ORIGINAL RESEARCH PAPER
Isolation and characterization of thermotolerant bacterium utilizing ammonium and nitrate ions under aerobic conditions Shinji Takenaka Æ Qi Zhou Æ Ampin Kuntiya Æ Phisit Seesuriyachan Æ Shuichiro Murakami Æ Kenji Aoki
Abstract A thermotolerant bacterium, identified as Bacillus licheniformis, completely utilized 0.1% (w/v) NH4NO3 at 30 and 50C under aerobic condition. The addition of 0.5 mM Fe2+ to the NH4NO3 medium markedly promoted the utilization of NH+4 and NO–3. At 50C, of total nitrogen originally provided, 24% was taken up into the cells and 20% remained in the culture supernatant. Residual nitrogen (56%) was probably removed into the atmosphere. The cell extracts contained enzymes involved in denitrification. GC-MS demonstrated that NH415NO3 had been converted to 15N2O. These results indicate that the strain has denitrification ability under aerobic condition.
S. Takenaka S. Murakami K. Aoki (&) Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Rokko, Kobe 657-8501, Japan e-mail: [email protected]
Q. Zhou Department of Biosystems Science, Division of Biosystems Chemistry Graduate School of Science and Technology, Kobe University, Rokko, Kobe 657-8501, Japan A. Kuntiya P. Seesuriyachan Department of Biotechnology, Faculty of Agroindustry, Chiang Mai University, Chiang Mai 50100, Thailand
Keywords Aerobic condition Ammonium nitrate Bacillus licheniformis Denitrification Ferrous ion Thermotolerant bacterium
Introduction As counter-measures against eutrophication in lakes, wetlands, and enclosed sea areas, the removal of nitrogen-containing compounds from industrial and domestic wastewaters has become urgent. Legally enforceable environmental quality standards for water pollutants have been established to maintain the quality of environmental waters and to prevent eutrophication under the fundamental environment law (Ministry of the Environment, Japan 2005). Two processes are involved in the microbial removal of NH+4 and NO–3: NH+4 aerobic oxidation to NO–3 by ammonia-oxidizing bacteria and anaerobic denitrification of NO–3 by denitrifying bacteria (Bock et al. 1992; Robertson and Kuenen 1984; Zumft 1997). For this, two batch processes under aerobic and anaerobic conditions have to be prepared. Alternatively, anaerobic ammonium oxidation (Anammox) process can be used in nitrogen elimination (Jetten et al. 2003). However, heterotrophic bacteria that can simultaneously perform heterotrophic nitrification and aerobic denitrification would be useful for removing NH+4 and NO–3 aerobically in a wastewater
treatment system (Frette et al. 1997; Joo et al. 2005; Lukow and Diekmann 1997; Wehrfritz et al. 1993). Such heterotrophic bacteria could be utilized to reduce the cost of maintaining an anoxic tank or decreasing its size. We have isolated heterotrophic microorganisms that utilize both NH+4 and NO–3 as nitrogen sources. Although the isolate, Klebsiella pneumoniae strain F-5-2, can carry out both functions (Kim et al. 2002), it cannot grow above 40C. Other bacteria that can grow well above 40C and remove NH+4 and NO–3 simultaneously are thus required. Here, we report the isolation and identification of a soil bacterium that can utilize NH4NO3 at 30 and 50C. The features of the removal of nitrogen compounds and denitrification by the isolate are described.
Vials, 10 ml, capped with a butyl rubber stopper and an aluminum seal, test tubes, and 500 ml flasks were used. Preculture was performed for 36 h using a test-tube; 1 ml preculture was inoculated into 100 ml fresh NH4NO3 medium. Morphological and phenotypic characterization Strain T-7-2 was identified on the basis of morphological and biochemical characteristics using methods described previously (Komagata 1985) and by analysis of the 16S rRNA gene. The nucleotide sequence of the 16S rRNA gene of strain T-7-2 (1501 bp, accession no. AB275356) was 99.8 and 99.6% identical to those of Bacillus licheniformis strain KL-185 (AY030337) and Bacillus licheniformis strain ATCC 14580 (CP000002), respectively.
Materials and methods Analytical methods Isolation of microorganism that can utilize NH+4 and NO–3 The screening medium (NH4NO3 medium) containing 3% (w/v) D-glucose, 0.1% (w/v) NH4NO3 (12.5 mM), 0.1 mM FeSO47H2O, and 8 pM Na2MoO42H2 O was prepared as reported previously (Kim et al. 2002). Soil samples, 1 g, from rice fields and farms in Hyogo and Osaka, Japan and in Chiang Mai, Thailand were suspended in 7 ml 0.8% (w/v) NaCl and 1 ml was transferred to NH4NO3 medium. Incubation was performed in a tube capped with a butyl rubber stopper at 50C without shaking. From a culture that showed a loss of NH+4 and NO–3 without NO–2 accumulation, one drop was taken and spread on an NH4NO3 medium plate. The plate was incubated at 50C; organisms that grew well were selected and transferred to the medium.
The NH+4 , NO–3, and NO–2 concentrations in the culture were measured by the Indophenol Blue method (Weatherburn 1967), a micro-salicylate method (Bhandari and Simlot 1986) and a diazo-coupling reaction method (Aoki et al. 1981), respectively. Total nitrogen in the culture supernatant and the cells was determined by Bu¨chi Kjeldahl units according to the instructions provided by the manufacturer (Bu¨chi Labortechnik AG, Zu¨rich, Switzerland). N2O gas was measured using a gas chromatograph (column: Porapak Q 50/80 mesh, 3 mm · 2 m) equipped with a photon ion detector. 15N2O and 15N2 were measured by GC-MS (Kim et al. 2002). Nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase were assayed according to published procedures (Aoki et al. 1981; Kakutani et al. 1981; Heiss et al. 1989; Kristjansson and Hollocher 1980).
Cultural condition Bacillus licheniformis strain T-7-2 was cultivated on 0.1% (w/v) NH4NO3 medium containing 0.5 mM FeSO47H2O under aerobic condition with shaking at 140 rpm, in order to examine the effects of various factors on the removal of NH+4 and NO–3 and to reveal denitrification steps.
Results and discussion Isolation and identification of strain T-7-2 Of eight isolated NH4NO3-removing bacteria, strain T-7-2 grew well on 0.1% (w/v) NH4NO3
medium and could utilize NH+4 and NO–3 at 30 and 50C (Table 1). The maximum NO–2 concentration was 3 mM after 1 day of cultivation; NO–2 disappeared completely with the growth of strain T-7-2. The isolate could remove nitrogen compounds at 30 and 50C. This indicates that it is a thermotolerant bacterium that can utilize and remove NH+4 and NO–3. Effects of various factors on growth of strain T-7-2 and NH4NO3 utilization by strain T-7-2 K. pneumoniae strain F-5-2 grew well on 0.4% (w/v) NH4NO3 medium containing 0.1 mM Fe2+ + and 8 pM MoO2– 4 and could remove NH4 and – NO3 aerobically (Kim et al. 2002). The effects of metal ions on the cell growth of strain T-7-2 and utilization of NH4NO3 were examined by culturing strain T-7-2 on 0.2% (w/v) NH4NO3 medium containing 0.1 mM metal ions (i.e., Mn2+, Zn2+, Co2+, Cu2+, Fe2+, and Ni2+). The strain could grow well on the medium with Fe2+ and effectively remove NH+4 and NO–3 at 30 and 50C. Although the growth of strain T-7-2 reached on OD660 values of 4.6 (at 30C) and 3.5 (at 50C) in the test medium containing Mn2+, NO–2 accumulated. Mn2+ enhances glutamine synthetase and glutamyl transferase activities in crude extract from Bacillus licheniformis (Leonard et al. 1962). In
3.6 9.7 8.0 7.8 9.5 6.0 4.9 10
Each isolated strain was incubated in the screening medium containing 0.1% (w/v) NH4NO3 at 30 and 50C with shaking at 140 rpm. After 96 h of cultivation, cell growth [mg (wet wt.)/ml] and remaining NH+4 and NO–3 were measured
G rowt h (O D6 6 0 )
66 62 16 58 80 77 54 45
3.2 2.4 4.0 2.4 4.8 2.4 2.4 4.0
3.6 8.1 9.2 7.9 9.5 6.0 4.9 10
53 53 6.4 48 53 47 20 38
2.4 2.4 0.8 1.6 1.6 2.4 0.8 1.6
by isolated strains
% Remaining Growth % Remaining Growth (mg/ml) (mg/ml) NO–3 NH+4 NO–3 NH+4 S-2-1 T-1-1 T-7-2 T-12-1 T-18-1 C-6-1 F-2-3 F-17-2
Figure 2a–d show utilization of NH4NO3 and total nitrogen in the culture. NH+4 and NO–3 were removed completely without NO–2 accumulation (Fig. 2a, c). Twenty-five percent of the total nitrogen originally provided was observed in the cells and 44% of that was in the cultural supernatant at 30C. Residual nitrogen (31% of total nitrogen) was removed from the culture. Twenty percent of the total nitrogen originally provided
Table 1 Utilization of under aerobic condition
Analysis of total nitrogen concentration in cultural supernatant and cells
Residual + NO3 -and NH4 ( % )
strain T-7-2, the supplementation of Mn2+ promoted effective ammonium assimilation and resulted in good growth. Zn2+, Co2+, Cu2+, and Ni2+ markedly inhibited the growth of strain T-72. Among the tested metal ions, Fe2+ was the most effective for strain T-7-2 to utilize nitrogen compounds at 30 and 50C (Fig. 1). Growth of Bacillus licheniformis strain 40-2 at 37C was enhanced in a medium containing Fe2+ under aerobic conditions (Konohana et al. 2000). Strain F-5-2 could remove aerobically 1.29% (w/v) NaNO3 at maximum (Kim et al. 2002). Although strain T-7-2 could hardly remove NO–3 at the same speed, a decrease of shaking speed (to 100 rpm) or addition of 0.01% (w/v) NH4Cl to 0.1% (w/v) NaNO3 medium brought complete removal of NO–3 at 30 and 50C by strain T-7-2.
Fig. 1 Effect of Fe2+ concentration on NH4NO3 utilization by B. licheniformis strain T-7-2 at 50C. The bacterium was incubated in a test tube under aerobic condition (shaking at 140 rpm) in 0.2% (w/v) [25 mM] NH4NO3 medium (7 ml/tube) containing 0.01, 0.1, 0.5, or 1.0 mM Fe2+. As a control, the strain was cultivated without Fe2+. After 72 h of cultivation, residual nitrate (white bar) and ammonium (black bar) concentration and the OD660 of the culture (hatched bar) were measured
G r owt h (O D660)
C on cen t r a t ion of N- com p ou n d s (m M ) N (m m ol / 10 m l)
0.2 0.1 0
48 Time (h)
48 Time (h)
Fig. 2 Utilization of NH4NO3 by B. licheniformis strain T-7-2 at 30C (A) and 50C (C) and total nitrogen concentration in the culture at 30C (B) and 50C (D). (A and C) The bacterium was incubated in a 500 ml flask under aerobic condition in 0.1% (w/v) [12.5 mM] NH4NO3 medium (70 ml/flask) containing 0.5 mM Fe2+ at 30C and
50C. Residual nitrate (h) and ammonia (m) concentrations, amount of accumulated nitrite (e) and the growth (s) were measured. Total nitrogen concentrations in the cells (solid bar) and cultural supernatant (open bar) were converted to nitrogen per 10 ml of culture
was observed in the cells and 24% of that was in the cultural supernatant at 50C. Residual nitrogen (56% of total nitrogen) would probably be removed into the atmosphere.
from NH415NO3 (Fig. 3). After NO–2 accumulation of nitrite in the culture, 15N2O gas also accumulated in each vial. Crude extracts of strain T-7-2 showed a high nitrite reductase activity, when NO–2 or NO–3 was present in the medium. Cells of strain T-7-2 were harvested whilst NO–2 and NO–3 still remained; the activities of respiratory nitrate reductase, nitrite reductase, and NO reductase in the crude extracts prepared from the
Analysis of enzymes involved in denitrification Strain T-7-2 was cultivated using a 10 ml vial to identify and estimate the amount of the gases
N2O ga s ( m icr o mole / via l )
G r o w t h ( O D660 )
C o nce nt rat ion of N - C ompo unds (m M)
Time ( h ) Fig. 3 Utilization of NH4NO3 by B. licheniformis strain T-7-2 and gas analysis from the culture. The bacterium was incubated in a 10 ml vial with shaking at 140 rpm in 0.1% (w/v) [12.5 mM] NH415NO3 medium (1 ml/vial) containing 0.5 mM Fe2+ at 50C. Residual nitrate (h) and ammonia
(m) concentrations, amount of accumulated nitrite (e) and the growth (s) were measured. Amount of 15N2O gas (hatched bar) was measured and converted to lmol per vial
cells were determined. The specific activities of these enzymes were 6.5, 6.7, and 2.5 nmol of product min–1 mg protein–1, respectively. These results indicate that strain T-7-2 synthesizes enzymes involved in denitrification (NO–3 ﬁ NO–2 ﬁ NO ﬁ N2O) under aerobic condition. Potential application for removal of nitrogen compounds by thermotolerant bacteria Pichinoty et al. (1978) reported gas production by 15 strains of Bacillus licheniformis from peptone medium containing nitrate. Although some of the strains showed denitrification, gas production was irregular and quite slow. Strain T-7-2 produced 15N2O from the culture; however, the amount of released 15N2O gas always decreased during shaking (Fig. 3). N2O returned into the medium was probably converted to N2 by strain T-7-2. To our knowledge, denitrification by B. licheniformis under aerobic condition has not been previously observed. Although strain F-5-2 is a manageable bacterium for removal of NH4NO3, K. pneumoniae is a notorious pathogen. B. licheniformis is generally considered non pathogenic to humans (de Boer et al. 1994). Strain T-7-2 utilizes NH4NO3 above at 40C. These indicate that thermotolerant strain T-7-2 has potential for application in wastewater treatment. Acknowledgements Part of this work was carried out through collaboration in a Core University and supported by the Japan Society for the Promotion of Science and the National Research Council of Thailand.
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