High Rate of N2 Fixation by East Siberian Cryophilic Soil Bacteria as Determined by Measuring Acetylene Reduction in Nitrogen-Poor Medium Solidified with Gellan Gum

For evaluating N₂ 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 N₂-fixing soil bacteria generally show only trace acetylene reduction activity. Here, we report that use of a N₂ 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 N₂-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 N₂ 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 N₂-fixing rhizobium of soybean can reduce acetylene to produce ethylene (C₂H₄) (32), a reaction analogous to the conversion of the natural substrate N₂ into ammonia. Shortly afterwards, it was shown that this acetylene reduction activity parallels N₂ reduction by nitrogenase (13), and since then, acetylene reduction assays have been widely used in the evaluation of biological N₂ 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 N₂-fixing bacteria, which have been regarded as notoriously difficult to culture. To solve this problem, Döbereiner and her group developed a soft gel method (7), which used 0.2% agar as a gel matrix for the medium. Due to a vertical gradient of dissolved oxygen concentrations, these microaerobes formed a thin layer at the particular depth of the medium that contained an ideal level of dissolved oxygen (10). Also, significant activities in acetylene reduction assays were observed for N₂-fixing microaerobes, particularly those from the rhizoplane of monocotyledonous crop plants (e.g., Azospirillum and Herbaspirillum spp.) (1, 9, 40). To date, these soft gel media solidified with 0.2% agar have been widely used as the most basic method for the screening of free-living or difficult-to-culture N₂-fixing bacteria (2, 16).In an agar composed of soft gel, however, the layer formation of highly transparent colony-forming bacteria is often obscured and is more difficult to observe than comparable layer formation in water due to the higher turbidity of the agar gel, and some members of the soil bacterial community do not show any positive response in acetylene reduction assays under these conditions. These drawbacks to the usage of agar as a soft gel matrix delayed the recognition that free-living N₂ fixers make a potent contribution to the support of ecosystems under adverse soil conditions. Hashidoko et al. developed an improved soft gel medium for growth of N₂-fixing bacteria in 2002 (15). In their study, 0.2% agar was replaced with 0.3% gellan gum, a bacterial extracellular polysaccharide (EPS) produced by Sphingomonas elodea (a synonym of Sphingomonas paucimobilis) ATCC 31461 (12, 17, 18). Initially, gellan gum was used for the purpose of preparing a highly transparent soft gel medium that was better for culturing microaerobic N₂-fixing rhizobacteria. It had other favorable physical properties: when 0.3% gellan gum containing Winogradsky''s mineral mixture was autoclaved, the medium remained in a liquid form over a period of several hours while cooling to room temperature. Even after the gellan gum had been solidified, the soft gel was easily liquefied upon mechanical agitation. The liquefied medium was able to resolidify after a short period of time, so it was easy to uniformly disperse inoculants into the soft gel medium. The outstanding transparency (14) and other properties of this gel matrix enable easy visualization of transparent colony-forming N₂-fixing bacteria and also allow observation of their responses to various concentrations of dissolved oxygen and cell motilities (15).In many preliminary experiments, nitrogen-poor gellan gum media allowed high growth of diazotrophs, but this study was needed to compare gellan gum with agar as a gel matrix for N₂ fixation. Because Siberian boreal forest soils have been noted for their low N₂-fixing capability (3), we first cultured bacterial microbiota from the eastern Siberian Taiga forest bed in gellan gum medium. A quantitative comparison of N₂ fixation behaviors of free-living soil bacteria was attempted to investigate gellan gum as a potential N₂ fixation-promoting soft gel matrix. We here first report on the efficacy of gellan gum as a soft gel matrix for monitoring acetylene reduction by the use of free-living N₂-fixing soil bacteria.