“99%难培养”微生物的概念与初步评价：以固氮菌为例

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

摘要:

[目的] 以固氮菌为例，厘清“99%难培养”的概念，定量评价土壤中可培养固氮菌的比例。[方法] 直接提取土壤中所有微生物DNA，同时，利用传统微生物富集技术获得固体和液体培养基第一代和第二代菌体及其DNA，高通量测序nifH和16S rRNA基因，通过系统发育分类方法，明确固氮菌富集物的物种组成及可培养比例。[结果] 文献分析表明，“99%难培养”并未有严格的定量实验证据，是“平板计数异常”的同义词，即采用显微计数法的微生物数量远高于平板计数法。针对典型旱作潮土中的固氮菌，微生物属水平的nifH基因分析发现，可培养固氮菌占比为（22.4±4.5）%-（28.4±6.3）%，而16S rRNA的结果为（31.6±3.4）%-（41.4±13）%。nifH基因分析发现土壤中固氮菌共67属，其中39属可在固体和液体培养形成菌落，但仅有4属得到显著富集，固体培养基富集了Proteobacteria门Azotobacter属，相对丰度高达（98.2±0.94）%；而液体培养基极显著富集了Firmicutes门的Paenibacillus和Clostridium属，相对丰度高达（76.7±3.9）%和（21.7±4.0）%。16S rRNA基因分析发现，土壤中所有固氮菌共计14门、255属，其中248属可在固体和液体培养基形成菌落被培养，但高达6门、226属尚未获得纯菌株或固氮生理报道，并且固体培养基仅显著富集了Proteobacteria门6个属；液体培养基则富集了Firmicutes门5个属。[结论] “99%难培养”是“平板计数异常”的同义词。nifH基因发现土壤中58.2%固氮菌属可培养；而16S rRNA基因则发现高达97.3%固氮菌属可在培养基上形成菌落，但其中91.1%的固氮菌尚未获得纯菌株或固氮生理报道。同时，绝大部分固氮菌为数量占弱势的稀有属，69.5%的nifH基因属丰度<0.1%；而78.5%的16S rRNA基因属丰度<0.1%，导致传统富集培养传代过程中，定向富集了常见的Proteobacteria和Firmicutes门的固氮菌，遗漏了绝大部分已形成菌落但尚未被分离纯化的固氮菌属。“不可培养”微生物的表述并不合理，未来亟需创新培养策略，定向分离传统培养基上被遗漏的微生物，获得更多“难培养”或“尚未培养”微生物。

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.