Robust at-line quantification of poly(3-hydroxyalkanoate) biosynthesis by flow cytometry using a BODIPY 493/503-SYTO 62 double-staining

Karmann, Stephanie (School of Engineering, HES-SO Valais-Wallis, HEI, HES-SO // University of Applied Sciences Western Switzerland ; ETH Zurich, Basel, Switzerland) ; Follonier, Stéphanie (School of Engineering, HES-SO Valais-Wallis, HEI, HES-SO // University of Applied Sciences Western Switzerland) ; Bassas-Galia, Monica (School of Engineering, HES-SO Valais-Wallis, HEI, HES-SO // University of Applied Sciences Western Switzerland) ; Panke, Sven (ETH Zurich, Basel, Switzerland) ; Zinn, Manfred (School of Engineering, HES-SO Valais-Wallis, HEI, HES-SO // University of Applied Sciences Western Switzerland)

Poly(3-hydroxyalkanoates) (PHAs) are bio-based and biodegradable polyesters which have been considered as a promising alternative to petrol-based plastics. Their bacterial production is a dynamic process in which intracellular polymerization and depolymerization are closely linked and depend on the availability of carbon substrates and other nutrients. These dynamics require a fast and quantitative method to determine the optimal harvest-time of PHA containing cells or to adjust carbon supply. In principle, flow cytometry (FCM) is an ideal tool that suits these requirements and, in addition, provides data on the PHA content of different cell populations. However, FCM-based PHA quantification methods have often relied on laborious sample preparation including washing steps and long incubation times. Here, we introduce a fast method based on double-staining using BODIPY 493/503 for PHA staining and SYTO 62 for DNA that allows acquiring reliable fluorescence and cell count data in < 10 min. Finally, fed-batch experiments with Pseudomonas putida KT2440 and Rhodospirillum rubrum S1 revealed that the method was robust and independent of the strain and type of PHA (medium-chain-length [mcl-] and short-chain-length [scl-] PHA, respectively). Interestingly, the specific PHA fluorescence was in case of mcl-PHA larger than for scl-PHA, probably reflecting the different material properties (e.g., specific density, hydrophilicity and crystallinity).


Article Type:
scientifique
Faculty:
Ingénierie et Architecture
School:
HEI-VS
Institute:
Institut Technologies du vivant
Date:
2016-12
Pagination:
6 p.
Published in:
Journal of Microbiological Methods
Numeration (vol. no.):
2016, vol. 131, pp. 166-171
DOI:
ISSN:
01677012
Appears in Collection:

Note: The status of this file is: restricted


 Record created 2021-06-18, last modified 2021-07-05

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