THESE Anne POSTEC Diversité de populations microbiennes ...

Résultats - Chapitre 3
µm) occurring singly or in pairs, and occasionally elongated, mixed with Thermotogales-like
cells. The detection of H 2 S in the gas phase of a medium containing sulfate and lacking
sulfur as electron acceptor strongly suggests the growth of a sulfate-reducing microorganism.
A partial 16S rDNA sequence was obtained from this culture and shared 96% of similarity
with the one of Thermodesulfatator indicus.
Positive cultures were also obtained on the medium DS containing sulfur. The culture was
successfully subcultured several times on the DS medium, and then several times on the DN
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medium depleted in sulfur and containing NO 3 as terminal electron acceptor. The
morphologies observed included few cocci and dominant motile, straight- to vibrio-shaped
organisms, detected in both media. Two strains were identified by 16S rDNA sequencing and
were affiliated to the hydrothermal species Deferribacter abyssi (Miroshnichenko, et al.
2003b) (99% of identity) probably corresponding to the rod-vibrio shaped cells, mixed with a
Thermococcus sp. strain probably corresponding to the coccoid cells.
Discussion
To access the cultivable microbial community inhabiting a hydrothermal black smoker,
enrichment cultures were performed in a bioreactor and in batch vials at 60°C and pH 6.5.
Molecular techniques based on the 16S rRNA gene gave snapshot images of the microbial
diversity examining the clone libraries and regular data on the microbial community structure
through the use of DGGE and whole-cell hybridization.
Biases inherent to molecular techniques must be kept in mind. However, the molecular
monitoring of the continuous culture in bioreactor showed that sequences closely related to
Thermosipho MV1063 could be detected by DGGE at T7 and were not retrieved by the
cloning method. Moreover, 29 and 35 16S rDNA sequences related to Marinitoga camini
were recovered respectively from the T7 and T28 clone libraries, and the corresponding
strain could be isolated from T7. However, only one sequence related to M. camini could be
detected by the DGGE analysis from the T31 sample. Also, no DGGE sequence related to
Thermodesulfatator was detected. The use of different molecular methods is therefore
complementary. The possible limitations of primer selectivity and cloning biases (Theron &
Cloete 2000) or PCR biases (Suzuki & Giovannoni 1996) are well established and can
explain the differences between results issued from various methods. Indeed, clone
abundance in a library does not necessarily represent the rDNA sequence abundance in the
corresponding nucleic acid extracts. Yet molecular methods generally do detect the
numerically dominant phylotype (Head et al. 1998).
Before discussing the results of molecular analysis and subcultures/isolations in vials from
culture samples of the bioreactor, it should be noted that the dilution rate applied after 34 h of
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