Previous Article | Next Article 
Applied and Environmental Microbiology, May 2009, p. 2811-2819, Vol. 75, No. 9
0099-2240/09/$08.00+0 doi:10.1128/AEM.02660-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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
,
Shintaro Hara,1
Yasuyuki Hashidoko,1*
Roman V. Desyatkin,2
Ryusuke Hatano,1 and
Satoshi Tahara1
Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan,1 Institute of Biological Problems of Cryolithzone, Russian Academy of Sciences, 41 Lenin Avenue, Yakutsk 677891, Russia2
Received 20 November 2008/ Accepted 3 March 2009
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.
* Corresponding author. Mailing address: Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. Phone: 81-117063839. Fax: 81-117064182. E-mail: yasu-h{at}abs.agr.hokudai.ac.jp
Published ahead of print on 13 March 2009.
Supplemental material for this article may be found at http://aem.asm.org/.
Applied and Environmental Microbiology, May 2009, p. 2811-2819, Vol. 75, No. 9
0099-2240/09/$08.00+0 doi:10.1128/AEM.02660-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»