Development of a quantitative real‐time PCR assay for direct detection of growth of cellulose‐degrading bacterium Clostridium thermocellum in lignocellulosic degradation

AIMS: Currently, there is no direct method for detecting Clostridium thermocellum in the insoluble medium. In this study, a quantitative real‐time PCR assay was developed for the direct growth detection of C. thermocellum at the single‐cell level in lignocellulosic biomasses. METHODS AND RESULTS: Th...

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Bibliographic Details
Published in:Journal of applied microbiology 2015-06, Vol.118 (6), p.1333-1344
Main Authors: Tang, H, Ou, J.F, Zhu, M.J
Format: Article
Language:English
Subjects:
Bacteria
Bacterial Proteins - genetics
Bioassays
Biodegradation
Biomass
cipA gene
Clostridium thermocellum
Clostridium thermocellum - genetics
Clostridium thermocellum - growth & development
Clostridium thermocellum - metabolism
coculture
detection
detection limit
Fermentation
genes
Lignin - metabolism
lignocellulose
lignocellulosic biomass
Microbiology
Molecular Sequence Data
Polymerase chain reaction
quantitative polymerase chain reaction
quantitative real‐time PCR
Real-Time Polymerase Chain Reaction - methods
Reproducibility of Results
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Summary:AIMS: Currently, there is no direct method for detecting Clostridium thermocellum in the insoluble medium. In this study, a quantitative real‐time PCR assay was developed for the direct growth detection of C. thermocellum at the single‐cell level in lignocellulosic biomasses. METHODS AND RESULTS: The assay targeted the cipA gene and was able to distinguish C. thermocellum from other species with good reproducibility which quantitative detection limit was 10 cell equivalents (CE) per reaction. OD600‐based counting and qPCR quantification of C. thermocellum cultured in soluble medium were compared and an excellent consistency was revealed, indicating the appropriateness of the developed qPCR method. Analysis based on yellow affinity substrate and fermentation products may incorrectly estimate its population. CONCLUSIONS: The developed assay can serve as a specific, sensitive and reproducible method for the detection of C. thermocellum in lignocellulosic biomass at the single‐cell level. SIGNIFICANCE AND IMPACT OF THE STUDY: With the ability to rapidly detect C. thermocellum, this method will contribute substantially to the understanding of the lignocellulosic biomass degradation mechanism. Moreover, it can also be applied to detect C. thermocellum growth in certain co‐culture system for the understanding of the metabolic interactions.
ISSN:1364-5072
1365-2672
DOI:10.1111/jam.12801