THESE Anne POSTEC Diversité de populations microbiennes ...

Résultats - Chapitre 3
Introduction
It has been established that microbial diversity could be explored by application of molecular
biology (Pace et al. 1986). In this respect, organisms could be identified without cultivation by
retrieving and sequencing macromolecules from nature, many technologies being based on
the molecular phylogeny of rRNA sequences. The widespread application of 16S rRNA gene
cloning and sequencing methods to identify microorganisms in natural samples has revealed
an extensive and, in many cases, unsuspected microbial diversity. Within deep-sea
hydrothermal environments, the microbial communities associated with in situ colonizers
(Alain et al. 2004), sediments (López-García 2003), chimneys (Schrenk et al. 2003), animals
(Alain et al. 2002a), or fluids (Huber et al. 2003) was reported in recent molecular surveys.
The rise of molecular microbial ecology has resulted in new knowledge about
microorganisms that have never been cultured. Indeed, Marine Crenarchaeota Group I (MGI)
(the most abundant and widely distributed Archaea in the global ocean biosphere) (DeLong
1992), Deep-sea Hydrothermal Vent Euryarchaeota (DHVE) groups I and II (Takai &
Horikoshi 1999) and Korarchaeota (Barns et al. 1994) still remain to be cultivated.
Consequently, their physiological features and ecological significance are still unclear.
Beside molecular techniques, classical cultural approaches are necessary to be improved to
better assess the microbial diversity of ecosystems as growth conditions routinely used only
revealed a small fraction of the global microbial community.
As an alternative to batch cultures in vials, a gas-lift bioreactor was developed to grow
anaerobic hyperthermophilic microorganisms (Raven et al. 1992). The bioreactor permits a
continuous substrate supply, elimination of the volatile metabolic end-products by N 2
sparging, pH and temperature regulation, and incubation for several weeks. It has been used
especially to investigate the metabolism of members of the Thermococcales including
Pyrococcus abyssi (Godfroy et al. 2000) and to develop minimal media and to optimize the
growth conditions of Pyrococcus furiosus and Thermococcus hydrothermalis (Postec et al.
2005a, Raven & Sharp 1997). Beside the study of pure culture, studying enrichment cultures
using a gas-lift bioreactor under controlled conditions might be helpful to detect
microorganisms poorly represented, exhibiting a long latency phase, or otherwise not
previously cultivated. In a previous study (Postec et al. 2005b), the gas-lift bioreactor was
used to enrich microorganisms from a black smoker collected at a depth of 2275 m on the
Rainbow hydrothermal field (Mid-Atlantic Ridge). The 50-day continuous culture was
performed at 90°C, on an rich medium containing sulfur under anaerobic conditions. In these
conditions, a large microbial diversity was detected, including hyperthermophilic members
but also moderatly thermophilic members of the orders Clostridiales and Thermotogales, and
members of the epsilon-Proteobacteria, that were not retrieved from cultures in vials. In the
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