Cultivatingin situtriggers growth of previously uncultivated microorganisms via a growth initiation factor in nature

Abstract

AbstractMost microorganisms resist cultivation under standard laboratory conditions. On the other hand, to cultivate microbes in a membrane-bound device incubated in nature (in situcultivation) is an effective approach. In the present study, we appliedin situcultivation 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 clarify the key and unknown mechanism ofin situcultivation by focusing on growth triggering via growth initiation factor. We hypothesized that majority of environmental microorganisms are in nongrowing state and requiring “growth initiation factor” for the recovery and that can be provided from environments. Consequently, significantly more novel and diverse microbial types were isolated viain situcultivation than by standard direct plating (SDP). Next, the effect of the sponge extract on starvation recovery was compared between strains derived fromin situand SDP cultivation. Adding small amounts of the sponge extracts to the medium elevated the colony-formation efficiencies of thein situstrains at the starvation recovery step, while it showed no positive effect on that of SDP strains. Conversely, specific growth rates or carrying capacities of all tested strains were not positively affected. These results indicate that, 1) the sponge extract contains chemical compounds that facilitate starvation recovery, these substances selectively worked on thein situstrains, and 2) growth initiation factor in the sponge extract did not continuously promote growth activity but worked as triggers for regrowth (resuscitation from dormancy).ImportanceMost microbial species resist cultivation under laboratory condition. This is critical impediment for both academic and applied microbiology, and thus clarification of the mechanism of microbial uncultivability is highly demanded. Several evidences have been reported that to cultivate microbes in a membrane-bound device incubated in nature (in situcultivation) is an effective approach. However, the mechanism behind this approach has not been clarified. The present study shows the evidence that 1) initiating growth is a key for cultivating previously uncultivated microbes rather than simple growth promotion, and 2) growth initiation factor (signaling-like compounds) in natural environments stimulate microbial resuscitation from a nongrowing state. Since no study has focused on growth initiation for cultivation of previously uncultivated microorganisms, the discovery shown in the present study provides a new insight into microorganisms previously considered uncultivable and a microbial growth controlling system in nature.