New Method of Denitrification Analysis of Bradyrhizobium Field Isolates by Gas Chromatographic Determination of ¹⁵ N-Labeled N ₂

Abstract

To evaluate the denitrification abilities of many Bradyrhizobium field isolates, we developed a new 15 Nlabeled N 2 detection methodology, which is free from interference from atmospheric N 2 contamination. 30 N 2 ( 15 N 15 N) and 29 N 2 ( 15 N 14 N) were detected as an apparent peak by a gas chromatograph equipped with a thermal conductivity detector with N 2 gas having natural abundance of 15 N (0.366 atom%) as a carrier gas. The detection limit was 0.04% 30 N 2 , and the linearity extended at least to 40% 30 N 2 . When Bradyrhizobium japonicum USDA110 was grown in cultures anaerobically with 15 NO 3 ؊ , denitrification product ( 30 N 2 ) was detected stoichiometrically. A total of 65 isolates of soybean bradyrhizobia from two field sites in Japan were assayed by this method. The denitrification abilities were partly correlated with filed sites, Bradyrhizobium species, and the hup genotype.Denitrification is anaerobic respiration using nitrate (NO 3 Ϫ ), nitrite (NO 2 Ϫ ), nitric oxide (NO), and nitrous oxide (N 2 O) as terminal electron acceptors. Denitrifying species are distributed over a broad variety of bacteria, including Proteobacteria spp., gram-positive bacteria, and archaea (31). Most denitrifying bacteria reduce the electron acceptors sequentially as NO 3 Ϫ 3 NO 2 Ϫ 3 NO 3 N 2 O 3 N 2 , and each step of these reactions is catalyzed by specific respective reductases (31). To evaluate denitrification capability, the acetylene inhibition assay (30) has been widely used in pure cultures (13) as well as for environmental samples such as soils (8,14,27) and water (24) because of sensitivity and atmospheric N 2 contamination (13). In this assay, the excess of N 2 O emission in the presence of acetylene over the level of N 2 O emission in the absence of acetylene is regarded as the N 2 evolution (because acetylene inhibits N 2 O reductase). N 2 O can be detected by a gas chromatograph (GC) equipped with a 63 Ni electron-capture detector possessing high-level sensitivity for N 2 O. The amount of N 2 O is calculated on the basis of the measured headspace concentration and corrected for dissolved gas by using the Bunsen coefficient (29).However, the acetylene inhibition assay has some drawbacks for evaluating bacterial denitrification. The most serious disadvantage is that it is impossible to measure N 2 O reductase activity directly. For this reason, the activities and kinetic parameters of N 2 O reductase have been studied directly by measurement of the N 2 O decrease (4) or by spectrophotometric assay of reduced benzyl viologen for nitrous oxide reductase activity in vitro (12, 23). Moreover, the underestimation of denitrification capability may occur because of incomplete blockage of N 2 O reductase at a low nitrate concentration or in the presence of sulfide (24). In practice, two determinations of N 2 O concentration per sample-in the presence and absence of acetylene-are required for estimation of the amount of N 2 evolved by N 2 O reductase.Acetylene inhibition assays have revealed the diversity of the...

Symbiotic Bradyrhizobium japonicum Reduces N 2 O Surrounding the Soybean Root System via Nitrous Oxide Reductase

“…Natural populations of B. japonicum that lacks N 2 O reductase activity were often dominant in soybean fields (44). To generate bradyrhizobial inoculants with high N 2 O reductase activity in soybean nodules, one can adopt several strategies: (i) introduction of nos genes by using stable vectors or chromosome integration with strong promoters, (ii) selection of natural isolates with high N 2 O reductase activity, and (iii) simple use of USDA110 and other wild-type strains carrying nos genes as inoculants.…”

“…Strains of B. japonicum can apparently be classified into three denitrification types: full denitrifiers (up to N 2 ), truncated denitrifiers (up to N 2 O), and nondenitrifiers (44,49). Their incidence depends on the field site and the phylogeny (44). We tested whether introduction of the nos gene cluster into the naturally occurring nosRZD-deficient strain T7 would induce N 2 O reductase activity.…”

“…The corresponding structural gene is nosZ and is assembled in the nosRZDFYL gene operon (54). Several species capable of denitrification are also nitrogen-fixing bacteria, including rhizobia such as Bradyrhizobium japonicum (4,6,44,49) and Sinorhizobium meliloti (7,20). Indeed, genes responsible for denitrification have been found in rhizobial genomes (15,23).…”

“…After the N 2 gas replacement, 15 N-N 2 O ( 15 N, Ͼ98 atom%; Cambridge Isotope Laboratories Inc., Andover, MA) was introduced. After incubation at 30°C for 67 h, the concentration of 15 N-N 2 in the gas phase was determined by TCD gas chromatography (44).…”

Effect of Rj Genotype and Cultivation Temperature on the Community Structure of Soybean-Nodulating Bradyrhizobia

“…This strain also has nitrous oxide (N 2 O) reductase activity (25,26,27). The N 2 O that is developed by defective denitrification is a strong greenhouse gas (2,11).…”