- Email: [email protected]
A simple screening procedure for heterotrophic nitrifying bacteria with oxygen-tolerant denitrification activity
JOURNALOF BIOSCIENCE AND BIOENGIKEERING Vol.
95, No. 4,409-41 I. 2003
A Simple Screening Procedure for Heterotrophic NitrifLing Bacteria with Oxygen-Tolerant Denitrification Activity EMIKO
MATSUZAKA,’ NOBUHIKO NOFUHISA OKADA,’
NOMURA,‘*
TOSHIAKI NAKAJIMA-KAMBE,’ NAKAHARA’
AND TADAATSU
Institute qfApplied Biochemistry. University oj’Tsukuba. Ibaraki 3058582, Japan’ Received 28 August 2002/Accepted 19 November 2002
Various naturally occurring strains of heterotrophic nitrifying bacteria were isolated by enrichment culture using acetamide as the C and N source, and 21 strains were identified as heterotrophic nitrifiers. Using a new simple procedure, these 21 strains were also investigated for the ability to carry out denitrification in the presence of oxygen. Several of the nitrifying strains were found to exhibit a distinct activity that allows for denitrification via nitrite (NO,-) in the presence of oxygen, indicating that they have an oxygen-tolerant denitrification system. A wide variety of bacteria possessing both nitrifkation and denitrification capabilities in the presence of oxygen were isolated and partially characterized by using the simple screening combinatorial procedure described in this paper. [Key words: nitrification, heterotrophic
nitrifying bacteria, denitrification]
Heterotrophic nitrification and aerobic denitrilication have recently received increased attention because of their contribution to the nitrogen cycle in the environment and the possibility of their application for wastewater treatment. Several studies of heterotrophic nitrifiers have been reported (l-4). Thiosphaera pantotropha is reportedly capable of simultaneous heterotrophic nit&cation and aerobic denitrification (5, 6). It has also been shown that Alcaligenes faecalis has the ability to nitrify and denitrify under aerobic conditions (7, 8). Many heterotrophic nitrifying bacteria are also aerobic denitrifiers, and have been isolated from soil and wastewater treatment systems. These organisms may oxidize ammonia via NO,- to NO,-, and also reduce NO,ultimately to dinitrogen (N,) gas. From reported results, it can be concluded that while there are many similarities between bacteria with the ability to simultaneously nitrify and denitrify, there are also many differences. It is not yet possible to generalize due to the small number of species tested, but the outlines of a pattern are emerging, indicating that a wide variety of bacteria possessing both nitritication and aerobic denitrification capabilities must be analyzed. In this work, we isolated various heterotrophic nitrifying bacteria and denitrifying bacteria in the presence of oxygen by combining simple screening procedures as described in this paper. Verstraete and Alexander (VA) medium (l), used as the screening medium in this study, was composed of the following (per liter): 2.0 g of acetamide, 8.2 g of KH,PO,, 1.6 g of NaOH, 0.5 g of MgSO,.7H,O, 0.5 g of KCl, and 0.0005 g each of CaSO,.2H,O, CuSO,.5H,O, and FeC1,.6H,O, and 0.0005 g of ZnS0,.H20. The final pH was adjusted to 7.0
with NaOH. Soil samples collected from various locations in Tsukuba City, Japan, were used to screen for heterotrophic nitrifying bacteria. Soil was added to test tubes (28 mm diameter) containing 5 ml of VA medium, then tubes were incubated at 30°C with shaking (220 rpm). Subculturing (5 times) was performed weekly by adding 0.2 ml of culture broth to fresh VA medium. Samples were diluted lOO-fold in saline solution (0.8% NaCl) before spreading on VA medium solidified with 1.5% agar. The plates were incubated at 30°C until colonies appeared. Separate colonies were picked and individually tested for nitrifying activity by growing the cultures for 3 d. The isolated strains were tested for the ability to carry out aerobic denitrification using Giltay and nitrite (GN) medium modified by replacing nitrate with nitrite Giltay medium (9). GN medium contained the following components per liter: 0.06 g of NaNO,, 1.0 g of L-asparagine.H>O, 5.0 ml of 1% alcoholic solution of brom thymol blue (BTB), 8.5 g of Na citrate, 1.O g of KH,PO,, 1.O g of MgSO,. 7H20, 0.2 g of CaCl,.6H,O, and 0.05 g of FeCl,.6H,O. The final pH was adjusted to 7.0. Each strain was inoculated into a test tube containing 5 ml of GN medium and an inverted Durham tube. Cultures were incubated for 3 d at 30°C with shaking (220 rpm) to ensure adequate oxygenation. The ability to carry out denitrification in the presence of oxygen was indicated by a change in color of the GN medium from green to blue and the formation of a bubble in the Durham tube. An analysis of the denitrification activity was carried out using Na”N0,. We used the nitrite nutrient (NN) medium of Sakai et al. (10) but with the pH increased to 7.8 NN medium contained the following components per liter: 0.06 g of NaNO?, 10 g of glucose, 10 g of peptone, 0.1 g of MgSO,. 7H,O, 2.0 g of K,HPO,, and 1.O g of yeast extract. Sterile
* Corresponding author. e-mail: [email protected] phone: +81-(0)29-853-7486 fax: +81-(0)29-853-4605 409
-110
MATSlJ%Ah;A
ET- Al..
stock solutions of NaNO, and glucose were added aseptically to the medium after the solution was autoclaved separately. When necessary, a solution with a 9:1 ratio of Na14N0, and Na15N0, was added to the NN medium. For these studies, each str& was inoculated into a Hungate tube containing 2 ml of NN medium and tubes were incubated for 3 d at 30°C with shaking (220 rpm) to eusure adequate oxygenation. Na15N0, (99 atom%) was obtained from Shoko-Tsusho (Tokyo): After incubation, an aliquot of gas in the upper space was collected and analyzed for oxygen and dinitrogen concentrations by gas chromatography (GC) and gas chromatography-mass spectrometry (CC-MS). respectively. A thermal conductivity detector (TCD) mounted on a HP-6890 gas chromatograph (Hewlett Packard, Palo Alto, USA) with a molecular sieve (5A, 3 mmx2 m; GL Science, Tokyo) column was used for analysis of O?. An electron capture detector (ECD) mounted on a GC-14B gas chromatograph (Shimadzu, Kyoto) with an OV- 17lm column was used for analysis of N,O. Dinitrogen was analyzed by isotope mass spectrometry using a Finnigan DELTA plus isotope mass spectrometer (Finnigan Delta, Bremen, Germany). Twenty-seven soil samples were examined for the presence of microorganisms exhibiting nitrification activity as measured by acetamide uptake and NO,- and NO,- production. NO? and NO,- were quantified calorimetrically as described by Donald et al. (11). Twenty-one bacterial strains were able to convert acetamide to NO?- and NO,- (Table 1). Furthermore, to investigate whether the heterotrophic nitrifiers possessed denitrification activity, tests were performed using Durham tubes in GN medium in the presence of oxygen. The color change of the GN medium from green to blue and the formation of bubbles in the Durham tubes were observed for six strains, NH-2, NH-7, NH-l 1, NH-14, NH15, and NH- 17 (Table 1). It was suggested that these heteroTABLE
1.
Nitrification
and denitrification
activities Color change and bubble formation
Production” of Strain NO,
NO;
NH-I
++
++
_
NH-2
+
++
NH-3
++
+
+ _
NH-4
+
+
_
NH-5
+
+
-
NH-6
+
++
_
NH-7
++
+
NH-8
+++
+
+ _
NH-9
+++
+
_
+ +
+ +
+
+
NH-13
+
+
NH-14 NH-15
+ + +
+
+
+ +
+ _
++
++ + + + +
+ _
NH-IO N!I-1 I NH-12
Nf1-I6 NH-l 7 NH-18 NH-19 NH-20 NH-21 “ +,-l-30
+ t + + pM; ++, 31-60
pM; +++. 61~100
+ _
_ _ PM.
TABLE
2.
Dinitrogcn evolution and Q concentration culturesofN~I-14.NH-lj,andNfI-17
Strain
“NL (pmol)
in gro\sing
(4 (mm)
NH-14
0.1 5
2.86
NH-15
0.73
I .?7
NH-17
0 (I .72)8
I .70
a NzO amount (pmoi).
trophic nitrifying strains possessed denitrification ability in the presence of oxygen. We focused on NO,~ conversion because it is an important intermediate not only of nitrification but also of denitrification. To confirm and further characterize the oxygen-tolerant denitrification activity observed in 2 1 strains, a pairedincubation technique was employed where the NO,- pool was labeled with 15N in the presence of oxygen. The results were used in 15N pool dilution calculations. The 21 strains were examined for the ability to generate 2WZ gas from a stable isotope of NO,- in NN medium. The results showed that the ability to carry out denitrification was present in 4 of the 2 1 strains, NH-7, NH- 11, NH- 14, and NH- 15. Only two strains, NH-14 and NH-l 5, generated N2 in the presence of oxygen (Table 2). The other two strains, NH-7 and NH- 11, generated N? in anaerobic conditions (0, concentration under 1 ppm). In addition, NH-17 was found to exhibit a distinct activity that allows the conversion of NO,- to N,O in the presence of oxygen (Table 2). It should be noted that the three strains NH- 14, NH-15, and NH-l 7 were among the six strains which demonstrated bubble formation and a change in color of the GN medium (Table 2). Thus, by this procedure, organisms capable of denitrification to N,O or N, in the presence of oxygen could be identified (Table 1). These results clearly showed that the ability to carry out oxygen-tolerant denitritication was present in three of the strains (NH- 14, NH-l 5, and NH-17), indicating that the simple procedure for enrichment of oxygen-tolerant denitrification was useful. For genetic analysis, the genomic DNAs of NH-14, NH15 and NH- 17 were extracted using the QIAGEN Blood Cell & Culture DNA kit (Qiagen, Hilden, Germany). Bacterial universal primer sets 27F (5’-AGAGTTTGATCCTGG CTCAG-3’) and 1492R (5’-TGACTGACTGAGGYTACCT TGTTACGACTT-3’) were used for amplification of bacterial 16s rDNA (12). Reaction conditions using the 27F and 1492R primers were as follows: after initial denaturation at 94°C for 2 min, 25 temperature cycles were performed at 94’C for 0.25 min, 50°C for 0.5 min, and 68°C for 1.5 min. Amplified 16s rDNA fragments were used as sequencing templates. DNA sequences of the PCR products were determined using the BigDye terminator cycle sequence kit (Applied Biosystems, Foster City, CA, USA). The products of the sequencing reaction were analyzed using an automated DNA sequencer (Applied Biosystems). Databases in the DNA Data Bank of Japan (DDBJ) were searched for sequences similar to the 16s rDNA sequence. A distance matrix tree was constructed by using the neighbor-joining method (13) contained in the Clustal X program (14). Matrix distances were calculated using the method of Kimura (15). The confidence level of tree topologies was evaluated by applying 100 bootstrap resamplings.
NOTES
VOL. 95,2003
* Ochrobucfnun
NH-15
(OCR242581)
grignonenre
Rhizobium
Mesorhi:obium
LNH-l4
(OAN
(AFO41444)
amorphae
(AF041442)
(ABO8.8414) Pamcoccus denitrijicons Ocherbhia
(AFW4720) (AF06321Y)
stutzeri
(AFO94736)
P.wudomonasputido
Burkholderia
vremamiens#s
Burkholderia
ceposia
Rurkhnlderro
Nurkholderro
bw
(PLI 16SRRNA)
(GBlCCC01)
coli
oeruginora
Pscudomonor
1
IESRRNA)
(AF008126)
geNicum
ipMmhbhsp
Pxudomonas
nology Corporation
(JST).
malla
(AFM3302)
(AB0514OX) (AFI 10187)
mulrivornnr (AFW7531)
INH_nABO8682?) Alcalrgenex
Kingella
Clostridium
REFERENCES
(RSITXSM)
genap
Rhizobium
(
of this work was supported by CREST (Core Research for Evolutionary Science and Technology) of the Japan Science and Tech-
(ABOSS415)
Ochrobactrum anfhropi
i”
411
demlrjicam
butvricum
jaecolis
(AFl55147)
(KINRRIMB) (AB0757h8)
FIG. 1. Unrooted distance matrix tree based on the 16s rDNA sequences of strains NH-14, NH-l 5 and NH-17 and other closely related bacteria obtained from the DDBJ. The scale bar represents two nucleotide substitutions per 100 positions.
Figure 1 shows the positions of NH-14, NH-15, and NH-17 in relation to other closely related bacteria. NH- 14 had 98% homology with Mesorhizobium amorphae and NH-15 had 98% homology with Ochrohactrum grignonense. NH- 17 and Alcaligenes faaecalis as a known heterotrophic nitrifying bacterium were shown to have a close relationship (97%). By analysis of the physiological properties, it appears that NH- 14 and NH- 15 qualify as new species (data not shown). These results showed that a wide range of bacteria that possess both nitrification and oxygen-tolerant denitrification activities were obtained by enrichment culture using this new simple procedure. Further investigations are in progress to identify the genes required for denitrification and nitrification as well as the regulatory mechanisms controlling these activities in NH- 14, NH- 15, and NH- 17. We thank Dr. Hirohumi Shoun and Dr. Naoki Takaya of University of Tsukuba for performing isotope mass spectrometry. A part
1. Verstraete, W. and Alexander, M.: Heterotrophic nitrification by Arthrobacter sp. J. Bacterial., 110,955-961 (1972). 2. Daum, H., Zimmer, W., Papen, H., Kloos, K., Nawrath,
K., and Bothe, H.: Physiological and molecular biological characterization of ammonia oxidation of the heterotrophic nitrifier Pseudomonas putida. CUT. Microbial., 37, 281-288 (1998). 3. Gunner, I-X.B.: Nitrification
by Arthrobacter globiformis. Nature, 197, 1127-1128 (1963). 4. Focht, D. D. and Verstraete, W.: Biochemical ecology of nitrification and denitrification. Adv. Microbial. Ecol., 1, 135214 (1977). 5. Robertson, L.A., Dalsgaard, T., Reusbecb, N. P., and Kuenen, J. G.: Comfirmation of aerobic denitrification in batch cultures, using gas chromatography and “N mass spectrometry. FEMS Microbial. Ecol., 18, 113-120 (1995). 6. Arts, P. A. M., Robertson, L. A., and Kuenen, J. G.: Nitrification and denitrification by Thiosphaera pantotropha in aerobic chemostat culture. FEMS Microbial. Ecol., 18, 305316 (1995). 7. Papen, H., Berg, R., von Hinkel, I., Thoene, B., and Rennenberg, H.: Heterotrophic nitrification by Alcaligenes faecalis. NO,-, NO,-, N,O, and NO production in exponentially growing cultures. Appl. Environ. Microbial., 55, 20682072 (1989). N. G., Robertson, L. A., Jetten, 8. Otte, S., Grobben, M. S. M., and Kuenen, J. G.: Nitrous oxide production by Alcaligenes faecalis under transient and dynamic aerobic and anaerobic conditions. Appl. Environ. Microbial., 62, 242 I2426 (1996). bacteria, p. 1484-1486. In 9. Alexander, M.: Denitrifying Black, C. A. (ed.), Methods of soil analysis, part 2. American Society of Agronomy, Wisconsin (1965). 10. Sakai, K., Ikebata, Y., Ikenaga, Y., Wakayama, M., and Moriguchi, M.: Nitrite oxidation by heterotrophic bacteria under various nutritional and aerobic conditions. J. Ferment. Bioeng., 82,613417 (1996). II. Donald, D. J. and Nicholas, A. N.: Determination of nitrate and nitrite, p. 981-984. In Colowick, S. P. and Kaplan, N. 0. (ed.), Methods in enzymology, vol. 3. Academic Press, New York (1957). 12. Amann, R. I., Ludwig, W., and Schleifer, K. H.: Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbial. Rev., 59, 143-169
(1995). 13. Saito, N. and Nei, M.: The neighbor-joining method and new method for constructing phylogenetic trees. Mol. Biol. Evol., 4,406-425 (1987). 14. Thompson, J.D., Higgins, D. G., and Gibson, T. G.: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific penalties and weight matrix choice. Nucleic Acids Res., 22,4673-4680 (1994). IS. Kimura, M.: A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol., 16, 11l-120 (1980).
95, No. 4,409-41 I. 2003
A Simple Screening Procedure for Heterotrophic NitrifLing Bacteria with Oxygen-Tolerant Denitrification Activity EMIKO
MATSUZAKA,’ NOBUHIKO NOFUHISA OKADA,’
NOMURA,‘*
TOSHIAKI NAKAJIMA-KAMBE,’ NAKAHARA’
AND TADAATSU
Institute qfApplied Biochemistry. University oj’Tsukuba. Ibaraki 3058582, Japan’ Received 28 August 2002/Accepted 19 November 2002
Various naturally occurring strains of heterotrophic nitrifying bacteria were isolated by enrichment culture using acetamide as the C and N source, and 21 strains were identified as heterotrophic nitrifiers. Using a new simple procedure, these 21 strains were also investigated for the ability to carry out denitrification in the presence of oxygen. Several of the nitrifying strains were found to exhibit a distinct activity that allows for denitrification via nitrite (NO,-) in the presence of oxygen, indicating that they have an oxygen-tolerant denitrification system. A wide variety of bacteria possessing both nitrifkation and denitrification capabilities in the presence of oxygen were isolated and partially characterized by using the simple screening combinatorial procedure described in this paper. [Key words: nitrification, heterotrophic
nitrifying bacteria, denitrification]
Heterotrophic nitrification and aerobic denitrilication have recently received increased attention because of their contribution to the nitrogen cycle in the environment and the possibility of their application for wastewater treatment. Several studies of heterotrophic nitrifiers have been reported (l-4). Thiosphaera pantotropha is reportedly capable of simultaneous heterotrophic nit&cation and aerobic denitrification (5, 6). It has also been shown that Alcaligenes faecalis has the ability to nitrify and denitrify under aerobic conditions (7, 8). Many heterotrophic nitrifying bacteria are also aerobic denitrifiers, and have been isolated from soil and wastewater treatment systems. These organisms may oxidize ammonia via NO,- to NO,-, and also reduce NO,ultimately to dinitrogen (N,) gas. From reported results, it can be concluded that while there are many similarities between bacteria with the ability to simultaneously nitrify and denitrify, there are also many differences. It is not yet possible to generalize due to the small number of species tested, but the outlines of a pattern are emerging, indicating that a wide variety of bacteria possessing both nitritication and aerobic denitrification capabilities must be analyzed. In this work, we isolated various heterotrophic nitrifying bacteria and denitrifying bacteria in the presence of oxygen by combining simple screening procedures as described in this paper. Verstraete and Alexander (VA) medium (l), used as the screening medium in this study, was composed of the following (per liter): 2.0 g of acetamide, 8.2 g of KH,PO,, 1.6 g of NaOH, 0.5 g of MgSO,.7H,O, 0.5 g of KCl, and 0.0005 g each of CaSO,.2H,O, CuSO,.5H,O, and FeC1,.6H,O, and 0.0005 g of ZnS0,.H20. The final pH was adjusted to 7.0
with NaOH. Soil samples collected from various locations in Tsukuba City, Japan, were used to screen for heterotrophic nitrifying bacteria. Soil was added to test tubes (28 mm diameter) containing 5 ml of VA medium, then tubes were incubated at 30°C with shaking (220 rpm). Subculturing (5 times) was performed weekly by adding 0.2 ml of culture broth to fresh VA medium. Samples were diluted lOO-fold in saline solution (0.8% NaCl) before spreading on VA medium solidified with 1.5% agar. The plates were incubated at 30°C until colonies appeared. Separate colonies were picked and individually tested for nitrifying activity by growing the cultures for 3 d. The isolated strains were tested for the ability to carry out aerobic denitrification using Giltay and nitrite (GN) medium modified by replacing nitrate with nitrite Giltay medium (9). GN medium contained the following components per liter: 0.06 g of NaNO,, 1.0 g of L-asparagine.H>O, 5.0 ml of 1% alcoholic solution of brom thymol blue (BTB), 8.5 g of Na citrate, 1.O g of KH,PO,, 1.O g of MgSO,. 7H20, 0.2 g of CaCl,.6H,O, and 0.05 g of FeCl,.6H,O. The final pH was adjusted to 7.0. Each strain was inoculated into a test tube containing 5 ml of GN medium and an inverted Durham tube. Cultures were incubated for 3 d at 30°C with shaking (220 rpm) to ensure adequate oxygenation. The ability to carry out denitrification in the presence of oxygen was indicated by a change in color of the GN medium from green to blue and the formation of a bubble in the Durham tube. An analysis of the denitrification activity was carried out using Na”N0,. We used the nitrite nutrient (NN) medium of Sakai et al. (10) but with the pH increased to 7.8 NN medium contained the following components per liter: 0.06 g of NaNO?, 10 g of glucose, 10 g of peptone, 0.1 g of MgSO,. 7H,O, 2.0 g of K,HPO,, and 1.O g of yeast extract. Sterile
* Corresponding author. e-mail: [email protected] phone: +81-(0)29-853-7486 fax: +81-(0)29-853-4605 409
-110
MATSlJ%Ah;A
ET- Al..
stock solutions of NaNO, and glucose were added aseptically to the medium after the solution was autoclaved separately. When necessary, a solution with a 9:1 ratio of Na14N0, and Na15N0, was added to the NN medium. For these studies, each str& was inoculated into a Hungate tube containing 2 ml of NN medium and tubes were incubated for 3 d at 30°C with shaking (220 rpm) to eusure adequate oxygenation. Na15N0, (99 atom%) was obtained from Shoko-Tsusho (Tokyo): After incubation, an aliquot of gas in the upper space was collected and analyzed for oxygen and dinitrogen concentrations by gas chromatography (GC) and gas chromatography-mass spectrometry (CC-MS). respectively. A thermal conductivity detector (TCD) mounted on a HP-6890 gas chromatograph (Hewlett Packard, Palo Alto, USA) with a molecular sieve (5A, 3 mmx2 m; GL Science, Tokyo) column was used for analysis of O?. An electron capture detector (ECD) mounted on a GC-14B gas chromatograph (Shimadzu, Kyoto) with an OV- 17lm column was used for analysis of N,O. Dinitrogen was analyzed by isotope mass spectrometry using a Finnigan DELTA plus isotope mass spectrometer (Finnigan Delta, Bremen, Germany). Twenty-seven soil samples were examined for the presence of microorganisms exhibiting nitrification activity as measured by acetamide uptake and NO,- and NO,- production. NO? and NO,- were quantified calorimetrically as described by Donald et al. (11). Twenty-one bacterial strains were able to convert acetamide to NO?- and NO,- (Table 1). Furthermore, to investigate whether the heterotrophic nitrifiers possessed denitrification activity, tests were performed using Durham tubes in GN medium in the presence of oxygen. The color change of the GN medium from green to blue and the formation of bubbles in the Durham tubes were observed for six strains, NH-2, NH-7, NH-l 1, NH-14, NH15, and NH- 17 (Table 1). It was suggested that these heteroTABLE
1.
Nitrification
and denitrification
activities Color change and bubble formation
Production” of Strain NO,
NO;
NH-I
++
++
_
NH-2
+
++
NH-3
++
+
+ _
NH-4
+
+
_
NH-5
+
+
-
NH-6
+
++
_
NH-7
++
+
NH-8
+++
+
+ _
NH-9
+++
+
_
+ +
+ +
+
+
NH-13
+
+
NH-14 NH-15
+ + +
+
+
+ +
+ _
++
++ + + + +
+ _
NH-IO N!I-1 I NH-12
Nf1-I6 NH-l 7 NH-18 NH-19 NH-20 NH-21 “ +,-l-30
+ t + + pM; ++, 31-60
pM; +++. 61~100
+ _
_ _ PM.
TABLE
2.
Dinitrogcn evolution and Q concentration culturesofN~I-14.NH-lj,andNfI-17
Strain
“NL (pmol)
in gro\sing
(4 (mm)
NH-14
0.1 5
2.86
NH-15
0.73
I .?7
NH-17
0 (I .72)8
I .70
a NzO amount (pmoi).
trophic nitrifying strains possessed denitrification ability in the presence of oxygen. We focused on NO,~ conversion because it is an important intermediate not only of nitrification but also of denitrification. To confirm and further characterize the oxygen-tolerant denitrification activity observed in 2 1 strains, a pairedincubation technique was employed where the NO,- pool was labeled with 15N in the presence of oxygen. The results were used in 15N pool dilution calculations. The 21 strains were examined for the ability to generate 2WZ gas from a stable isotope of NO,- in NN medium. The results showed that the ability to carry out denitrification was present in 4 of the 2 1 strains, NH-7, NH- 11, NH- 14, and NH- 15. Only two strains, NH-14 and NH-l 5, generated N2 in the presence of oxygen (Table 2). The other two strains, NH-7 and NH- 11, generated N? in anaerobic conditions (0, concentration under 1 ppm). In addition, NH-17 was found to exhibit a distinct activity that allows the conversion of NO,- to N,O in the presence of oxygen (Table 2). It should be noted that the three strains NH- 14, NH-15, and NH-l 7 were among the six strains which demonstrated bubble formation and a change in color of the GN medium (Table 2). Thus, by this procedure, organisms capable of denitrification to N,O or N, in the presence of oxygen could be identified (Table 1). These results clearly showed that the ability to carry out oxygen-tolerant denitritication was present in three of the strains (NH- 14, NH-l 5, and NH-17), indicating that the simple procedure for enrichment of oxygen-tolerant denitrification was useful. For genetic analysis, the genomic DNAs of NH-14, NH15 and NH- 17 were extracted using the QIAGEN Blood Cell & Culture DNA kit (Qiagen, Hilden, Germany). Bacterial universal primer sets 27F (5’-AGAGTTTGATCCTGG CTCAG-3’) and 1492R (5’-TGACTGACTGAGGYTACCT TGTTACGACTT-3’) were used for amplification of bacterial 16s rDNA (12). Reaction conditions using the 27F and 1492R primers were as follows: after initial denaturation at 94°C for 2 min, 25 temperature cycles were performed at 94’C for 0.25 min, 50°C for 0.5 min, and 68°C for 1.5 min. Amplified 16s rDNA fragments were used as sequencing templates. DNA sequences of the PCR products were determined using the BigDye terminator cycle sequence kit (Applied Biosystems, Foster City, CA, USA). The products of the sequencing reaction were analyzed using an automated DNA sequencer (Applied Biosystems). Databases in the DNA Data Bank of Japan (DDBJ) were searched for sequences similar to the 16s rDNA sequence. A distance matrix tree was constructed by using the neighbor-joining method (13) contained in the Clustal X program (14). Matrix distances were calculated using the method of Kimura (15). The confidence level of tree topologies was evaluated by applying 100 bootstrap resamplings.
NOTES
VOL. 95,2003
* Ochrobucfnun
NH-15
(OCR242581)
grignonenre
Rhizobium
Mesorhi:obium
LNH-l4
(OAN
(AFO41444)
amorphae
(AF041442)
(ABO8.8414) Pamcoccus denitrijicons Ocherbhia
(AFW4720) (AF06321Y)
stutzeri
(AFO94736)
P.wudomonasputido
Burkholderia
vremamiens#s
Burkholderia
ceposia
Rurkhnlderro
Nurkholderro
bw
(PLI 16SRRNA)
(GBlCCC01)
coli
oeruginora
Pscudomonor
1
IESRRNA)
(AF008126)
geNicum
ipMmhbhsp
Pxudomonas
nology Corporation
(JST).
malla
(AFM3302)
(AB0514OX) (AFI 10187)
mulrivornnr (AFW7531)
INH_nABO8682?) Alcalrgenex
Kingella
Clostridium
REFERENCES
(RSITXSM)
genap
Rhizobium
(
of this work was supported by CREST (Core Research for Evolutionary Science and Technology) of the Japan Science and Tech-
(ABOSS415)
Ochrobactrum anfhropi
i”
411
demlrjicam
butvricum
jaecolis
(AFl55147)
(KINRRIMB) (AB0757h8)
FIG. 1. Unrooted distance matrix tree based on the 16s rDNA sequences of strains NH-14, NH-l 5 and NH-17 and other closely related bacteria obtained from the DDBJ. The scale bar represents two nucleotide substitutions per 100 positions.
Figure 1 shows the positions of NH-14, NH-15, and NH-17 in relation to other closely related bacteria. NH- 14 had 98% homology with Mesorhizobium amorphae and NH-15 had 98% homology with Ochrohactrum grignonense. NH- 17 and Alcaligenes faaecalis as a known heterotrophic nitrifying bacterium were shown to have a close relationship (97%). By analysis of the physiological properties, it appears that NH- 14 and NH- 15 qualify as new species (data not shown). These results showed that a wide range of bacteria that possess both nitrification and oxygen-tolerant denitrification activities were obtained by enrichment culture using this new simple procedure. Further investigations are in progress to identify the genes required for denitrification and nitrification as well as the regulatory mechanisms controlling these activities in NH- 14, NH- 15, and NH- 17. We thank Dr. Hirohumi Shoun and Dr. Naoki Takaya of University of Tsukuba for performing isotope mass spectrometry. A part
1. Verstraete, W. and Alexander, M.: Heterotrophic nitrification by Arthrobacter sp. J. Bacterial., 110,955-961 (1972). 2. Daum, H., Zimmer, W., Papen, H., Kloos, K., Nawrath,
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