Exploring active chemolithoautotrophic microorganisms thriving at deep-sea hydrothermal vent chimney structures in the Mid-Okinawa Trough by using RNA-based microbial community analysis and a new culture method.

概要

Since the discovery of a deep-sea hydrothermal field in 1977, hydrothermal
chimneys have attracted much attention as a research target due to the diversity of their
microbial communities, their potential as an analogue for the sub-seafloor environment
and as a mineral resource. Physiological ecology and phylogeny of chimney
microorganisms have been studied using a combination of culture-dependent and cultureindependent methods, but there are some challenges due to methodological issues. For
example, it is technically difficult to extract microbial RNA, which is essential for
analyzing microbial activity in deep-sea hydrothermal chimneys using cultureindependent methods, and biogeographical studies that require numerous isolates remain
laborious because there is no consideration of the solid media incubation technique for
efficient isolation of deep-sea hydrothermal vent living microorganisms.
In chapter 2, I assessed RNA extraction methods for deep-sea vent chimneys that
had complex mineral compositions. Mineral-RNA adsorption experiments were
conducted using mock chimney and Escherichia coli total RNA solution and showed that
detectable RNA significantly decreased possibly due to adsorption onto minerals. This
RNA decrease was prevented by adding sodium tripolyphosphate (STPP),
deoxynucleotide triphosphates (dNTPs), salmon sperm DNA, and NaOH. The addition
of STPP was also effective for RNA extraction from the mixture of E. coli cells and mock
chimney minerals when TRIzol reagent and the RNeasy column were used. A
combination of STPP, TRIzol reagent, the RNeasy column, and sonication resulted in the
highest RNA yield from a natural chimney sample. This new method may extend
analytical methods for microbial communities within deep-sea hydrothermal vent
chimneys, and thus may further our understanding of microbial activities in deep-sea
hydrothermal fields.
In chapter 3, first, a simple and efficient solid media cultivation method for
hydrogen- / sulfur-oxidizing microorganisms that are dominant in deep-sea hydrothermal
vents was investigated. Combination of pouch bags, oxygen absorbent, and H2/CO2
mixture gas, I succeeded in solid media cultivation of hydrogen- / sulfur-oxidizing
microorganisms, including that had never reported colony formation on solid media. ...