Physiology and formation of lipid particles during growth of Yarrowia lipolytica IMUFRF 50682 yeast.

Author(s)

Fernanda Bacciotti

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Escola Politécnica (EP/BC)
• Defense date: 2015-08-06

Examining board members

Andreas Karoly Gombert; Monica Monteiro Lomeli; Everson Alves Miranda

• Advisor: Andreas Karoly Gombert

Abstract

The yeast Yarrowia lipolytica has been intensively investigated, especially for being an oleaginous microorganism, thus possessing the capacity of accumulating high amounts of lipids, which mainly takes place in organeles known as lipid bodies. These lipids present several potential biotechnological appications, as in the production of single cell oil or biodiesel. During this research project, we aimed at investigating the physiology of two Y. lipolytica strains: w29, traditionally investigated by the international scientific community, and IMUFRJ 50682, isolated from the Guanabara Bay in Rio de Janeiro. Shake-flask cultivations in baffled Erlenmeyer flasks (total volume 500 mL, liquid volume 100 mL, 28 oC and 200 rotations per minute) with cotton stoppers were carried out and allowed us to: 1) choose a fully defined cultivation medium, in which tiamine is the sole growth factor, suitable for quantitative physiological studies with this yeast; 2) verify that Y. lipolytica is not capable of growing on sucrose or xylose as the sole carbon source; 3) observe that Y. lipolytica grows with a maximum specific growth rate (MAX) of 0.49 h-1 in a complex medium containing glucose, yeast extract and peptone (YPD medium), 0.31 h-1 in a defined medium with glucose as the sole carbon source, and 0.35 h-1 in the same medium, but with glycerol as the sole C-source, without excreting metabolites to the cultivation medium; 4) verify that oxygen limitation took place during our shakeflask cultivations and that this caused cells to leave the exponential growth phase; 5) verify that urea can substitute ammonium as the sole nitrogen-source for Y. lipolytica, keeping pH variations less pronounced, without compromising cell growth; 6) observe that cells presented an altered morphology and higher amounts of lipid bodies, when less nitrogen was added to the medium (C/N ratio increased from 12.6 to 126); 7) determine an elemental composition for the biomass of Y. lipolytica (CH1,98O0,58N0,13), in which the average carbon atom was more reduced with respect to the biomass of yeasts such as Dekkera bruxellensis and Saccharomyces cerevisiae. Bioreactor cultivations in batch mode (working volume 1 L, 28 oC, full aerobiosis and pH controlled at 5.0) were also carried out, which allowed us to: a) define a protocol for the cultivation of Y. lipolytica in this system (which involves mechanical agitation, aeration and the use of anti-foam, among other differences with respect to shake-flask cultivations); b) confirm the main physiological parameters presented by this yeast, previously obtained from shake-flask cultivations; c) confirm that the biomass yield on substrate (Yx/s) is higher on glycerol than on glucose (0.53 g/g and 0.48 g/g, respectively). Finally, N-limited chemostat cultivations with glycerol as the carbon and energy source and ammonium as the N-source were also performed (dilution rates of 0.25 h-1 and 0.15 h-1, C/N ratio in the medium of 126), allowing us to verify that the number of lipid particles per cell is around 2 under both conditions and that there was a decrease in the N content in the cells when the specific growth rate decreased from 0.25 to 0.15 h-1. (AU)

• FAPESP's process: 11/16417-2 - Influence of the specific growth rate on lipid metabolism in the yeast Yarrowia lipolytica
• Grantee: Fernanda Bacciotti
• Support Opportunities: Scholarships in Brazil - Master