title: Improved quantification of farnesene during microbial production from Saccharomyces cerevisiae in two-liquid-phase fermentations.
abstract: Organic solvents are widely used in microbial fermentations to reduce gas stripping effects and capture hydrophobic or toxic compounds. Reliable quantification of biochemical products in these overlays is highly challenging and practically difficult. Here, we present a significant improvement of identification and quantification methods for farnesene produced by Saccharomyces cerevisiae in two-liquid-phase fermentations using GC-MS and GC-FID. By increasing the polarity of the stationary phase introducing a ZB-50 column (50%-phenyl-50%-dimethylsiloxane) peak intensity could be increased and solvent carryover could be minimized. Direct quantification of farnesene in dodecane was achieved by GC-FID whereas GC-MS demonstrated to be an excellent technique for identification of known and unknown metabolites. The GC-FID is a suitable technique for direct quantification of farnesene in complex matrices as shown by the good calibration curve (R(2)>0.998, N=5) within the tested concentration range of 1-50 µg/mL and the reproducibility of the intensity (intraday; <10% RSD at each concentration; N=5). The limit of detection (LOD) and limit of quantification (LOQ) of the method were 0.24 and 0.80 µg/mL, respectively. Furthermore, the FID method proved to be highly stable with regard to the intensity of the calibration (N=6) when the measurements were performed across 250 samples that were derived from a dodecane overlay.
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, SE41296 Gothenburg, Sweden; Chalmers Metabolomics Centre, Chalmers University of Technology, Gothenburg, Sweden; Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE41296 Gothenburg, Sweden.
authors.forename: Stefan
authors.initials: S
authors.lastname: Tippmann
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, SE41296 Gothenburg, Sweden; Chalmers Metabolomics Centre, Chalmers University of Technology, Gothenburg, Sweden; Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE41296 Gothenburg, Sweden; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK2970 Hørsholm, Denmark.
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, SE41296 Gothenburg, Sweden; Chalmers Metabolomics Centre, Chalmers University of Technology, Gothenburg, Sweden. Electronic address: sakda@chalmers.se.
authors.forename: Sakda
authors.initials: S
authors.lastname: Khoomrung
mesh_terms: D000473:Alkanes / Q000378:metabolism; D019149:Bioreactors / Q000382:microbiology; D001709:Biotechnology / Q000379:methods*; D005285:Fermentation; D008401:Gas Chromatography-Mass Spectrometry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D012717:Sesquiterpenes / Q000378:metabolism*; D012997:Solvents / Q000737:chemistry
chemical_list: D000473:Alkanes; D012717:Sesquiterpenes; D012997:Solvents; C007548:n-dodecane
keywords: Biofuels; GC–FID; GC–MS; Isoprenoids; Metabolic engineering