Optimization of Production Parameters for Single Cell Oil and Metabolite Production from Yarrowia lipolytica Strains

Nowadays, microbial oil and metabolite production have gained increasing interest due to the foreseeable limitation of plant and animal sources. Microbial lipids and metabolites are promising raw materials for food, fuel and oleo-chemical industries. Many reports of past few years indicated the potential of microorganisms to convert low value substrates to value added products such as microbial lipids, enzymes, citric acid, microbial mass, and polyols. Microbial oil production presents a potential industrial interest due to the capacity of various oleaginous microorganisms (these microorganisms can accumulate more than 20%, wt/wt, of lipid in their dry matter) to store lipids. Enzymes and organic acids are principal industrial metabolites of microorganisms. Citric acid represents 60% of all food acidulates. Among the enzymes, lipases are important in lipid technology, bio-industry, and they catalyze reactions such as hydrolysis (of ester bonds of tri-, di- and mono-glycerides of long-chain fatty acids into fatty acids and glycerol), esterification, interesterification, acidolysis, alcoholysis, and trans-esterification. Lipases constitute about 5% of the enzyme market and they have many applications in the food, dairy, detergent, and pharmaceutical industries. The model oleaginous yeast Yarrowia lipolytica has been used in lipid and metabolite production due to its biotechnological ability and intense secretory activity. Yarrowia lipolytica is known to have the ability to assimilate hydrophobic substrates like triglycerides, fats, and oils and produce single cell oils, lipases and organic acids. Non-conventional yeast Y. lipolytica produces intracellular and extracellular lipases which can be utilized as biocatalyst in the pharmaceutical, food and environmental industries. The objectives of this Ph.D. thesis were (i) to investigate the effects of different oil sources (borage, canola, sesame, Echium and trout oils) and oil industry residues (olive pomace oil, hazelnut oil press cake, and sunflower seed oil cake) on the growth and morphology of Yarrowia lipolytica YB 423-12; (ii) to determine lipid accumulation and composition of cellular lipids produced using Y. lipolytica YB 423-12 strain; (iii) to investigate citric acid and lipase production by Y. lipolytica YB 423-12 strain; (iv) to improve the extracellular lipase activity from Y. lipolytica YB 423-12 by optimizing fermentation conditions using hazelnut oil press cake as a substrate; (v) to characterize and purify microbial lipase enzyme; (vi) to determine the effects of lipid, protein, cellulose on lipase enzyme production; (vii) to evaluate processing costs for lipase enzyme in large scale. In Chapter 1, research framework and objectives of this Ph.D. thesis are introduced. Following that, in Chapter 2, literature review on microbial lipids and microbial metabolites are presented. Then, studies investigating the lipid accumulation, fatty acids and different microorganisms are reviewed. Afterwards, optimization studies, strain improvement by genetic manipulation and metabolic engineering are discussed. Lastly, microbial metabolites such as citric acid and lipase enzyme are described. In Chapter 3, materials and methods of the analysis are introduced. To achieve these goals, five different experiments (Chapters 4-8) were conducted. Firstly, Y. lipolytica YB 423-12 strain was cultivated in media containing glucose and various oils (borage, Echium, linseed, rapeseed, sesame, trout oils) as carbon and energy sources. These substrates were investigated for biomass and morphology of cells (Chapter 4). Lipid accumulation and fatty acid profiles of microbial oil produced using different substrates were investigated in Chapter 5. Following that, microbial metabolites such as citric acid and lipase production were determined using the same oils and oil industry residues such as olive pomace oil, hazelnut oil and sunflower seed oil cakes (Chapter 6). After that, considering the high efficiency of lipase enzyme production by Y. lipolytica using hazelnut oil press cake, studies focused on enzyme production. In this context, experimental designs such as Plackett-Burman design and Response Surface Methodology (RSM) were conducted to improve enzyme activity (Chapter 7). Finally, protein determination, enzyme purification and characterization studies of lipase enzyme were conducted. Also the effects of lipid, protein, and cellulose on lipase enzyme production were determined to increase the enzyme activity.Cost analysis of lipase enzyme production were determined in large-scale production (Chapter 8). Mycelial growth have been observed in all mediums containing different substrate oils (borage, Echium, linseed, rapeseed, sesame, trout oils). Addition of oils to medium caused a significant increase in dry biomass of the strain (p<0.05). The maximum and minimum dry biomass yields were obtained with linseed oil (26.67±0.09 g/L), and trout oil (20.48±0.03 g/L), respectively (Chapter 4). It was observed that cellular lipid yields were in the range of 31.99 – 61.67 g/100 g dry biomass. The increase in lipid content compared to the control was 92.78% in linseed oil medium (61.67±0.26%, g/g). Also, maximum lipid accumulation was observed as 16.45 and 14.86 g/L with linseed and Echium oils, respectively (after 5 days of incubation period). The results of fatty acid profiles indicated that GLA containing medium favored the production of microbial oil with GLA content. A similar result was observed for trout oil regarding the eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) content in the accumulated oil (Chapter 5). The highest citric acid (CA) concentration (5.34±0.938 g/L) was found with borage oil containing medium. Echium seed (5.19±0.20 g/L), trout (4.62±0.14 g/L), rapeseed (4.61±0.19 g/L), linseed (4.58±0.83 g/L) and sesame oils (4.49±0.66 g/L) and sunflower seed oil cake (4.85±0.13 g/L) had also statistically important effects on CA production as well as borage oil (Chapter 6). Among the tested oil sources and oil industry residues, hazelnut oil press cake was the best medium for lipase production. The Y. lipolytica YB 423-12 strain produced 12.32±1.54 U/mL (lipase activity) of lipase on hazelnut oil press cake medium supplemented with glucose. In order to verify the efficacy of oleic acid contribution to the lipase production, three different substrates (hazelnut oil, oleic acid and hazelnut oil press cake) containing the same amount of oleic acid were used as substrates for cultivation media for three different Y. lipolytica strains. Maximum lipase activity was obtained in the presence of hazelnut oil press cake with Y. lipolytica CBS 6303 strain on the 3rd day as 15.67 U/mL. A two-level Plackett-Burman design revealed that fermentation time and KH2PO4 significantly affected lipase activity (p<0.05) of Y. lipolytica YB 423-12 strain among the parameters tested. Further optimization studies Central Composite Circumscribed Design (CCC) and Response Surface Methodology (RSM) indicated that all first-order parameters were statistically significant (p<0.05) and the optimum conditions were 22.1 g/L of KH2PO4 and 4.8 days of incubation period. Under these optimum conditions, highest lipase activity (9.18 U/mL) was obtained in the presence of 10% of hazelnut oil press cake at 28 °C (Chapter 7). Purification studies indicated that maximum lipolytic activity was 332.55 U/mg with 60% ammonium sulfate precipitation and this activity stems from a single 41 kDa protein. After conducting characterization studies of lipase enzyme at different temperatures, pH, and metal ionsoptimum enzyme activity was observed between the pH ranges of 7.0 and 9.0 and at 40°C. Among the tested metal ions (CaCl2, MgCl2, CuSO4) at 100mM concentration, the lipase activity increased with Ca2+ and Mg2+ ions, while Cu2+ ions decreased the enzyme activity. Afterwards, the lipase activity analysis were made with the control (30 g/L glucose), protein, cellulose, lipid freee and untreated hazelnut oil press cake and without glucose mediums. The highest lipase activity (16.89±0.44 U/mL) was achieved for untreated hazelnut oil press cake medium. The results showed that hazelnut oil press cake medium increased the lipase activity of Y. lipolytica. However, this effect cannot only be attributed to oleic acid present in the cake, because lipid-free hazelnut oil press cake also increased lipase activity. Finally, feasibility of producing lipase enzyme using hazelnut oil press cake by Y. lipolytica strain was evaluated (Chapter 8).According to the results of cost analysis, microbial lipase production is feasible in the presence of hazelnut oil press cake in the fermentation medium. Furthermore, valorization of various low-cost raw materials, residues, wastes and renewable resources as substrates would be of such interest in production of value added products.