title: Assessing an effective feeding strategy to optimize crude glycerol utilization as sustainable carbon source for lipid accumulation in oleaginous yeasts.
abstract: BACKGROUND
  Microbial lipids can represent a valuable alternative feedstock for biodiesel production in the context of a viable bio-based economy. This production can be driven by cultivating some oleaginous microorganisms on crude-glycerol, a 10% (w/w) by-product produced during the transesterification process from oils into biodiesel. Despite attractive, the perspective is still economically unsustainable, mainly because impurities in crude glycerol can negatively affect microbial performances. In this view, the selection of the best cell factory, together with the development of a robust and effective production process are primary requirements.
  
  
  RESULTS
  The present work compared crude versus pure glycerol as carbon sources for lipid production by three different oleaginous yeasts: Rhodosporidium toruloides (DSM 4444), Lipomyces starkeyi (DSM 70295) and Cryptococcus curvatus (DSM 70022). An efficient yet simple feeding strategy for avoiding the lag phase caused by growth on crude glycerol was developed, leading to high biomass and lipid production for all the tested yeasts. Flow-cytometry and fourier transform infrared (FTIR) microspectroscopy, supported by principal component analysis (PCA), were used as non-invasive and quick techniques to monitor, compare and analyze the lipid production over time. Gas chromatography (GC) analysis completed the quali-quantitative description. Under these operative conditions, the highest lipid content (up to 60.9% wt/wt) was measured in R. toruloides, while L. starkeyi showed the fastest glycerol consumption rate (1.05 g L(-1) h(-1)). Being productivity the most industrially relevant feature to be pursued, under the presented optimized conditions R. toruloides showed the best lipid productivity (0.13 and 0.15 g L(-1) h(-1) on pure and crude glycerol, respectively).
  
  
  CONCLUSIONS
  Here we demonstrated that the development of an efficient feeding strategy is sufficient in preventing the inhibitory effect of crude glycerol, and robust enough to ensure high lipid accumulation by three different oleaginous yeasts. Single cell and in situ analyses allowed depicting and comparing the transition between growth and lipid accumulation occurring differently for the three different yeasts. These data provide novel information that can be exploited for screening the best cell factory, moving towards a sustainable microbial biodiesel production.
authors.affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Signori
authors.affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy.|Department of Physics, University of Milano-Bicocca, Piazza della Scienza 3, 20126, Milan, Italy.|Consorzio Nazionale Interuniversitario per le Scienze fisiche della Materia (CNISM), UdR Milano-Bicocca, Via R. Cozzi 53, 20126, Milan, Italy.
authors.forename: Diletta
authors.initials: D
authors.lastname: Ami
authors.affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Posteri
authors.affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Giuzzi
authors.affiliation: Center for Nanotechnology Innovation@NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127, Pisa, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Mereghetti
authors.affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy.
authors.forename: Danilo
authors.initials: D
authors.lastname: Porro
authors.affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy. paola.branduardi@unimib.it.
authors.forename: Paola
authors.identifier: http://orcid.org/0000-0003-4115-7015
authors.initials: P
authors.lastname: Branduardi
mesh_terms: D001487:Basidiomycota / Q000254:growth & development / Q000378:metabolism*; D056804:Biofuels / Q000032:analysis; D018533:Biomass; D002244:Carbon / Q000378:metabolism*; D002849:Chromatography, Gas; D005227:Fatty Acids / Q000032:analysis / Q000737:chemistry; D005434:Flow Cytometry; D005990:Glycerol / Q000378:metabolism*; D008055:Lipids / Q000096:biosynthesis*; D008856:Microscopy, Fluorescence; D025341:Principal Component Analysis; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D056804:Biofuels; D005227:Fatty Acids; D008055:Lipids; D002244:Carbon; D005990:Glycerol
keywords: Crude glycerol; Cryptococcus curvatus; Fatty acids methyl esters (FAME); Flow-cytometry; Fourier transform infrared (FTIR) microspectroscopy; Lipomyces starkeyi; Principal component analysis (PCA); Rhodosporidium toruloides