Accessing previously uncultured marine microbial resources by a combination of alternative cultivation methods.

Few microbes can grow under laboratory conditions, highlighting the fact that the majority of microbes in environment are still uncultured and untapped resources. This study used alternative cultivation methods, diffusion chambers (DC), dilution-to-extinction culture (DTE) and modified agar preparation step (PS media) to cultivate previously uncultured marine bacterial species. These methods were applied to samples from a coastal intertidal zone, and the results were compared with those from standard direct plating (SDP) cultivation. Among the strains isolated with DC, DTE and PS media methods, 28%, 48% and 33% were novel species, respectively, while the SDP method resulted in the isolation of only 9% of novel species. Most isolates were unique to the method used for their cultivation. This implies that each method is selective in its own way, which is different from SDP, thus able to access species that are difficult to obtain using conventional approaches. Comparing the diversity showed that 75 genera were recovered by the alternative methods, 2.7 times higher than that of the SDP cultivation, which constituted 45% of total diversity from culture-independent sequencing. We conclude that combining alternative cultivation methods represents a highly promising key for accessing 'microbial dark matter'.