Development of an effective method for isolating and culturing
non-cultureable microbes using a specialized cellulose sheet
Hideki AOYAGIFaculty of Life and Environmental Sciences, University of Tsukuba
Research aims:
In recent years, it has been determined that the conventional method for culturing
microbes (agar plate culture method) can cultivate less than 1% of microorganisms existingin nature1), and its limitations have been pointed out. The remaining 99% of uncultured
microorganisms are called “microbial dark matter”2). These uncultured microorganisms areexpected to be used in various strata of research and industry. So, a comprehensive,
world-wide environmental genome analysis is being carried out. If the remaining 99% of
microorganisms can be cultured and their specific attributes can be efficiently applied, thenthey may solve current challenges. Base sequence information of microbial genomes is
helpful in the prediction of microbial functions, although it is always necessary to confirm
simulated predictions via wet lab experiments of the isolation and cultivation of uncultured
microorganisms (dark matter microbes). Against this background, various culture methods
have been developed but have not been generally and widely used.
In this study, a culture substratum was developed using a specialized cellulose sheet(CS-SCS) for the isolation and cultivation of uncultured
microorganisms in attempt to eliminate the problems ofthe conventional agar plate culture method. Applicationof the CS-SCS led to the isolation and cultivation of
uncultured microorganisms from various environmental
samples; the successful growth of uncultured microbes
demonstrated the practicability and flexibility of theCS-SCS.
Methods:
Various environmental samples isolated from soil, freshwater, seawater and plants
were treated appropriately, and cultured at 25 °C for about 2 weeks using the CS-SCS (Fig.
1) and a conventional agar plate medium. The extracted DNA of the cultured
microorganisms was analyzed using the denaturing gradient gel electrophoresis(PCR-DGGE) method and compared comprehensively using next-generation sequencing.

The grown colonies were randomly isolated, and the entire length of the 16S rDNA wasamplified by direct colony PCR. Sequence analysis was performed to identify the isolated
microorganism species.
An attempt was also made to use the CS-SCS under acidic cultivation conditions
where it was difficult to use an agar plate.
Results:
Cultivation of various microbes using theCS-SCS were performed to optimize the specification of
the CS-SCS. Soil samples collected at the University ofTsukuba were cultivated on the CS-SCS and agar platemedium. Any cultured microbial communities werecompared using the PCR-DGGE method. The results
indicated that the cultured microbial community wasdistinct in the CS-SCS compared to the agar plate medium(Fig. 2). These results suggested the applicability of theCS-SCS in the isolation and cultivation of novel
microorganisms. A similar trend was observed in studies
using environmental samples such as those isolated from
freshwater, seawater, etc. The soil samples were cultured on the CS-SCS and agar plate
media using R2A medium; later, the cultured microbial communities and the existing
microbial communities in the soil samples were analyzed using a next-generation sequencer.
These results showed that compared to the agar plate medium, the CS-SCS supported acultured microbial community with a structure close to that of a soil sample.
While culturing various samples from plants (Wasabia japonica and Chamaecyparisobtuse, etc.) and using the CS-SCS and an agar plate medium, it was found that the cultured
microorganism communities obtained by the CS-SCS and the agar plate medium weregreatly different (as per the result of statistical analysis, the similarity was low among theobtained microorganisms). Some novel microorganisms (less than 97% of 16S rDNA) could
be isolated and cultivated using the CS-SCS.
The CS-SCS was further explored for its applicability under acidic conditions
where agar plates cannot be used. The elastic value of the agar plate medium, in which
Escherichia coli can maintain the shape of the colony, was measured (the limiting elasticvalue was 27.8 kPa). Incubation of agar plates with various pH values and the change inelasticity over time showed that the agar plates with pH 1 could be used within 3 d ofcultivation. The comparison of cultivation on the agar plate medium and the CS-SCS withpH <3.0 revealed a great variation among the cultured microbial communities. The CS-SCScould withstand the strong acidic conditions and supported the cultivation of acidophilic
microorganisms. Thus, novel acid-resistant microorganisms could be isolated and cultured
from environmental samples using the CS-SCS under pH 1.
Conclusion
The culture substratum developed in this study (CS-SCS) could solve the problemsof conventional agar plate medium. The CS-SCS was found to be effective for the isolationand cultivation of novel microorganisms. The CS-SCS could be generally and widely used
and is expected to acquire novel microbial resources.
References1) Amann, R. I., Ludwig, W., Schleifer, K. H. (1995) Phylogenetic identification and in situ
detection of individual microbial cells without cultivation. Microbiol. Rev. 59:143-169.
2) Ling, L. L. et al. (2015) A new antibiotic kills pathogens without detectable resistance.
Nature 517: 455-459.