Data_Sheet_1_Triggering Growth via Growth Initiation Factors in Nature: A Putative Mechanism for in situ Cultivation of Previously Uncultivated Microorganisms.pdf

Most microorganisms resist cultivation under standard laboratory conditions. On the other hand, cultivating microbes in a membrane-bound device incubated in nature (in situ cultivation) can be an effective approach to overcome this limitation. In the present study, we applied in situ cultivation to isolate diverse previously uncultivated marine sponge-associated microbes and comparatively analyzed this method’s efficiencies with those of the conventional method. Then, we attempted to investigate the key and previously unidentified mechanism of growing uncultivated microorganisms by in situ cultivation focusing on growth triggering via growth initiation factor. Significantly more novel and diverse microbial types were isolated via in situ cultivation than by standard direct plating (SDP). We hypothesized that some of environmental microorganisms which resist cultivation are in a non-growing state and require growth initiation factors for the recovery and that these can be provided from the environment (in this study from marine sponge). According to the hypothesis, the effect of the sponge extract on recovery on agar medium was compared between strains derived from in situ and SDP cultivation. Adding small amounts of the sponge extracts to the medium elevated the colony-formation efficiencies of the in situ strains at the starvation recovery step, while it showed no positive effect on that of SDP strains. Conversely, specific growth rates or saturated cell densities of all tested strains were not positively affected. These results indicate that, (1) the sponge extract contains chemical compounds that facilitate recovery of non-growing microbes, (2) these substances worked on the in situ strains, and (3) growth initiation factor in the sponge extract did not continuously promote growth activity but worked as triggers for regrowth (resuscitation from non-growing state).

众多微生物在常规实验室培养条件下难以繁殖。然而，在自然环境中的膜状装置内（原位培养）繁殖微生物，可作为一种有效的途径以克服此限制。在本研究中，我们采用原位培养法，旨在分离出多种先前未培养的海洋海绵相关微生物，并对比分析了此方法与传统方法的效率。随后，我们尝试探究通过生长启动因子触发生长的原位培养中未培养微生物生长的关键且尚未识别的机制。通过原位培养分离出的新型微生物种类显著多于传统直接平板培养（SDP）。我们假设，某些抗培养的微生物处于非生长状态，需要生长启动因子以实现复苏，而这些因子可能来源于环境（在本研究中为海洋海绵）。据此假设，我们比较了源自原位和SDP培养的菌株在琼脂培养基上复苏效果的差异。向培养基中添加少量海绵提取物提高了原位菌株在饥饿复苏步骤中的菌落形成效率，而对SDP菌株则无此积极影响。相反，所有测试菌株的特定生长速率或饱和细胞密度均未受到积极影响。这些结果表明，（1）海绵提取物含有促进非生长微生物复苏的化学成分，（2）这些成分作用于原位菌株，（3）海绵提取物中的生长启动因子并非持续促进生长活性，而是作为生长复苏（从非生长状态复苏）的触发剂。