Vol. 20, No. 4, 208–215, 2005 http://wwwsoc.nii.ac.jp/jsme2/

Characterization of the Aerobic in sp. Strain NH-14 in Comparison with that in Related

NORIHISA OKADA1‡, NOBUHIKO NOMURA1‡, TOSHIAKI NAKAJIMA-KAMBE1 and HIROO UCHIYAMA1*

1 Graduate School of Life and Environmental Science, University of Tsukuba, Tsukuba, Ibaraki 305–8572, Japan

(Received May 9, 2005—Accepted September 9, 2005)

A novel aerobic denitrifier, designated strain NH-14, was isolated from soil collected in Tsukuba, Japan. − Strain NH-14 could reduce nitrite (NO2 ) to (N2O) under aerobic and anaerobic conditions. The − −1 conversion of NO2 to N2O occurred under extremely aerobic conditions (O2 concentration: 4.2 mmol flask ), − and was induced by NO2 , indicating aerobic denitrification. From the biochemical characterization and phylo- genic analysis of the complete 16S rRNA gene, strain NH-14 was shown to belong to the genus Mesorhizobium with M. loti and M. amorphae as its closest relatives. The strain was designated Mesorhizobium sp. strain NH-14. It was concluded that Mesorhizobium sp. strain NH-14 is capable of denitrification under both aerobic and anaer- obic conditions.

Key words: aerobic denitrification, gene, Mesorhizobium sp.

there have also been some reports on organisms that can Introduction 9,24,30,34) denitrify in the presence of O2 . This phenomenon Denitrification is anaerobic respiration using nitrate is called aerobic denitrification. Aerobic denitrification is − − − − (NO3 ), nitrite (NO2 ), nitric oxide (NO) and nitrous oxide defined as the co-respiration of O2 and NO3 (or NO2 ); co- − − (N2O) as terminal electron acceptors. Most denitrifying bac- respiration means that both O2 and NO3 (NO2 ) are used as − 22,36) teria reduce NO3 to gaseous forms such as N2O electron acceptors concurrently . Paracoccus pantotro- 2,21,23,24) and dinitrogen (N2), whereas some strains such as Alcali- phus (formerly Thioshaera pantotropha) is an aero- − 18,34) genes faecalis reduce only NO2 . Denitrifying bic denitrifier isolated from activated sludge; it is the most are distributed in various bacterial genera36). Denitrification extensively characterized aerobic denitrifier. More recent is a key process in the biogeochemical , and is studies have revealed some novel species of aerobic denitri- of great importance in agriculture, and fiers, such as Microvirgula aerodenitrificans19) and Thaurea environmental quality. mechernichensis27). Numerous studies have focused on organisms, pathways Previously, we reported a novel simple method of screen- 15) and , with particular attention to the control of de- ing O2-tolerant denitrifiers . As a result, several aerobic nitrification by O2. Denitrification commonly occurs only un- denitrifiers were isolated, and the relationship between − der -limiting conditions. NO3 is thermodynamically aerobic denitrification and heterotrophic nitrification in one less favorable as a terminal electron acceptor than oxygen of them, Burkholderia cepacia NH-17, was determined14). A 31) 11) (O2) , and results in a 40% lower cell yield . Hence, it is heterotrophic bacterium, designated strain NH-14, was also − not surprising that in many , the synthesis and activ- isolated; strain NH-14 produces N2 from NO2 in the pres- 36) ity of denitrifying enzymes are inhibited by O2 . However, ence of O2, though most previously reported denitrifiers, such as NH-17, produce N2O instead of N2 under aerobic * Corresponding author; E-mail: [email protected] ba.ac.jp, Tel & Fax: +81–29–853–6626 conditions. In this study, we determined those properties of ‡ N. Okada and N. Nomura contributed equally to the work present- strain NH-14 related to the reduction of nitrite under aerobic ed here. and anaerobic conditions, comparing them with those of Characterization of Denitrifier Strain NH-14 209

15 15 other denitrifiers. In most studies of aerobic denitrifiers, N N), and the amount of residual O2 in the headspace gas. − − only the consumption of NO3 and NO2 is measured, not To measure time-dependent anaerobic denitrification, a the production of denitrified gases (e.g., N2O and N2). portion (1 ml) of the preculture was transferred into 100 ml Therefore, we examined the formation of these products un- of LD medium in a 150-ml serum bottle with a butyl rubber der aerobic conditions using gas chromatography (GC) and stopper. Anaerobic conditions were initiated by flushing the GC-mass spectrometry. Moreover, we identified strain NH- vial with He several times. After inoculation, the vials were 14 and discussed its phylogenetic position on the basis of sealed with Teflon-silicon septa and aluminum rings. The the results of a 16S rRNA gene analysis and phenotypic vials were then incubated without shaking at 30 or 28°C. characterization. The aerobic denitrification activities of strains of the order Rhizobiale and strain NH-14 were evaluated as 15) Materials and Methods described previously . The organisms were cultured in 2 ml of LD medium with 5 mM NaNO2 in 16-ml test tubes Materials and incubated with shaking (200 rpm). The production of 15 15 14 15 15 15 The N-labelled stable isotope compounds K NO3 and N2O, N2 ( N N and N N) and O2 was determined. 15 Na NO2 were purchased from Shoko Co., Ltd., Tokyo, Ja- − pan. All the other compounds used were standard commer- Resting cell reactions with NO2 cial preparations. Strain NH-14 was cultured in 500-ml Erlenmeyer flasks containing 50 ml of LD medium with or without 5 mM Strains and Media NaNO2 and incubated on a rotary shaker at 200 rpm and Strain NH-14 was isolated from soil collected in Tsuku- 30°C for 48 h. The cells were harvested by centrifugation ba, Japan as reported previously15). Several well-known or- (8,000 × g, 10 min, 4°C) and washed twice with 20 mM ganisms of the order Rhizobiales (Mesorhizobium loti potassium phosphate buffer (pH 7.8). The cells were MAFF303099, M. huakui subsp. rengei B3, Rhizobium suspended in 20 mM potassium phosphate buffer (pH 7.8) T leguminosarum biovar Viceae IAM12609 , Sinorhizobium containing 1 mM NaNO2 to give an optimal density of 5 at fredii USDA191, S. meliloti 1021, Bradyrhizobium elknaii 660 nm. The reaction was carried out in a 16-ml test tube USDA94, B. japonicum USDA110 DJ2 and Azorhizobium with 1 ml of the resting cell mixture and incubated at 30°C caulinodans ORS571) were kindly provided by K. Mina- for 3 h with shaking (160 rpm). After the reaction, the N2O misawa of Tohoku University. Cells were grown at 30°C for concentration was determined. strain NH-14 and 28°C for the authentic test strains of the order Rhizobiales in liquid denitrification (LD) medium (pH Sequencing of 16S rRNA gene 7.8) containing (per liter) 10 g of mannitol, 10 g of peptone The sequence of the strain NH-14 16S rRNA gene was (Becton, Dickinson and Company, Sparks, USA), 0.1 g of analyzed by the National Collection of Industrial and MgSO4·7H2O, 2.0 g of K2HPO4 and 1.0 g of yeast extract Marine Bacterial Ltd. (NCIMB Japan Ltd., Shizuoka, (Becton, Dickinson and Company), with 5 mM NaNO2 or Japan), using a Microseq full gene 16S rRNA gene bacterial 10 mM KNO3 respectively. sequencing kit (Microseq system; Applied Biosystems, Foster City, USA) and an ABI PRISM 377 automated DNA Culture conditions sequencer (Applied Biosystems). The complete 16S rRNA The following method was used to measure time-depen- gene sequence of strain NH-14 was compared to available dent aerobic denitrification. Precultures were prepared by sequences using MicroSeq bacterial identification software inoculating into test tubes (18 mm diameter) containing 4 V.1.4.1 (Applied Biosystems). ml of nutrient broth (Becton, Dickinson and Company) and The nucleotide sequences were also analyzed using the incubating the test tubes aerobically. A portion (0.5 ml) of GENETYX-MAC program ver. 8 (Software Development each preculture was transferred into 50 ml of LD medium in Co., Ltd., Tokyo, Japan), the BLAST program from the a 500-ml Erlenmeyer flask. The flask was sealed with a bu- National Center for Biotechnology Information (http:// tyl rubber stopper and agitated using a rotary shaker at 200 www.ncbi.nlm.nih.gov/) and the Ribosomal Database rpm. Atmospheric air in the headspace was not replaced; Project1,13). Evolutionary distance10) was calculated and a thus, the initial condition was aerobic. The aerobic denitrifi- neighbor-joining phylogenic tree25) was constructed using cation activity of the bacteria was evaluated by determining the CLUSTAL W program32). 14 15 the time-dependent production of N2O and N2 ( N N and 210 OKADA et al.

and injected into a gas chromatograph (model GC-8A, PCR amplification of nitrite reductase genes Shimadzu) equipped with a TCD detector. A stainless steel The genomic DNA of strain NH-14 was extracted with column (3 mm × 2 m) packed with Molecular Sieve 5A the Genomic-tip System (QIAGEN, Hilden, Germany). The (Shimadzu) was used. The injector, oven and detector primers for the nitrite reductase genes (nirS and nirK) were temperatures were 50°C. described previously5). The Cu-type nitrite reductase gene nirK was PCR-amplified with the primers nirK1F (GG(A/ Measurement of labeled N2 with stable isotope C)ATGGT(G/T)CC(C/G)TGGCA) and nirK5R (GCCTC- N2 gas with a stable isotope label was measured by GC- GATCAG(A/G)TT(A/G)TGG). The cytochrome cd1-type mass spectrometry. The gas phase (0.5 ml) was collected nitrite reductase gene nirS was PCR-amplified with the using a gas-tight syringe and injected into a gas chromato- primers nirS1F (CCTA(C/T)TGGCCGCC(A/G)CA(A/G)T) graph equipped with a DELTA plus isotope mass spectro- and nirS6R (CGTTGAACTT(A/G)CCGGT). After a dena- meter (Finnigan Mat, Bremen, Germany). A Molsieve 5A turation step of 5 min at 95°C, touchdown PCR was per- PLOT capillary column (32 mm × 25 m, GL Science, formed. This consisted of a denaturation step of 30 sec at Tokyo, Japan) was used33). 95°C, a primer-annealing step of 40 sec, and an extension step of 40 sec at 72°C. During the first 10 cycles, the Nucleotide sequence accession numbers annealing temperature was decreased by 0.5°C every cycle, The nucleotide sequence data were deposited in the starting at 45°C until it reached the touchdown PCR DDBJ/EMBL/GenBank nucleotide sequence databases temperature of 40°C. During the subsequent 20 cycles, under accession numbers AB196496 (16S rRNA gene of the annealing temperature was 43°C. After 30 cycles, a final strain NH-14) and AB196786 (nirK of strain NH-14). 7-min incubation at 72°C was performed. PCR products were sequenced directly using a BigDye Results Terminator Cycle Sequencing kit (Applied Biosystems) and − analyzed with an automated DNA sequencer as described Time-dependence of NO2 conversion under aerobic or above. anaerobic conditions Strain NH-14 was inoculated into LD medium containing Phenotypic characterization − − 5 mM NaNO2, and the conversion of NO2 to NO3 , N2O 14 15 15 The phenotypic characteristics and ubiquinone were and N2 ( N N and N15N) was investigated in growing cul- determined by NCIMB Japan Ltd. Tests on carbon nutrition tures. Batch cultures in the flasks had a headspace initially − were performed using an API 20 NE kit (bioMerieux, Lyon, filled with air to generate aerobic conditions. The NO2 France). Colony size was investigated using nutrient broth concentration decreased rapidly after 24 h of incubation, −1 (Becton, Dickinson and Company) supplemented with 1.5% and N2O accumulated (0.02 mmol flask ) after 48 h (Fig. − agar. 1A). The conversion of NO2 to N2O occurred at an oxygen concentration of 4.2 mmol flask−1, indicating that strain DNA base composition − NH-14 could denitrify NO2 under nearly full atmospheric The G + C content of genomic DNA was estimated by oxygen tension. In addition, N2 production was monitored HPLC as described by Mesbah et al.16). under initially aerobic condition, and the strain produced a −1 significant amount of N2 (0.01 mmol flask ) after 84 h. Analytical methods 14 15 15 15 However the evolved gas was N2 ( N N), not N2 ( N N), − − 14 15 − NO3 and NO2 concentrations were measured color- indicating that N2 ( N N) was produced from N in NO2 7) 15 14 imetrically . N2O production was measured by GC. The gas ( N) and N in the other nitrogen compounds ( N), such as phase (0.5 ml) was collected using a gas-tight syringe and peptone and yeast extract, in the medium. In this study, − injected into a Shimadzu model GC-8A gas chromatograph NO3 production was not observed, conflicting with the (Shimadzu, Kyoto, Japan) equipped with a TCD detector. A result of our previous study, in which strain NH-14 pro- − 15) stainless steel column (3 mm × 2 m) packed with Shincarbon duced NO3 by nitrification . In our previous study, we − ST (Shimadzu) was used. The injector, oven and detector measured the production of NO3 from acetamide but not − temperatures were 200°C. from NO2 , unlike the case in this study. This contradiction The degree of O2 reduction was measured by GC. The might be due to the difference in the type of nitrogen − gas phase (0.1 ml) was collected using a gas-tight syringe substrate for NO3 production. Further study is necessary to Characterization of Denitrifier Strain NH-14 211

− Fig. 1. Denitrification by strain NH-14 in batch cultures. Cell growth, NO2 and gas phase O2 and N2O were monitored periodically as described −1 in Materials and Methods. (A) The strain was cultured in flasks containing LD medium with 5 mM NaNO2 (0.25 mmol flask ) under aerobic −1 conditions. (B) The strain was cultured in vials containing LD medium with 5 mM NaNO2 (0.5 mmol vial ) under anaerobic conditions. − Symbols: , O2; , optical density at 660 nm (OD660); , NO2 ; , N2O. Typical results from more than three independent experiments are shown.

− draw a conclusion regarding the capability for nitrification resting cells cultured without NO2 did not produce N2O of strain NH-14. within 3 h, even when the reaction was prolonged up to 12 Under anaerobic conditions, growing cells demonstrated h. These results indicate that strain NH-14 is capable of − − concomitant NO2 consumption and N2O production (0.03 denitrification and that its activity is induced by NO2 under −1 − mmol flask ) (Fig. 1B), and NO2 reduction was linked to aerobic and anaerobic conditions. ATP production. These results suggest that strain NH-14 − Identification of nitrite reductase gene(s) can convert NO2 to N2O regardless of the O2 tension and 14 15 this conversion is a step in denitrification. But, N2 ( N N To identify the gene(s) for nitrite reductase (nir), we used and 15N15N) production was not detected. sets of primers developed to detect nirK or nirS from − We also examined NO3 conversion by strain NH-14 with a touchdown PCR method using under aerobic and anaerobic conditions. Strain NH-14 was total DNA preparations from strain NH-14. A 514-bp DNA incubated aerobically in LD medium with 10 mM KNO3; fragment was observed using a set of primers for nirK, but although the strain NH-14 cells grew, they did not reduce no products were obtained with the primers for nirS (data − 14 15 15 15 NO3 (Fig. 2). N2O and N2 ( N N and N N) were not not shown). The PCR-amplified products were sequenced, identified and apparently were not produced. Growth was and the strain NH-14 sequence was compared with other − not apparent in anaerobic cultures with NO3 (data not bacterial gene sequences using the BLAST program against shown). DDBJ/GenBank/EMBL nucleotide sequence databases. The analyzed sequence was found to have 84% identity with a − Induction of denitrification activity by NO2 region of the nirK gene from Mesorhizobium sp. 4FB11 − 29) NO2 is an inducer of gene expression for denitrification (AY078254) . in various denitrifiers36). Using resting cells of strain NH-14 Phenotypic and phylogenetic analyses cultured in LD medium with or without 5 mM NaNO2, the − induction of denitrification by NO2 was examined. The As shown in Table 1, strain NH-14 had gram-negative, −1 production of 0.01 µmol tube of N2O was confirmed in rod-shaped cells, produced oxidase and catalase, and was − the resting cells cultured with NO2 . On the other hand, the capable of growth only on D-mannitol and N-acetyl-D-glu- 212 OKADA et al.

Table 1. Phenotypic characteristics of strain NH-14

Characteristic Observationa Size (µm) 0.5–0.6 × 1.2–1.5 Colony size (mm) <1.0b Spore − Motility + Catalase + Oxidase + − − NO3 reduction to NO2 − Indole production − Utilization as sole carbon and energy source: Glucose − L-Arabinose − D-Mannose − D-Mannitol + N-Acetyl-D-glucosamine + Maltose − Fig. 2. Denitrification by strain NH-14 in initially aerobic batch cul- Gluconate − tures. The strain was cultured in flasks containing LD medium −1 n-Capric acid − with 10 mM KNO3 (0.5 mmol flask ). Symbols: , O2; , − Adipic acid − OD660; , NO3 ; , N2O. Typical results from more than three independent experiments are shown. DL-Malic acid − Citrate − Phenyl acetate − cosamine among the organic compounds used as the carbon Ubiquinone Q-10 − and energy sources. In addition, no NO3 reductase activity G + C content (mol%) 61.7 was detected (Fig. 2). The major ubiquinone was Q-10. The a Symbols: +, reactions positive; −, reactions negative. G + C content of the genomic DNA was 61.8 mol% as b After 6 days of aerobic incubation on nutrient broth supplemented determined by HPLC. with 1.5% agar. The nucleotide sequence of the 16S rRNA gene (1460 bp) was determined for strain NH-14. Sequence analysis of 15N15N) were not detected (Fig. 4). Furthermore, the capa- the nearly complete 16S rRNA gene of strain NH-14 was bility of other rhizobia strains to carry out aerobic denitrifi- carried out with the MicroSeq bacterial identification cation was investigated as described previously15). Strain system. The 16S rRNA gene sequence of strain NH-14 was NH-14 cultured in LD medium in the presence of O2 (0.45 −1 −1 similar to sequences of species in the genus Mesorhizobium: mmol tube ) produced N2O (1.3 µmol tube ). All eight 98.2% similarity to M. loti (NC 002678), 98.2% to M. rhizobium strains (M. loti MAFF303099, M. huakui subsp. amorphae (AF041442) and 97.8% to M. tianshanense rengei B3, R. leguminosarum biovar Viceae IAM12609T, S. (AF041447). The phylogenetic analysis showed that strain fredii USDA191, S. meliloti 1021, B. elknaii USDA94, B. NH-14 belongs to Mesorhizobium, a genus of the order japonicum USDA110 DJ2 and A. caulinodans ORS571) 14 15 15 15 Rhizobiales (Fig. 3). Therefore, the test organism was grew, but did not produce N2O and N2 ( N N and N N) designated as Mesorhizobium sp. strain NH-14. (data not shown). These results suggest that the ability − to convert NO2 to N2O under aerobic and anaerobic Analysis of aerobic denitrification by organisms of the conditions is specific to strain NH-14. order Rhizobiales The high similarity of strain NH-14’s 16S rRNA gene Discussion sequence to those of the genus Mesorhizobium prompted us − to examine the aerobic time-dependent NO2 conversion by Our results showed that strain NH-14 produces N2O (0.02 −1 − M. loti MAFF303099. Cell growth and a decrease in the O2 mmol flask ) from NO2 under a nearly full atmospheric − −1 concentration were observed, but the decrease in the NO2 oxygen tension (O2 concentration, 4.3 mmol flask ) (Fig. 14 15 concentration was minimal, and N2O and N2 ( N N and 1A). Strain NH-14 was capable of denitrification at O2 con- Characterization of Denitrifier Strain NH-14 213

Fig. 3. Neighbor-joining phylogenic tree showing the relationship between strain NH-14 and related species of the order Rhizobiales, on the ba- sis of nearly complete 16S rRNA gene sequences. Gaps were excluded and 1460 nt were compared. The scale bar denotes the number of sub- stitutions per nucleotide position.

(14N15N) was formed by combining two nitrogen atoms, one − 15 14 from NO2 ( N) and one from another source ( N). The 14 15 production pathway for this hybrid N2 species ( N N) is called codenitrification, and it has been reported for fungi 12,28) and actinomycetes . In this report, N2O was detected by GC, therefore nitrogen atoms were unidentified. Further research is necessary to clarify this phenomenon. Besides, 14 15 the amount of N2O (0.02 mmol) and N2 ( N N) (0.01 mmol) produced in 84 h were not stoichiometrically − equivalent to the amount of NO2 used (0.25 mmol) in − a similar period. It is presumed that almost all NO2 was − assimilated by strain NH-14. NO2 metabolism in strain NH-14 remains to be investigated and further research is being planned to clarify this point. Interestingly, strain NH-14 consumed O2 during the − conversion of NO2 to N2O (Fig. 1A). This process is known as ‘aerobic denitrification’. Aerobic denitrification is de- − − Fig. 4. Denitrification by M. loti MAFF303099 in initially aerobic fined as the co-respiration of O2 and NO3 (or NO2 ). Co- − − batch cultures. The strain was cultured in flasks containing LD respiration means that both O2 and NO3 (NO2 ) are used as −1 medium with 5 mM NaNO2 (0.25 mmol flask ). Symbols: , 22,36) − electron acceptors at the same time . 2 660 2 2 O ; , OD ; , NO ; , N O. Typical results from more − than three independent experiments are shown. NO2 is an inducer of the expression of denitrification- related genes36). It was observed that the activity of strain − NH-14 to convert NO2 to N2O under aerobic conditions is 23) − centrations as high as those reported for P. pantotrophus . induced by NO2 . Furthermore, strain NH-14 possesses In a previous study, strain NH-14 was cultured in LD nirK, which encodes a copper-containing nitrite reductase; 14 15 medium with NaNO2 (9:1 ratio of Na NO2 and Na NO2) sequence comparisons revealed 84% identity with a region 14 15 15) 29) and generated N2 ( N N) . We considered strain NH-14 to of the nirK gene from Mesorhizobium sp. 4FB11 . Thus, − have produced N2 by aerobic denitrification. However, N2 it was concluded that the conversion of NO2 to N2O by (15N15N) was not detected in batch cultures and it was strain NH-14 under aerobic conditions proceeds by aerobic 14 15 confirmed that strain NH-14 produced N2 ( N N) (0.01 denitrification. These results indicate that strain NH-14 is an mmol flask−1). Denitrification is defined as a process in aerobic denitrifier. Under anaerobic conditions, strain NH- − which both nitrogen atoms in the N2 product are derived 14 grew and consumed NO2 while producing N2O (Fig. − − from NO2 (NO3 ), however our result suggested that N2 1B). This result suggests that strain NH-14 is coupled with 214 OKADA et al.

− the reduction of NO2 to conserve energy. Our study showed In this study, we showed that Mesorhizobium sp. strain − that this strain has no nitrate reductases (Fig. 2 and Table 1), NH-14 could denitrify NO2 to N2O regardless of the O2 − 6) but is able to reduce NO2 the same as A. faecalis, a typical concentration. Casella et al. reported that Rhizobium 18,34) − aerobic denitrifier . “hedysari” strain HCNT 1 reduces NO2 to N2O under On the basis of the phylogenetic analysis of its 16S rRNA aerobic conditions the same as Mesorhizobium sp. strain gene sequence, strain NH-14 was shown to belong to the ge- NH-14. They described that nitrite reductase activity in − nus Mesorhizobium with M. loti and M. amorphae as its strain HCNT 1 cells functions as a NO2 detoxification closest relatives (98.2% similarity). Some of its phenotypic mechanism. Our results suggest that the growth of strain − characteristics (i.e., Gram negative, motile, rod shaped) NH-14 is suppressed by NO2 under aerobic conditions were identical to those of known Mesorhizobium strains4) (Fig. 1A and Fig. 2). In view of the results of this study, we (Table 1). In addition, the quinone profile with Q-10 as the plan to investigate the function of aerobic denitrification in sole component and the G+C content (61.7 mol%) suggest Mesorhizobium sp. strain NH-14. Further study is needed to that it belongs to the genus Mesorhizobium8) but is different identify genes and enzymes involved in aerobic denitrifica- from other species of the genus in terms of carbon nutrition. tion in Mesorhizobium sp. strain NH-14. Almost all Mesorhizobium species could use glucose, L-ara- binose or maltose as the sole carbon source, whereas strain Acknowledgements NH-14 was capable of growth only on D-mannitol and N- acetyl-D-glucosamine8) (Table 1). Although strain NH-14 We are grateful to Masatoshi Matsumura and Naoki may represent a novel species of the genus Mesorhizobium, Takaya of our faculty, and Hirofumi Shoun of the Univer- we designate it Mesorhizobium sp. strain NH-14 simply sity of Tokyo, for performing GC and isotope mass spec- until there is sufficient data to warrant an official taxonomic trometry, respectively. We thank Kiwamu Minamisawa of proposal. Tohoku University for providing strains of the order In previous investigations of denitrification by Rhizobiales. We also thank Emiko Matsuzaka, Yukie diazotrophs3,17,35), some strains of the genera Rhizobium and Shigeno-Akutsu and Kyoko Ikegami of our laboratory for Sinorhizobium were confirmed to carry out denitrification technical assistance. under anaerobic conditions. 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