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

Abstract

Most 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.

Cultivation of previously uncultured sponge-associated bacteria using advanced cultivation techniques: A perspective on possible key mechanisms

“…The ratio was calculated using the average value from triplicate measurements (n=3) of the CFUs on each agar plate. Data for SDP strains have already been reported (Jung et al, 2021).…”

“…We refer to isolates from SDP cultivation performed in the same series of experiments (Jung et al, 2021) using the same sample. SDP cultivation resulted in isolates representing 13 species from three taxonomic groups (Bacteroidetes, Alphaproteobacteria and Gammaproteobacteria; Figure 3, Table S3).…”

“…Specific growth rate and saturated cell density of CF (continuous-flow bioreactor; filled circle), I-tip (cross), and SDP (standard direct plating; open circle) strains. Data for specific growth rate and saturated cell density of SDP strains have already been reported (Jung et al, 2021).…”

“…Previous studies using in situ cultivation methods aiming to better simulate the natural environment in a variety of environments such as activated sludge, sediments, soils, alkaline soda lakes, sponges, and thermal springs, have shown success for isolating novel microbes (Bollmann et al, 2007;Aoi et al, 2009;Jung et al, 2013;Jung et al, 2014;Jung et al, 2016;Jung et al, 2018;Chaudhary et al, 2019). Recently we clarified some of the principles of in situ cultivation methods including the presence of growth initiation factors (signaling-like compounds) in natural environments that can stimulate bacterial resuscitation from a nongrowing state (Jung et al, 2021).…”

Ecological and biotechnological aspects of Aplysina-associated microorganisms

“…The sponge holobiont evolved in intricate association with microorganisms and numerous modes of interaction have been identified that mediate between microorganisms and their sponge host. For example, sponge extract seems to contain chemical compounds that facilitate bacterial starvation recovery (Jung et al, 2019). Proteins involved in eukaryote-prokaryote interactions such as eukaryotic-like proteins (ELPs), ankyrin repeats (ANKs) and tetratricopeptide repeats (TPRs) are regularly detected in sponge-associated bacteria and have been shown to modulate phagocytosis by amoebal cells (Nguyen et al, 2014;Reynolds & Thomas, 2016).…”