High Rate of N ₂ Fixation by East Siberian Cryophilic Soil Bacteria as Determined by Measuring Acetylene Reduction in Nitrogen-Poor Medium Solidified with Gellan Gum

Abstract

For evaluating N 2 fixation of diazotrophic bacteria, nitrogen-poor liquid media supplemented with at least 0.5% sugar and 0.2% agar are widely used for acetylene reduction assays. In such a soft gel medium, however, many N 2 -fixing soil bacteria generally show only trace acetylene reduction activity. Here, we report that use of a N 2 fixation medium solidified with gellan gum instead of agar promoted growth of some gellan-preferring soil bacteria. In a soft gel medium solidified with 0.3% gellan gum under appropriate culture conditions, bacterial microbiota from boreal forest bed soils and some free-living N 2 -fixing soil bacteria isolated from the microbiota exhibited 10-to 200-fold-higher acetylene reduction than those cultured in 0.2% agar medium. To determine the N 2 fixation-activating mechanism of gellan gum medium, qualitative differences in the colony-forming bacterial components from tested soil microbiota were investigated in plate cultures solidified with either agar or gellan gum for use with modified Winogradsky's medium. On 1.5% agar plates, apparently cryophilic bacterial microbiota showed strictly distinguishable microbiota according to the depth of soil in samples from an eastern Siberian Taiga forest bed. Some pure cultures of proteobacteria, such as Pseudomonas fluorescens and Burkholderia xenovorans, showed remarkable acetylene reduction. On plates solidified with 1.0% gellan gum, some soil bacteria, including Luteibacter sp., Janthinobacterium sp., Paenibacillus sp., and Arthrobacter sp., uniquely grew that had not grown in the presence of the same inoculants on agar plates. In contrast, Pseudomonas spp. and Burkholderia spp. were apparent only as minor colonies on the gellan gum plates. Moreover, only gellan gum plates allowed some bacteria, particularly those isolated from the shallow organic soil layer, to actively swarm. In consequence, gellan gum is a useful gel matrix to bring out growth potential capabilities of many soil diazotrophs and their consortia in communities of soil bacteria.In 1967, Schöllhorn and Burris discovered that nitrogenase from an N 2 -fixing rhizobium of soybean can reduce acetylene to produce ethylene (C 2 H 4 ) (32), a reaction analogous to the conversion of the natural substrate N 2 into ammonia. Shortly afterwards, it was shown that this acetylene reduction activity parallels N 2 reduction by nitrogenase (13), and since then, acetylene reduction assays have been widely used in the evaluation of biological N 2 fixation. An acetylene reduction assay is generally performed under the following conditions: precultured bacterial cells are suspended into N-free or -deficient liquid medium containing a carbon source, usually D-glucose or D-mannitol (35) at 0.5 to 2.0%, and exposed for 24 h or less at a representative room temperature, e.g., 25°C (2). However, this method is not applicable to free-living, microaerobic N 2 -fixing bacteria, which have been regarded as notoriously difficult to culture. To solve this problem, Döbereiner and her group developed a soft g...

Development of a Direct Isolation Procedure for Free-Living Diazotrophs under Controlled Hypoxic Conditions

“…In contrast to the molecular assessment results, only 39% of isolates displayed acetylene reduction activity. Previously, it has been reported that the N 2 -fixing ability of the different diazotrophs varied depending upon a number of factors, such as incubation time and temperature, growth medium, O 2 concentration, and type of solidifying agent (7,8,14,21,24,26). Therefore, it is likely that the absence of nitrogenase activity can be attributed to the experimental conditions used in this study.…”

“…4) highlights the potential bias that might occur during attempts to isolate N 2 fixers using traditional methods, regardless of their dominance in the environment. Previously, Hara and coworkers (21) reported that 80 to 100% of the N 2 -fixing isolates from surface and subsurface soil samples from a Siberian forest belonged to the genus Pseudomonas. We reason that the high dominance of this group might be associated with the use of a semisolid N-free medium.…”

“…This method was based upon the traditional enrichment strategy, in which repeated series of soil dilutions were transferred or subcultured on the N-free medium before plating on N-containing medium. This classical approach has led to the isolation of a number of diazotrophic strains (2,6,12,21,34). However, the final outcome of any bacterial enrichment-based method is known for being influenced by differential growth rates and negative interactions among microorganisms (45).…”

Variation in N2Fixation in Subarctic Tundra in Relation to Landscape Position and Nitrogen Pools and Fluxes

“…Photosynthesis constitutes the more abundant source of energy in tundra compared to easily decomposable organic compounds and therefore the majority of studies on N 2 fixation in high-latitude ecosystems are focused on autotrophic N 2 fixation by cyanobacteria (Liengen and Olsen, 1997;Liengen, 1999b;Solheim et al, 2002;Sorensen et al, 2006;Stewart et al, 2011a), while the contribution of heterotrophic bacteria has been considered insignificant. However, recent studies presented evidence that also diazotrophic soil bacteria can substantially contribute to the local soil N pool by fixing atmospheric N 2 (Nosko et al, 1994;Hara et al, 2009;Zadorina et al, 2009). As such, there is a paucity of studies on N 2 fixation in highlatitude ecosystems and our understanding of the role of diazotrophs for N budgets in tundra is poor.…”

The Effects of Sap of Nitraria Plant on the Growth of Its Endogenous Nitrogen-fixing Microbes under Saline-Alkaline Stress

“…Culture Media: Preparation of Winogradsky's N-free medium referred to Hara's method, N-free plates with 1.6% agar [14].…”