131NISR RESEARCH GRANT
2003 Young Investigator Research Grant

Characterization of Aerobic Denitrification Mechanism inKlebsiella pneumoniae Strain F-5-2 That Can Utilize Ammonium and Nitrate Ions Simultaneously and Application of Nitrifying and Denitrifying Processes to Waste Water Treatment
Shinji TAKENAKA
Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University
Research aims
As countermeasures against eutrophication in lakes, wetlands, rivers, and enclosed areas of a sea, the removal of nitrogen compounds (NH4 and NO3) from industrial and domestic waste waters has become an urgent and crucial subject. In the microbial removal of NH4 and NO3, twoprocesses have been thought to participate: oxidation ofNH4 to NO3 by nitrifying autotrophic bacteria under aerobic conditions and denitrification of nitrate by denitrifying
bacteria under anaerobic conditions. We isolated a heterotrophic bacterium, Klebsiella pneumoniae F-5-2, which
can utilize both NH4 and NO3 simultaneously. This strain
removed 4 mg/ml (1,400 ppm as N) of NH4NO3 completelyin 48 h of aerobic cultivation with controlled Fe2 andMoO42 concentrations. GC-MS analysis indicated that theN2 gases were derived from the nitrogen atoms of bothNH4 and NO3. For these reasons, we concluded that K.
pneumoniae F-5-2 has pathways for heterotrophic nitrification and aerobic denitrification. Our aim is to reveal themechanism of nitrification and denitrification in this strain
under aerobic condition and to investigate the effects of factors on the removal of NH4 and NO3 simultaneously forapplication.
Methods
1. Functional analysis of the genes involved in the nitrification and denitrification steps.
On the basis of conserved region of previous reported nitrate reductases, the primers were synthesized. The amplified DNA fragment was used as a colony hybridization
probe. The gene library of strain F-5-2 was constructed andscreened by colony hybridization using the labeled probe.
2. Effects of various factors on the removal of NH4 andNO3.
i) Growth condition: Strain F-5-2 was cultured in thebasal medium containing 0.1% (w/v) NH4NO3.
ii) Analytical tests: NH4, NO2, and NO3 were measured by a modification of the indophenol method, the diazo-coupling reaction, and nitration of salicylic acid, respectively.
iii) Effect of phosphate concentration on the removal ofNH4 and NO3: Media containing 0 to 128 mM sodiumpotassium phosphate were prepared. The cultures weretaken every 12 hours. Growth and concentrations of Ncompounds were determined.
iv) Effect of carbon sources on the removal of NH4and NO3: Test medium was a modified NH4NO3 medium
containing either sugar (8 kinds), organic acid (4 kinds),
amino acid (7 kinds), or nutrient broth (4 kinds). The cultures were taken every 12 hours. Growth and concentrationsof N-compounds were determined.
Results
1. Functional analysis of the genes involved in the nitrification and denitrification steps.
Sequence analysis of the PCR product showed high identities to those of the previous reported nitrate reductase
genes. In colony hybridization using the labeled PCR product, three positive clones (p68D7, p24A12, and p6E12)
were selected in the gene library of strain F-5-2. The insertsof these plasmids p68D7, p24A12, and p6E12 covered12 Kb and 6 ORFs were found in the determined sequence.
Each ORF was high identity to previous reported enzymes
involved in denitrification step in the deduced amino acidsequence (Table 1).
2. Effects of various factors on the removal of NH4 andNO3.
Strain F-5-2 could grow well and remove NH4 and NO3completely in 60 h of aerobic cultivation in a medium containing more than 64 mM of sodium potassium phosphate.
Among the tested carbon sources, sucrose, lactose, D-mannose, maltose, D-fructose, and D-glucose promoted thegrowth and the removal of NH4 and NO3 (Table 2). The
removal of NH4 and NO3 was inhibited in the medium
containing L-arabinose, D-galactose, or sodium acetate. Larabinose, D-galactose, or sodium acetate was added to themedium containing effective sugar (sucrose, lactose, D-132
Table 1.

Comparison of primary amino acid sequences deduced from ORF 1 to 6.
Homology

ORF

12
3
4
5
6
a

Predicted function

Amino acid identity(%)

ProteinSourceAccession
No.

66
89
79
9380
—aNasC
NasB
NasA
NarL
NarX
NarK

Klebsiella oxytocaKlebsiella oxytoca
Klebsiella oxytoca
Salmonella typhimurium
Salmonella typhimurium
Salmonella typhimurium

L27431
L06800
L06800AE008778
AE008778
AE008778

Nitrate reductase (small subunit)
Nitrite reductase
Nitrate reductase (large subunit)
Nitrate/Nitrite response regulator
Nitrate/Nitrite sensor protein
Nitrite extrusion protein

partial sequence

Table 2. Effects of carbon sources on the removal of NO3 and NH4from 0.2% (w/v) (25 mM) NHNO3 medium under aerobic conditions.
Carbonsource
D-Glucose

Lactose
L-ArabinoseD-Mannose
Maltose
D-Galactose
D-Fructose
Sodium acetate
DL-Malic acid
Disodiumsuccinate

Remaining (mM, after 60 h cultivation)

Growth(OD660)

NO3

NO2

NH4

00
15.5
0
0.4
16.8
0
18
7.5

00
1.5
0
0
1.0
0
3.0
1.4

00
0.65
00
4.25
0
23
0

13
11
9.1
1313
8.7
110.2
3.1

20

1.5

3.0

4.3

mannose, maltose, D-fructose, or D-glucose). Strain F-5-2
was cultivated in these media. The removal of NH4 andNO3 was inhibited in the medium containing sodium acetate.
Conclusion
A 12 Kb-region, which contained several genes involvedin the nitrification and denitrification steps, was cloned

from K. pneumoniae F-5-2. The region contained six ORFs.
Some of the deduced amino acid sequences of these ORFs
showed high identities with those of putative nitrate reductase or nitrite reductase. Further genetic studies are needed
to clarify the denitrification and nitrification in strain F-5-2.
Effects of various factors on the removal of NH4 andNO3 simultaneously were examined. With the growth ofstrain F-5-2, pH of the medium was changed to weak acidic
condition. This condition affected the removal of NH4 andNO3 and further growth. Among the tested carbon sources,
the sugars which promoted the growth and the removal ofNH4 and NO3 are generally metabolized to intermediatesof glycolytic route or TCA cycle via fructose 6-phospahte.
Disaccharides, which contained D-glucose as a constituent
promoted the removal of both N-compounds. Strain F-5-2
is a heterotrophic bacterium; the important thing to lead tomore effective denitrification and nitrification is not only tocontrol concentration of metal ions (Fe2 and MoO42) but
also to select these sugars.
Reference
Kim, Y-J., Yoshizawa, M., Takenaka, S., Murakami, S., and Aoki, K., Isolation and culture conditions of a Klevsiella pneumoniae strain thatcan utilize ammonium and nitrate ions simultaneously with controlled iron and molybdate ion concentrations. Biosci. Biotechnol.
Biochem., 66, 996–1001 (2002).