Μεταβολισμός της γλυκερόλης στη ζύμη Yarrowia lipolytica και προοπτικές ανάπτυξης νέων βιοδιεργασιών

Abstract

In this thesis the metabolism of glycerol in Yarrowia lipolytica ACA–DC 50109, with emphasis on glycerol conversion into value–added biotechnological products, such as single cell oils and citric acid, was studied.The growth of Y. lipolytica was studied in bioreactor batch cultures in multiple limited medium and three distinct phases were identified during growth cycle. In each phase, yeast cells were characterized by specific morphological and biochemical features: biomass formation phase (in which 4–4.5 g/l of biomass were synthesized), lipogenic phase (in which 20–22% lipids wt/wt in dry weight were accumulated in biomass, containing 90% wt/wt neutral lipids) and citric acid production phase (in which 14–30 g/l of citric acid were secreted in the growth environment). Distinct cellular forms of Y. lipolytica were developed during the above phases: in biomass formation phase short true mycelia and pseudo–mycelia were predominant while a few yeast–like cells were observed, in lipogenic phase large obese cells were predominant and in citric acid production phase cells size was diminished.Glycerol passes into the microbial cell by facilitated diffusion. Y. lipolytica successfully converts glycerol via phosphorylation pathway, in which glycerol kinase (GK) and glycerol–3–P–dehydrogenase are implicated. Though high activity of NAD+ dependent isocitric dehydrogenase (NAD+–ICDH) was detected during biomass formation phase, this activity was significantly decreased afterwards inducing lipogenesis. Surprisingly, storage (neutral) lipid turnover and synthesis of glycolipids, sphingolipids and phospholipids – Ρ simultaneously occurred with citric acid production, and happened when GK activity was considerably reduced and NAD+–ICDH activity was minimised. Oleic acid was the major fatty acid in all lipid fractions and phosphatidylcholine – PC was the main Ρ.In continuous culture in nitrogen limited medium Y. lipolytica accumulated low quantities of lipids (~10% w/w, in dry weight), maybe due to the fact that there was not a region of specific dilution rate (D, h–1) in which the key–enzymes that are implicated in lipogenesis (i.e. ΑΣΡ:citrate lyase – ATP:CL and malic enzyme – ME) presented simultaneously high activity while NAD+–ICDH activity was insignificantly decreased in low D. ATP:CL presented high activity (60–300 Units/mg DW) in D 0,033 h–1 while ME presented maximum activity (650 Units/mg DW) inD=0,104 h–1. Lipids were more unsaturated in intermediate D values whilephosphatidylethanolamine – PE, phosphatidylinositol – PI and PC are the main Ρclasses. As far as the morphology is concerned, in D<0,055 h–1 short true mycelia andpseudo–mycelia were predominant in culture medium while in D 0,055 h–1 onlyyeast cells were observed.In experiments performed in nitrogen limited medium in D=0,026 h–1 indifferent dissolved oxygen – DO concentrations, it was found that in extreme DOvalues ( 70% and 7%) the percentage of P was increased. Independently the DOconcentration PC was the main class followed by PI and PE. The morphology of Y.lipolytica was influenced by the different concentration of DO and it was observedthat in DΟ 50% short true mycelia and pseudo–mycelia were predominant in culturemedium while in DΟ 50% more yeast cells were appeared.In experiments performed in D=0,026 h–1, it was found that the absence ofmicronutrients from the growth medium, i.e. magnesium and calcium that areimplicated in multiple cellular functions, had severe effects in yeast physiology, whilethe fatty acid composition of cellular lipids was not affected by the nature of thegrowth limiting factor.The present thesis aspires to contribute in the study of oleaginousmicroorganisms’ physiology and in use of glycerol as substrate in futurebiotechnological applications.