History and definition of the “99% unculturability paradigm”-case analysis with nitrogen-fixing bacteria

Abstract:

[Objective] To clarify the source references of the paradigm that 99% of microorganisms are noncuturable; and to quantitatively evaluate the proportion of culturable diazotrophs in an agricultural soil. [Methods] Total microbial DNA was directly extracted from soil. Classic cultivation methods were used to obtain diazotrophic colonies from the first and second generation of solid and liquid culture medium for DNA extration. The abundance and composition of microbiome were analyzed by high-throughput sequencing of nifH and 16S rRNA genes. Community composition and proportions of culturable diazotrophs were then identified through phylogenetic classification.[Results] At the genus level, phylogenetic analysis of nifH gene showed the proportion of culturable diazotrophs in the soil was (22.4±4.5)%-(28.4±6.3)%, and result of 16S rRNA genes was (31.6±3.4)%-(41.4±13.1)%. Phylogeny of nifH genes revealed a total of 67 genera in soil; 39 genera could grow in liquid or solid medium, but only 4 genera were significantly enriched. The solid meidum led to significant enrichment of Azotobacter of Proteobacteria as the dominant nitrogen-fixing bacteria with a relative abundance of (98.2±0.94)%. Meanwhile, liquid medium enriched members within two genera of Paenibacillus and Clostridium of Firmicutes significantly, and the relative abundance of the first generation was as high as (76.7±3.9)% and (21.9±4.0)%, respectively. As for 16S rRNA gene-based result, a total of 255 genera within 14 phyla were obtained in soil; 248 genera could grow in liquid or solid medium, but the sequences of up to 226 genera and 5 phya showed high similariry to bacterium in which N₂-fixing physiology has not been demonstrated. Members within 6 genera of Proteobacteria were significantly enriched in solid medium, while liquid medium encirhed significantly 5 genera of Firmicutes. [Conclusion] Literature survey reveals that 99% unculturability paradigm is not yet experimentally verified; and it is more a synonym for "the plate count anomaly", depsite it is widely accpeted in past 30 years. It indicates that direct microscopic counts of total microorganisms in environment is often much more than dilution plate counts of culturable microorganisms. Using N₂-fixer as an emxple, we show that up to 58.2% (nifH gene) and 97.3% (16S rRNA gene) of diazotrophs in soil can form colony in solid or liquid medium; but 91.1% of these colony have not been isolated yet with known physiology of N₂-fixing activity. Meanwhile, the majority of diazotrophs are rare taxa (with relative abundance<0.1%) at genus level on the basis of nifH and 16S rRNA genes, representing 69.5% and 78.5% of the total diazotrophs in soil, respectively. Desite of the clony forming in medium, these numerically less dominant taxa could thus liekly escaped isolation, due to the continous enrichment and long-term subcutlure strategies, and the members within the phyla of Proteobacteria and Firmicutes were particularly enriched. Our study thus highlight that the term “unculturable” should be avoided, and the difficult-to-cultivated term would be more appropriate because >90% of diazotrophs could form colony in medium, although they are not isolated yet. Cultivation effort is warranted for future studies with focused target of these rare taxa.