency of NHþ4 -N is shown. The removal effi-

ciency of NHþ4 -N by the isolates was higher at a C/N

ratio of 8 than that at C/N ratio of 4. At a C/N ratio

of 8, there was not any effect of the substrate compo-

sition on the removal efficiency of NHþ4 -N, but the ef-

fect of substrate composition was significant at the C/Nratio of 4. This indicates that the isolates required

higher concentrations of organic carbons (Mevel and

Prieur, 2000).

DO (% sat0 5 10 15

Rem

oval

effi

cien

cy o

f NH

4 -N (%

)

0

20

40

60

80

100

120

At C/N= 8

Fig. 1. The effect of DO on removal efficiency of NHþ4 -N and c

3.3. Taxonomic identification and characteristics of

isolates

The five isolates outgrown over the other isolates in

the experiment of change of cell population (Section

3.2) were taxonomically identified on the basis of theirbiochemical characteristics. The results are presented

in Table 3. Those species have been reported in waste-

water treatment systems (Kubo et al., 2001; Matsui

et al., 1998; Zissi and Lyberatos, 1996), but the result

of identification did not exhibit very high similarity

(78% to 94%). All three isolates (PK8, PK11 and

PK15) were identified to be Bacillus subtilis. Although

the microscopic features of PK8 and PK11 were verysimilar (Table 1), they showed a different colony type

in elevation and surface on an agar plate. Some features

of the isolates were in agreement with those written in

the Bergey�s Manual (Claus and Berkeley, 1986), but

further taxonomic study such as phylogeny analysis

achieved by homology comparison of 16S rDNA se-

quence in available gene bank is necessary for their exact

identification.Flask experiments for the study of effects of growth

factors on the growth of PK15 that was largely present

in the cell population (Table 2) and mixture culture of

the five isolates was conducted, and the results are tab-

ulated in Tables 4 and 5. The addition of 0.8% peptone

increased both the growth of PK15 isolate and the re-

moval of NHþ4 -N significantly (Table 4). The addition

of 0.3% yeast extract resulted in some effect on thegrowth and the removal of ammonia as well, while the

additions of 0.1% glucose and 0.1% vitamin solution

did not. A similar trend was observed in the mixed

uration)20 25 30 35

Cel

l pro

duct

ion

(g/l)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Removal efficiency of NH4-NCell production

(800 ppm TOC/100 ppm NH4-N)

+

+

ell production. Error bars: mean ± S.D. of two replicates.

C/N Ratio

C/N = 4 C/N = 8

Rem

oval

effi

cien

cy o

f NH

4 -N (%

)

0

20

40

60

80

100

120

400 ppm TOC/100 ppm NH4 -N




Under the condition of 30% DO+

+

+

+

Fig. 2. The effect of substrate composition on removal efficiency of NHþ4 -N. Error bars: mean ± S.D. of two replicates.

Table 3

The result of identification for the dominant Bacillus species among

isolatesa

Dominant species Identification Similarity (%)

PK5 B. cereus 78 ± 2

PK8 B. subtilis 92 ± 0



PK16 B. licheniformis 94 ± 0

a Values represent mean ± S.D. of two replicates.

Table 4

The effects of growth factors on the growth of isolates cultivated at pH 7 an

Addition of growth factors PK15

DOD510 � initial OD510 % removal of NH

+0.1% glucose 0.47 ± 0.06c 9.3 ± 1.3c

+0.8% peptone 7.25 ± 0.19a 64.7 ± 1.6a

+0.3% yeast extract 2.56 ± 0.13b 29.9 ± 1.1b

+0.1% vitamin solution 0.27 ± 0.06c 3.2 ± 0.7c

a Means in the same column with different superscript are significantly diffb Mixture of PK5, PK8, PK11, PK15 and PK16 cultures.

Table 5

The effects of metal ions on the growth of PK15 and the removal of NHþ4 -N

Measurement Ca2+ Co2+ Cu2+ Fe2+

DOD510 � initial OD510 21.67 ± 0.44a 1.11 ± 0.11d 1.22 ± 0.11d 19.56

% removal of NHþ4 -N 87.9 ± 2.0a 4.8 ± 0.5d 5.3 ± 0.5d 81.2

a Means in the same row with different superscript are significantly differe


culture studies. Hence, in later experiments we used a

synthetic medium containing 0.8% peptone and 0.3%yeast extract additionally in the basal medium.

As shown in Table 5, 0.5-mM metal ions such as

Ca2+, Fe2+ and Mg2+ influenced the growth of PK15

and removal of NHþ4 -N as well. However, Co2+, Cu2+

and Zn2+ did not show their effects on either the growth

or the removal of ammonia, while Mn2+ and Mo2+ did

significantly only on the growth of PK15. These phe-

nomena can be explained in that the metal ions have

d 30 �Ca

Mixed cultureb

þ4 -N DOD510 � initial OD510 % removal of NHþ

4 -N

0.73 ± 0.09c 14.7 ± 1.3c

8.31 ± 0.31a 60.3 ± 2.0a

3.77 ± 0.15b 32.2 ± 1.7b

0.42 ± 0.08c 3.9 ± 0.6d

erent (P < 0.05). Values represent mean ± S.D. of two replicates.

a

Mg2+ Mn2+ Mo2+ Zn2+

± 0.44b 21.56 ± 0.33a 18.44 ± 0.23b 18.89 ± 0.33b 6.89 ± 0.22c

± 1.9a 85.5 ± 1.5a 28.5 ± 1.9c 40.1 ± 1.9b 9.2 ± 0.9d

nt (P < 0.05). Values represent mean ± S.D. of two replicates.

Culture time (h)0 2 4 6 8 10 12 14

OD

510

0.0

0.5

1.0

1.5

2.0

2.5

PK5PK8PK11PK15PK16

Fig. 3. Growth profiles of the five isolates obtained from each pure culture. Error bars: mean ± S.D. of two replicates.


been known to be involved in enzyme catalysis in a vari-

ety of ways and that trace metals Mo2+ and Fe2+ are

found in nitric-oxide reductase (Palmer, 1995).

To characterize the growth of each pure isolate, flask

cultures were incubated with the synthetic medium con-

taining 0.8% peptone and 0.3% yeast extract. Thegrowth profiles of five isolates were plotted in Fig. 3.

The data points represent average values for two repli-

cates. All isolates reached stationary phase in 8 h, and

the cultures of PK5 and PK16 exhibited longer lag times

than the cultures of PK8, PK11 and PK15. The values of

specific growth rates were calculated to be 0.51, 0.43,

0.47, 0.55 and 0.45 h�1 for PK5, PK8, PK11, PK15

and PK16, respectively. Among the three B. subtilis spe-cies that were found to be aerobes, the specific growth

rate of PK15 was highest. This fact might result in over-

growth of PK15 over the other two species, PK8 and

PK11 (described in the experiment in Section 3.2),

although cell interaction can be very complex in mixed

culture (Purtschert and Gujer, 1999).

3.4. Capacity of the isolates for aerobic denitrification

In Fig. 4, changes of various components in the flask

culture during the experiment of nitrogen removal under

an aerobic condition are shown. The main substrate for

this experiment was night soil because the isolates re-

quired high C/N ratio with a high concentration of or-

ganic carbon. The concentration of NHþ4 -N decreased

significantly in 1 h, and as did TN. The same trend couldbe seen in both the removal of COD and the decrease in

alkalinity. This indicates that active nitrification oc-

curred largely in 1 h with a decrease of COD. At this

time, nitrate began to accumulate by nitrification, and

remained as denitrification occurred simultaneously,

without any nitrite build-up. The similar result has been

seen in other studies using B. subtilis sp. (Sakai et al.,1996). It has been reported that during aerobic denitrifi-

cation by autotrophic Nitrosomonas europaea, dinitro-

gen was produced as an end product, whereby nitrous

oxide was observed as an intermediate (Shrestha et al.,

2002). Thus, the heterotrophic Bacillus strains possessed

less complex metabolic pathways for removal of ammo-

nia than did autotrophs. The initial DO was measured

to be 5.29 mg L�1, decreased to 1.93 mg L�1 in 1 hand then was reduced slowly to 0.44 mg L�1 (about

8% saturated) after 4 h. The amount of DO derived

from oxygen pressurized fully in the tightly sealed flask

did not match consumption of DO by Bacillus strains

due to the low solubility of oxygen (Nielsen and Villad-

sen, 1994). The concentrations of NHþ4 -N, TN, COD

and alkalinity did not decrease significantly after 2 h,

probably due to the decrease of DO in the flask. How-ever, the production of N2 increased steadily. This indi-

cated that a low partial oxygen pressure was favorable

for N2 production, and this result is in agreement with

that from study of autotrophic Nitrosomonas europaea

(Shrestha et al., 2002). From the results obtained in Sec-

tion 3.2, it can be inferred that PK15 was largely in-

volved in nitrification and PK5 and PK16 in

production of N2 during aerobic nitrification–denitrification.

Culture time (h)0 2 4

NH

4 -N

, NO

2 -N

, NO

3 -N

(mg/

l)

0

10

20

30

40

NH4+ -N

NO2- -N

NO3- -N


TN (m

g/l)

0

10

20

30

40

50

60

Org

-N, N

2, D

O (m

g/l)

0

2

4

6

8

10

12

14

16

18

TNOrg-NN2

DO


CO

D, A

lkal

inity

(mg/

l)

0

50

100

150

200

250

300

CODAlkalinity


TP (m

g/l)

0

2

4

6

8

Dry

cel

l wei

ght (

mg/

l)

0.40

0.45

0.50

0.55

0.60

TPDCW

(a) (b)

(c) (d)

4 514 51

1 3 5 4 51

Fig. 4. Changes of NHþ4 -N, NO

�2 -N and NO�

3 -N (a), TN, Org-N and N2 (b), COD and alkalinity (c), TP and DCW (d), in the flask culture of mixed

isolates. Error bars: mean ± S.D. of two replicates.


The organic nitrogen (Org-N) was almost depleted by

the isolates after 4 h. The removal efficiencies of

NHþ4 -N, TN, COD and TP for 4 h were found to be

72.0, 57.6, 89.5 and 21.4%, respectively. The low re-

moval efficiency of TP could result from low production

of cells (only 100 mg L�1 produced for 4 h) (Su et al.,2001a). The alkalinity destructed in the culture was

5.28 mg L�1 (as CaCO3) per each gram of NHþ4 -N oxi-

dized. In conventional active sludge, substantial quan-

tity of alkalinity is lost during nitrification, but the

alkalinity decreased in this study was only 74.6% of

the theoretical value (Grady et al., 1999). When the con-

centration of organic nitrogen decreased, cell produc-

tion was largely retained and substantial removal ofCOD and NHþ

4 -N did not continue. The ratio of milli-

gram of COD removed per milligram of NHþ4 -N re-

moved was calculated to be 7.46.

Table 6

Nitrogen balance for the flask culturea (units: mg L�1)

Initial TN Final NHþ4 -N Final NO�

2 -N Final NO�3 -N

50.0 ± 1.5 9.8 ± 0.1 0 ± 0 11.3 ± 0.1

a Values represent mean ± S.D. of triplicates.b Calculated value.c Biomass composition was assumed to be C5H7O2N.d % N lost = 100 · {(initial TN) � (final NHþ

4 -N) � (final NO�2 -N) � (fina

From the nitrogen balance (Table 6), 14.5 mg L�1

(33% of initial TN) of nitrogen in the medium disap-

peared after 4 h. The amount of N2 gas measured was

12.0 mg L�1 (24% of initial TN). Hence, 9% of initial

TN was missing during nitrogen removal by the isolate.

The difference might result from measurement errors,but the similar result could be found in the previous re-

port, showing that on an average 8% of influent nitrogen

did not appear in the effluent in nitrogen balance between

the influent and the effluent (Gonenc and Harremoes,

1985). From these results, the mixed Bacillus cells were

found to lead to aerobic denitrification. Heterotrophs

that have been reported to occur aerobic denitrification

includeAlcaligenes faecalis (vanNiel et al., 1992),Hypho-

microbium X (Meiberg et al., 1980), Paracoccus denitrifi-

cans (previously named Thiosphaera pantotropha)

(Robertson et al., 1988) and Pseudomonas stutzeri

Final Org-Nb N in Biomassc % N lostd

0.5 ± 0.2 12.4 ± 0.3 33.0

l NO�3 -N) � (final Org-N) � (N in biomass)}/(initial TN).


(Su et al., 2001b). In Bacillus strains, the possibility of

aerobic denitrification by B. licheniformis has been re-

ported (Brycki et al., 2000). However, microbial interac-

tion between mixed Bacillus strains and their

characteristics in removal of nitrogen have not been

reported.

4. Conclusions

To understand characteristics of Bacillus strains used

in a biological nitrification–denitrification process, 24

species were isolated from the sludge in a night soil

treatment system. Under different environmental condi-

tions, cell interactions took place among them and cell

population changed. Additions of 0.8% peptone and

0.3% yeast extract to a basal medium influenced thegrowth of isolates and the removal of NHþ

4 -N, and as

did metal ions such as Ca2+, Fe2+ and Mg2+. It was

found that PK15 (identified to be B. subtilis) was in-

volved largely in nitrification and PK5 (identified to be

Bacillus cereus) and PK16 (identified to be B. lichenifor-

mis) were involved in production of N2 during aerobic

nitrification–denitrification. A low partial oxygen pres-

sure was favorable for the N2 production. From thenitrogen balance, the percentage of nitrogen lost in a

nitrification–denitrification process was estimated to be

33.0%, which was presumed to have been converted to

N2 gas. The Bacillus strains were found to convert

ammonia to N2 without formation of nitrous oxide

under aerobic conditions. Thus, in removal of ammonia,

the heterotrophic Bacillus strains had less complex met-

abolic pathways than autotrophs. The ability to both ni-trify and denitrify makes Bacillus strains attractive

candidates for application in the removal of ammonia

nitrogen from wastewater. Thus, the B3 process using

Bacillus strains seemed to have some economic advanta-

ges, and the development of this aerobic denitrification

process may serve as an alternative to the costly nitrifi-

cation–denitrification processes.

Acknowledgements

This research has been supported by theKorea Science

Engineering Foundation (KOSEF) (Grant No. R12-

1996-009203-0) through the Institute for Environmental

Technology and Industry at Pusan National University.

Technical assistance of Mr. Ill-Ho Jung (Daekyong En-

Tech International Co., Seoul) is also acknowledged.

The students, Kyoung Joo Park and Kyoung SookCho, were supported by the Brain Korea 21 Project.

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