PK-190:

Letting Microorganisms escape loneliness and grow

Over 99% of environmental microorganisms remain as yet uncultured. Although this phenomenon is essentially important, reason or mechanism has not been well revealed, and there have been developed very limited number of new cultivation methods aiming to overcome above issue.
We hypothesized that 1) the majority of environmental microbial cells goes dormant when cell density is low (lower than the certain level); and 2) dormant cells resuscitate and start to grow when cell density is higher than certain level; and 3) these cell density dependent phenomenon is caused by the cell-cell interaction among inter- or intra species. However, it is principally impossible to realize such high cell density at the step of isolation. Thus we assumed that “this” can be the bottle neck of the conventional cultivation method.
In this study, the new cultivation method based on novel concept is developed. The method provides high cell density (over 10⁶cell / ml) condition from the start whereas each inoculums are separated each other till the end (keeping pure culture). This condition stimulates interactions among inoculated microorganisms. W/O emulsion consisting of 10⁶– 10⁷of hydro-gel particles (10 – 30 um in diameter) with the media and single cells inside is a key for this method. Hydro-gel particles are partly and softly aggregated in oil phase. We found that this involves essentially important properties; i) movement of microbial cells to other hydro gel particles are strongly restricted; ii) water-soluble molecules easily transfer among the hydro-gel particles by diffusion; iii) huge number (10^{6-10^7)}of individual containers with pure cultures. As a result of comparative experiment using the soil sample, cultivation efficiency in the new method showed much higher than the conventional method, and the diversity and novelty of isolates is also higher than those from conventional method.

keywords:Environmental microorganism,Unculturability,Cultivation technology