Vol. 20, No. 4, 208–215, 2005 http://wwwsoc.nii.ac.jp/jsme2/ Characterization of the Aerobic Denitrification in Mesorhizobium sp. Strain NH-14 in Comparison with that in Related Rhizobia 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 nitrous oxide (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, nitrite reductase 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 nitrogen 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 species 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 nitrogen cycle, and is studies have revealed some novel species of aerobic denitri- of great importance in agriculture, wastewater treatment 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 enzymes, 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 oxygen-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 bacteria, 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).