THESE Anne POSTEC Diversité de populations microbiennes ...

Résultats - Chapitre 3
while the number of clones related to Caminicella decreased from 50% to 7%. Only two
phylotypes were recovered in the T28 bacterial library: (i) 12 clones were closely related to
Thermosipho MV1063; the sequence type 840 shared 99% identity with this strain and the
closest relative species was T. melanesiensis (96% identity) and (ii) 3 clones were affiliated
to the genus Thermodesulfatator and the sequence type 850 shared 96% identity with T.
indicus.
DGGE and whole-cell hybridization analysis from the enrichment culture in bioreactor
As bacterial diversity was detected in the enrichment culture in the bioreactor by examining
the clone distribution among T7 and T28 libraries, it was further investigated with DGGE
analysis. The v3 hypervariable region of the bacterial 16S rDNA could be amplified using
DGGE primers from the following culture samples: T1, T4, T9, T13, T16, T24, T28, T31 and
T36. No amplification could be obtained from samples collected after T36, probably due to
the low cell density (1.8 10 7 cell ml -1 at T41). Representative DGGE bands corresponding to
various samples and displaying different migration distances were extracted from gels (not
shown) and could be successfully re-amplified directly with the DGGE primers used
previously. Sequences retrieved from representative bands were affiliated with phylogenetic
groups yet represented in the T7 and T28 bacterial clone libraries (Table 1). DGGE bands
corresponding to sequence types (approximately 500 pb) closely related to the strain
Thermosipho MV1063 (99% identity) were obtained from T1 to T36 samples, Marinitoga
camini (96%) was obtained from T31 sample, Caminicella sporogenes (99-98%) were
obtained from T4 to T31 samples, and Deferribacter abyssi (87%) were obtained from T1 to
T31 samples (Table 2). The relative proportions of archaeal and bacterial cells were
determined in eleven culture samples of the bioreactor, from T2 to T41 (Fig. 1).
Approximately 2000 cells were counted on filter for each sample. 99.0% of the cells detected
at T2 belonged to the Archaea ; Bacteria were dominant from T4 to T31 (between 94.8 to
99.8% until T24, then 60.0% at T28 and 54.6% at T31) (Fig. 3). At T36 and T41, Archaea
became predominant again, representing 61.6% of the cells at T36 and 98.8% of the cells at
T41. Temperature increased to 70°C at T31 and to 80°C at T36 and was associated with a
significant decrease in the cell density (2.2 10 9 cell ml -1 at T31 and 1.8 10 7 cell ml -1 at T41).
16S rDNA libraries from the enrichment cultures in vials
Total DNA was successfully extracted from A1, A2 and B culture samples. Archaeal 16S
rDNA could be amplified by PCR only from sample A1, corresponding to the primary
enrichment culture after 24 h of incubation. No archaeal 16S rDNA sequences could be
amplified neither after 41 h of culture (A2), nor after subculturing from A1 (B). All 55 archaeal
clones from the A1 library were related to the genus Thermococcus (Table 3). The sequence
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