title: Robust at-line quantification of poly(3-hydroxyalkanoate) biosynthesis by flow cytometry using a BODIPY 493/503-SYTO 62 double-staining.
abstract: 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 <10min. 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).
authors.affiliation: University of Applied Sciences and Arts Western Switzerland (HES-SO Valais), Institute of Life Technologies, Route du Rawyl 47, 1950 Sion, Switzerland; ETH Zurich, Department of Biosystems Science and Engineering, Mattenstrasse 26, 4058 Basel, Switzerland.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Karmann
authors.affiliation: University of Applied Sciences and Arts Western Switzerland (HES-SO Valais), Institute of Life Technologies, Route du Rawyl 47, 1950 Sion, Switzerland.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Follonier
authors.affiliation: University of Applied Sciences and Arts Western Switzerland (HES-SO Valais), Institute of Life Technologies, Route du Rawyl 47, 1950 Sion, Switzerland.
authors.forename: Monica
authors.initials: M
authors.lastname: Bassas-Galia
authors.affiliation: ETH Zurich, Department of Biosystems Science and Engineering, Mattenstrasse 26, 4058 Basel, Switzerland.
authors.forename: Sven
authors.initials: S
authors.lastname: Panke
authors.affiliation: University of Applied Sciences and Arts Western Switzerland (HES-SO Valais), Institute of Life Technologies, Route du Rawyl 47, 1950 Sion, Switzerland. Electronic address: manfred.zinn@hevs.ch.
authors.forename: Manfred
authors.initials: M
authors.lastname: Zinn
mesh_terms: D001419:Bacteria / Q000378:metabolism; D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D001896:Boron Compounds; D002244:Carbon / Q000378:metabolism; D002452:Cell Count / Q000379:methods; D003470:Culture Media; D005285:Fermentation; D005434:Flow Cytometry / Q000379:methods*; D011091:Polyesters / Q000032:analysis*; D016958:Pseudomonas putida / Q000254:growth & development / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000254:growth & development / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
chemical_list: C527198:4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene; C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D003470:Culture Media; D011091:Polyesters; D002244:Carbon
keywords: BODIPY 493/503; Flow cytometry; PHA; Pseudomonas putida; Quantification; Rhodospirillum rubrum