title: Rapid and qualitative fluorescence-based method for the assessment of PHA production in marine bacteria during batch culture.
abstract: The expansion of polyhydroxyalkanoates (PHAs) into the biodegradable polymers market is mainly prevented by their production process which is still complicated with a low efficiency, resulting in relatively expensive products. In this study, we developed a method that used the lipophilic fluorescent probe Nile Red (1 mg l(-1) solution in DMSO) directly into the culture broth to stain the PHA inclusions inside bacterial cells followed by detection of the emitted fluorescence by both microscopic and spectrometric techniques. Epifluorescence microscopy provides a rapid tool to distinguish producing from non-producing bacterial species and the relative fluorescence intensity (FI) determined at the maximum of emission spectra in the wavelength region of 560-710 nm (λ(ex): 543 nm), allows a fast assessment of the cultural conditions that may enhance PHA production yield. During two-step cultivation in 500-ml flasks with glucose as the sole carbon source, the method aimed to select bacterial strains efficient for PHA synthesis among a marine collection. Subsequently, the NR assay was used to determine the C0/N0 ratio of the producing media that may improve the polymer yield as well as to follow the time course of fermentation. Characterization by GC-MS and DSC confirmed the production of the P(3-HB) homopolymer.
authors.affiliation: Laboratoire d'Ingénierie des Matériaux de Bretagne (LIMATB), Université de Bretagne-Sud, Rue de Saint Maudé, 56321, Lorient Cedex, France. anne.elain@univ-ubs.fr.
authors.forename: Anne
authors.initials: A
authors.lastname: Elain
authors.affiliation: Laboratoire d'Ingénierie des Matériaux de Bretagne (LIMATB), Université de Bretagne-Sud, Rue de Saint Maudé, 56321, Lorient Cedex, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Le Fellic
authors.affiliation: Laboratoire d'Ingénierie des Matériaux de Bretagne (LIMATB), Université de Bretagne-Sud, Rue de Saint Maudé, 56321, Lorient Cedex, France.
authors.forename: Yves-Marie
authors.initials: YM
authors.lastname: Corre
authors.affiliation: Laboratoire d'Ingénierie des Matériaux de Bretagne (LIMATB), Université de Bretagne-Sud, Rue de Saint Maudé, 56321, Lorient Cedex, France.
authors.forename: Adélaïde
authors.initials: A
authors.lastname: Le Grand
authors.affiliation: Laboratoire d'Ingénierie des Matériaux de Bretagne (LIMATB), Université de Bretagne-Sud, Rue de Saint Maudé, 56321, Lorient Cedex, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Le Tilly
authors.affiliation: UMR CNRS, Institut des Sciences Chimiques de Rennes, Université Rennes 1, 263 Avenue du Général Leclerc, CS74205, Case 1011, 35042, Rennes Cedex, France.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Audic
authors.affiliation: Laboratoire d'Ingénierie des Matériaux de Bretagne (LIMATB), Université de Bretagne-Sud, Rue de Saint Maudé, 56321, Lorient Cedex, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Bruzaud
mesh_terms: D059001:Aquatic Organisms / Q000378:metabolism*; D001419:Bacteria / Q000378:metabolism*; D061249:Batch Cell Culture Techniques; D002244:Carbon / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis; D005470:Fluorometry / Q000379:methods*; D008401:Gas Chromatography-Mass Spectrometry; D005947:Glucose / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D010078:Oxazines / Q000032:analysis; D054813:Polyhydroxyalkanoates / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D010078:Oxazines; D054813:Polyhydroxyalkanoates; D002244:Carbon; D005947:Glucose; C044808:nile red
keywords: Batch culture; Fluorescent probe; Marine bacteria; Nile Red dye; Polyhydroxyalkanoates granules