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Fluorescent in situ hybridisation; Artificial consortium; Quantitative PCR
Abstract :
[en] The use of an artificial consortium composed of selected hydrogen-producing species,
instead of a natural anaerobic sludge, has been proposed for biohydrogen production. In
order to monitor such a consortium composed of different Clostridium spp., new protocols
were tested for two different assays, FISH and qPCR. New species-specific FISH probes and
qPCR primer sets were developed and optimised for three strains: Clostridium butyricum,
Clostridium felsineum and Clostridium pasteurianum, that were used in a consortium. Application
of a fast two-step FISH protocol, with pre-treatment step at 90 C for 5 min and
a subsequent hybridisation step at higher temperature (55 C) for 20 min resulted in a much
shorter analytical time compared to the standard FISH procedure (46 C for 2e3 h) and gave
a high hybridisation performance. Moreover, to accurately quantify each microorganism by
qPCR assay, two innovations were tested: the direct use of cell lysates (omitting the DNA
extraction step) and the use of two alternative molecular markers, recA and gyrA. These
markers are present in single copies in the genome, whereas there are multiple copies of
the ribosomal operons. This resulted in the development of accurate, reliable and fast FISH
and qPCR assays for routine monitoring of the dynamics of artificial hydrogen-producing
microbial consortia. Moreover, both techniques can be easily adapted to new Clostridium
strains.
Research Center/Unit :
CIP - Centre d'Ingénierie des Protéines - ULiège
Disciplines :
Microbiology
Author, co-author :
Savichtcheva, Olga
Joris, Bernard ; Université de Liège - ULiège > Département des sciences de la vie > Physiologie et génétique bactériennes
Wilmotte, Annick ; Université de Liège - ULiège > Département des sciences de la vie > Physiologie et génétique bactériennes
Calusinska, Magdalena ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Language :
English
Title :
Novel FISH and quantitative PCR protocols to monitor artificial consortia composed of different hydrogen-producing Clostridium spp.
Alternative titles :
[fr] Protocoles nouveaux de FISH et PCR quantitative pour suivre un consortium artificiel de clostridies productrices d'hydrogène
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