Photobioreactor design for isotopic non-stationary 13C-metabolic flux analysis (INST 13C-MFA) under photoautotrophic conditions.
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 (13) C-metabolic flux analysis (INST (13) C-MFA). To evaluate (13) C-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 (13) C labeling injection device based on mixing control in a torus-shaped photobioreactor with plug-flow hydrodynamics allowing a sudden step-change in the (13) C 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. (13) C-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). (13)C 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.
LUNAM Université, Université de Nantes, CNRS, GEPEA, UMR 6144, Bât. CRTT, 37 boulevard de l'Université, BP 406, F-44602 Saint-Nazaire Cedex, France.
Arnaud
A
Martzolff
Edern
E
Cahoreau
Guillaume
G
Cogne
Lindsay
L
Peyriga
Jean-Charles
JC
Portais
Emmanuel
E
Dechandol
Fabienne
F
Le Grand
Stéphane
S
Massou
Olivier
O
Gonçalves
Jérémy
J
Pruvost
Jack
J
Legrand
D018533:Biomass; D002247:Carbon Isotopes / Q000032:analysis* / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D005227:Fatty Acids / Q000032:analysis / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D058433:Photobioreactors; D015203:Reproducibility of Results; D049490:Systems Biology / Q000379:methods*
D002247:Carbon Isotopes; D005227:Fatty Acids