Reference : Novel FISH and quantitative PCR protocols to monitor artificial consortia composed of...
Scientific journals : Article
Life sciences : Microbiology
Novel FISH and quantitative PCR protocols to monitor artificial consortia composed of different hydrogen-producing Clostridium spp.
[fr] Protocoles nouveaux de FISH et PCR quantitative pour suivre un consortium artificiel de clostridies productrices d'hydrogène
Savichtcheva, Olga mailto [ > > ]
Joris, Bernard mailto [Université de Liège - ULiège > Département des sciences de la vie > Physiologie et génétique bactériennes >]
Wilmotte, Annick mailto [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 >]
International Journal of Hydrogen Energy
Pergamon Press - An Imprint of Elsevier Science
Yes (verified by ORBi)
[en] Fluorescent in situ hybridisation ; Artificial consortium ; Quantitative PCR
[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
Centre d'Ingénierie des Protéines - CIP
Communauté française de Belgique - CfB
Microbiological production of hydrogen: study of microalgal and bacterial processes
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