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

• Published in: Biotechnology and bioengineering Vol. 109; no. 12; pp. 3030 - 3040
• Main Authors: Martzolff, Arnaud, Cahoreau, Edern, Cogne, Guillaume, Peyriga, Lindsay, Portais, Jean-Charles, Dechandol, Emmanuel, Le Grand, Fabienne, Massou, Stéphane, Gonçalves, Olivier, Pruvost, Jérémy, Legrand, Jack
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-12-2012; Wiley Subscription Services, Inc
• Subjects: Biochemistry; Biomass; Biotechnology; Carbon; Carbon Isotopes - analysis; Carbon Isotopes - metabolism; carbon labeling experiment; Cells; Chlamydomonas reinhardtii; Chlamydomonas reinhardtii - metabolism; Devices; Enrichment; Fatty Acids - analysis; Fatty Acids - metabolism; Flux; Hydrogen-Ion Concentration; isotopic non-stationary 13C-metabolic flux analysis; isotopomer analysis; Labels; Marking; Metabolism; Microorganisms; photoautotrophic metabolism; photobioreactor; Photobioreactors; Reactors; Reproducibility of Results; Sampling; Symbols; Systems Biology - methods
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.24575

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.
The paper focuses on the experimental design of a photobioreactor especially conceived for transient isotopic 13C‐labeling experiments under photoautotrophic conditions. The experimental set‐up provided for (i) a rapid sampling and quenching procedure and (ii) rapid mixing throughout the reactor to ensure a sudden step‐change in the 13C‐labeled feed from liquid label injection without affecting uptake flux rate. The operating procedure and performance of the experimental device were tested and validated for continuous cultures of the unicellular green alga Chlamydomonas reinhardtii.