Microbi fastidiosi: coltivazione di non coltivabili (FASMIC)

Responsabili di progetto

Mikhail Iakimov, Elizaveta Bonch-osmolovskaya

Accordo

RUSSIA - RAS old - Russian Academy of Sciences old

Bando

CNR/RAS 2011-2013

Dipartimento

Terra e Ambiente

Area tematica

Scienze del sistema Terra e tecnologie per l'ambiente

Stato del progetto

Nuovo

Proposta di ricerca

The recent application of molecular phylogeny to environmental samples has resulted in the discovery of an abundance of unique and previously unrecognized microorganisms. Using this technique and 454 pyrosequencing, the number of existing microbial species is now estimated at 105 to 106, but only several thousand have been isolated in pure culture, because few microorganisms from environmental samples grow on nutrient media using conventional methods of cultivation. This number means that almost of 99,9% of prokaryotic genomic information is hidden for us and cannot be seen and utilized for any kind of biotechnological application. Attempts to improve the recovery of microorganisms from environmental samples by manipulating growth media have met with limited success, and the problem of uncultivability remains a major challenge. It is clear, that the cultivation of uncultured will enhance our understanding of microbial physiology and metabolic adaptation and will provide new sources of microbial metabolites for medical and biotechnological applications.
In the frame of proposed bilateral collaboration we are planning to apply and validate a sophisticated cultivation approach that provides access to this immense reservoir of untapped microbial diversity. This technique, developped in laboratory of Prof. E.A. Bonch-Osmolovskaya (Russian partner of the FASMIC Project), combines enrichment of cells in a fermenter simulating the environmental conditions followed by isolation of pure cultures using a diffusion chamber. The ability to grow and study of previously uncultured organisms using this method was successfully demonstrated by isolation of members of deeply-branched Thermotoga division, two novel orders of themophilic Crenarchaeota, novel phylum Caldothrix, etc. Recently, the new type of metabolism (the conversion of formate and water to bicarbonate and H2) was demonstrated by this laboratory in pure culture of hyperthermophilic archaea belonging to the genus Thermococcus (Nature 2010, 467(7313):352-5).
The aim of FASMIC project is to show that this technology can be applied to samples from several different environments, including (i) shallow hydrothermal vents and (ii) deep anoxic hypersaline lakes, both ecosystems located in Mediterranean Sea.
(i) Shallow hydrothermal systems of Panarea Island, Mediterranean Sea.
Microorganisms inhabiting shallow hydrothermal vents possess nutritional requirements and overall metabolic pathways ideally suited to such ecosystem that represents a clear example of the close connection between geosphere and biosphere. Isolation efforts of Bacteria and Archaea from thermal vents were often unsuccessful mostly due to the difficulties in reproducing the complex geochemical composition of the environment in growth media. This results in a limited knowledge of the real microbial metabolic diversity.
The most active Eolian submarine hydrothermal system is located off the coast of Panarea Island, where several gaseous emissions spread over the sea bottom up to a depth of 150 m. The island of Panarea is an example of volcanic edifice of Aeolian arc in the Southern Tyrrhenian Sea, Italy. Immediately off the islands there is a geothermal field of 4 km2, whose hydrothermal emissions have been observed since historical times. Indeed, old reports talk about a so-called “bollitore,” namely boiling water at the sea surface. Over the last two decades such manifestations have been limited to weak gas bubbling that could only be seen when the sea was very calm. The water in this zone exhibits typical composition of hydrothermal fluids suffering steam condensation and removal of acidic species (CO2 generally exceeds 95mol%, the rest being CO, CH4, H2, N2 and H2S in variable proportion). Such chemistry could support the chemolithoautotrophical style of life and, indeed, several aerobic and anaerobic, thermophilic, and hyperthermophilic microorganisms belonging to Bacteria and Archaea domains were detected at this site using molecular, culture-independent, techniques. In spite of observed vast diversity of prokaryotes, inhabiting Panarea hydrothermal vents, there are very few cultivable autotrophic hyperthermophilic prokaryotes known.
(ii) Deep-sea hypersaline anoxic lakes at the seabed of Eastern Mediterranean Sea
Unique the world's deepest and most hypersaline anoxic lakes (DHAL) found on depressions on the seafloor of the Eastern Mediterranean Sea, are resulted from complex geological processes that involve global and regional tectonics. Physical-chemical conditions in DHALs are very hostile to majority of known living organisms. A number of very initial attempts gained valuable and promising background information on the astonishing diversity in these extreme environments of novel and unique autochthonous pro- and eukaryotic organisms, called MSBL candidate divisions (MedSea Brine Lake), suggesting the existence of a very specific microbiota, highly adapted to thrive under anoxic conditions at elevated pressure and salinity. At the moment they are fifteen of such divisions, members of which were recovered either from similar environments or exclusively from MedSea DHABs. Taxonomic decomposition indicates the high abundance of members of Euryarchaeota related to MSBL1 division, members of Thermotoga and Halanaerobacter. None of them have ever been obtained and maintained in laboratories, thus, their metabolic features and roles in their natural ecosystems are still unknown.

Obiettivi della ricerca

The main objectives of FASMIC are to sample two extreme environments located in Mediterranean Sea and along with molecular characterization of microbial diversity to perform an enrichment and isolation of yet uncultured goups of thermo- and halophilc microorganisms.
(i)  Panarea Island, Mediterranean Sea.
Study the microbial communities thriving at two shallow hydrothermal vents characterised by different depth, temperature and chemical composition. Water and sediment samples will be collected close to the fluid emissions and initially characterized by conventional molecular and hydro-chemical techniques followed by the enrichment and isolation attempts performed with in situ fermenter. Special emphasis will be given to isolation of hyperthermo- and acidophilic prokaryotes.
(ii) DHALs at the seabed of Eastern Mediterranean Sea
Two different DHALs will be studied, thalassohaline lake Thetis, filled with saturated halite (5M of NaCl) and athalassohaline lake Kryos, filled with almost saturated bischoffite (4M of MgCl2). Both lakes were found in 2008 at the depth of 3500m by Italian partner of FASMIC project (Laboratory of Dr. M. Yakimov IAMC-ME, CNR). Brine and sediment samples will be collected from these lakes and initially characterized by conventional molecular and hydro-chemical techniques. Isolation attempts will be performed with special emphasis  to obtain cultivable members of Thermotoga-related KB1 division, the deepest branching lineage between Archaeota and Eubacteria.

Ultimo aggiornamento: 04/05/2024