Get access

Photobioreactor design for isotopic non-stationary 13C-metabolic flux analysis (INST 13C-MFA) under photoautotrophic conditions

Authors

  • Arnaud Martzolff,

    1. LUNAM Université, Université de Nantes, CNRS, GEPEA, UMR 6144, Bât. CRTT, 37 boulevard de l'Université, BP 406, F-44602 Saint-Nazaire Cedex, France; telephone: +33-2-40-17-26-11; fax: +33-2-40-17-26-18
    Search for more papers by this author
  • Edern Cahoreau,

    1. Université de Toulouse, INSA, UPS, INP, Toulouse, France
    2. INRA, UMR 792, Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France
    3. CNRS, Toulouse, France
    Search for more papers by this author
  • Guillaume Cogne,

    Corresponding author
    1. LUNAM Université, Université de Nantes, CNRS, GEPEA, UMR 6144, Bât. CRTT, 37 boulevard de l'Université, BP 406, F-44602 Saint-Nazaire Cedex, France; telephone: +33-2-40-17-26-11; fax: +33-2-40-17-26-18
    • LUNAM Université, Université de Nantes, CNRS, GEPEA, UMR 6144, Bât. CRTT, 37 boulevard de l'Université, BP 406, F-44602 Saint-Nazaire Cedex, France; telephone: +33-2-40-17-26-11; fax: +33-2-40-17-26-18
    Search for more papers by this author
  • Lindsay Peyriga,

    1. Université de Toulouse, INSA, UPS, INP, Toulouse, France
    2. INRA, UMR 792, Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France
    3. CNRS, Toulouse, France
    Search for more papers by this author
  • Jean-Charles Portais,

    1. Université de Toulouse, INSA, UPS, INP, Toulouse, France
    2. INRA, UMR 792, Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France
    3. CNRS, Toulouse, France
    Search for more papers by this author
  • Emmanuel Dechandol,

    1. LUNAM Université, Université de Nantes, CNRS, GEPEA, UMR 6144, Bât. CRTT, 37 boulevard de l'Université, BP 406, F-44602 Saint-Nazaire Cedex, France; telephone: +33-2-40-17-26-11; fax: +33-2-40-17-26-18
    Search for more papers by this author
  • Fabienne Le Grand,

    1. LUNAM Université, Université de Nantes, CNRS, GEPEA, UMR 6144, Bât. CRTT, 37 boulevard de l'Université, BP 406, F-44602 Saint-Nazaire Cedex, France; telephone: +33-2-40-17-26-11; fax: +33-2-40-17-26-18
    Search for more papers by this author
  • Stéphane Massou,

    1. Université de Toulouse, INSA, UPS, INP, Toulouse, France
    2. INRA, UMR 792, Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France
    3. CNRS, Toulouse, France
    Search for more papers by this author
  • Olivier Gonçalves,

    1. LUNAM Université, Université de Nantes, CNRS, GEPEA, UMR 6144, Bât. CRTT, 37 boulevard de l'Université, BP 406, F-44602 Saint-Nazaire Cedex, France; telephone: +33-2-40-17-26-11; fax: +33-2-40-17-26-18
    Search for more papers by this author
  • Jérémy Pruvost,

    1. LUNAM Université, Université de Nantes, CNRS, GEPEA, UMR 6144, Bât. CRTT, 37 boulevard de l'Université, BP 406, F-44602 Saint-Nazaire Cedex, France; telephone: +33-2-40-17-26-11; fax: +33-2-40-17-26-18
    Search for more papers by this author
  • Jack Legrand

    1. LUNAM Université, Université de Nantes, CNRS, GEPEA, UMR 6144, Bât. CRTT, 37 boulevard de l'Université, BP 406, F-44602 Saint-Nazaire Cedex, France; telephone: +33-2-40-17-26-11; fax: +33-2-40-17-26-18
    Search for more papers by this author

Abstract

Adaptive metabolic behavior of photoautotrophic microorganisms toward genetic and environmental perturbations can be interpreted in a quantitative depiction of carbon flow through a biochemical reaction network using isotopic non-stationary 13C-metabolic flux analysis (INST 13C-MFA). To evaluate 13C-metabolic flux maps for Chlamydomonas reinhardtii, an original experimental framework was designed allowing rapid, reliable collection of high-quality isotopomer data against time. It involved (i) a short-time 13C labeling injection device based on mixing control in a torus-shaped photobioreactor with plug-flow hydrodynamics allowing a sudden step-change in the 13C proportion in the substrate feed and (ii) a rapid sampling procedure using an automatic fast filtration method coupled to a manual rapid liquid nitrogen quenching step. 13C-substrate labeling enrichment was controlled through the total dissolved inorganic carbon concentration in the pulsed solution. First results were obtained from steady-state continuous culture measurements allowing the characterization of the kinetics of label incorporation into light-limited growing cells cultivated in a photobioreactor operating at the maximal biomass productivity for an incident photon flux density of 200 µmol m−2 s−1. 13C label incorporation was measured for 21 intracellular metabolites using IC-MS/MS in 58 samples collected across a labeling experiment duration of 7 min. The fastest labeling rate was observed for 2/3-phosphoglycerate with an apparent isotopic stationary state reached after 300 s. The labeling rate was consistent with the optimized mixing time of about 4.9 s inside the reactor and the shortest reliable sampling period assessed at 5 s. Biotechnol. Bioeng. 2012; 109: 3030–3040. © 2012 Wiley Periodicals, Inc.

Get access to the full text of this article

Ancillary