Development of a direct isolation procedure for free-living diazotrophs under controlled hypoxic conditions

Abstract

Free-living diazotrophs are diverse and ubiquitous in soil, contributing the nitrogen pool in natural ecosystems. The isolation of nitrogen-fixing microorganisms has relied on semisolid nitrogen-free medium enrichment, followed by multiple subculturing steps. These procedures limit the diversity of recovered isolates. In the current study, we investigated three different isolation strategies for free-living diazotrophs using a soil sample from the Amazon forest. The methods were (i) direct plating on solid nitrogen-free medium under a 2% O(2) concentration, (ii) enrichment in semisolid nitrogen-free medium before plating on solid nitrogen-free medium under 2% O(2), and (iii) enrichment followed by subculturing in the semisolid nitrogen-free medium before plating on nitrogen containing medium under a 21% O(2) concentration. A total of 794 isolates were differentiated by their genomic fingerprinting patterns, and strains with unique profiles were identified on the basis of sequencing of their 16S rRNA gene. Isolates belonged to four bacterial phyla: Proteobacteria, Firmicutes, Actinobacteria, and Bacteriodetes. The novel strategy of combining a solid N-free medium and hypoxic conditions showed an increase of 62.6% in the diversity of diazotrophs in comparison to that obtained by the conventional semisolid medium-based methods. All isolates grew on the nitrogen-free medium under a 2% O(2) concentration, 78% of them showed the presence of the nifH gene, and 39% tested positive for acetylene reduction activity. Our results suggest that direct plating of soil dilutions on nitrogen-free solid medium under a 2% O(2) concentration is a useful strategy for the isolation of the diverse diazotrophic communities.

Fig 1

Different strategies used for the isolation of free-living diazotrophs from an Amazon forest soil. (A) Strategy A, direct plating on solid N-free medium incubated inside a hypoxic chamber (2% O₂); (B) strategy B, inoculation into semisolid N-free medium with incubation in air for a week, followed by serial dilution and plating on solid N-free medium and incubation inside a hypoxic chamber 2% O₂; (C) strategy C, inoculation into semisolid N₂-free medium with incubation in air with three subsequent transfers in the same semisolid medium before plating on solid medium amended with NH₄Cl. The boxed area represents the 2% O₂ condition in the hypoxic chamber. The total number of isolates (n) obtained with each method is provided.

Fig 2

Percentage distribution of bacterial genera isolated from the Amazon forest soil using three different isolation strategies: direct plating (A), enrichment in semisolid N-free medium before plating (B), and enrichment followed by subculturing in semisolid N-free medium before plating (C). Isolates were identified on the basis of sequencing of the 16S rRNA gene. The total number of isolates (n) obtained with each method is provided.

Fig 3

Maximum likelihood phylogenetic tree based on partial sequences of the 16S rRNA gene of the 210 unique pure cultures obtained through three different isolation methods. Sequences belonging to the same bacterial genera were clustered together, and branches within clusters were collapsed to show the overall relationships of the clusters to one another. The size of the triangle is proportionate to the number and variation of the sequences within a cluster. Asterisks at the nodes reflect bootstrap support values above 70% or posterior probability values from at least three of four phylogenetic methods: maximum likelihood, maximum parsimony, neighbor joining, and Bayesian analyses. Numbers corresponding to each bacterial genus represent number of the unique strains within the genus/number of strains that yielded positive amplification for the nifH gene/number of strains that showed acetylene reduction activity. Clustering at the phylum level is shown on the right.

Fig 4

Genomic fingerprint profiles of 22 bacterial isolates obtained from enrichment and subculturing in semisolid N-free medium of six strains grown together. Lanes on the right represent results for individual strains used for the inoculation. Fragment sizes on the left indicate DNA size markers.