Lipid production by Yarrowia lipolytica growing on food waste

Abstract

Biodiesel production from plants and vegetable oils or different organic wastes as feedstock for microorganisms, can helps to decrease the consumption of fossil fuels and generation of greenhouse gases along with improve the economy. Applying organic wastes as feedstock for oleaginous yeasts is an economic technique in order to replace fossil-derived diesel with biodiesel as a clean and green fuel. Using food waste (FW) as a rich organic carbon source for cultivation of oleaginous yeast is considered as a promising environmentally friendly approach to achieve microbial lipid as the source of biodiesel. In this thesis, FW was collected from refectory of Istanbul Technical University. It contained cooked and uncooked food which dried and filtered after collection. Dark fermentation process was carried out with the collected FW and rumen microorganisms as inoculum. The rumen was taken from sheep stomach. The output of fermentation process was collected and applied as substrate for cultivation of oleaginous yeast Yarrowia lipolytica. The Soluble COD (SCOD) and TKN concentrations of fermented food waste (FFW) were 48.400 ± 0.49 and 0.907 ± 0.01 g/L, respectively and pH of the medium was 5.44 ± 0.05. Different concentrations of FFW (diluted with distilled water) were applied as growth medium of Y. lipolytica. The medium which was applied with no dilution was identified as the optimum one. In all applied mediums the growth was monitored as optical density (OD600) and the highest OD of 36.11 was observed in medium with FFW with no dilution. Nitrogen is considered as an essential nutrient for synthesis of cell materials and metabolites by microorganism. In terms of nitrogen depletion along with excess amount of carbon in the culture, carbon uptake rate is limited that causes metabolic activities shift towards lipid storage instead of cell proliferation. This approach was used in this thesis to increase lipid content of Y. lipolytica cultivated on FFW. Different carbon sources of glucose, glycerol, and potassium acetate along with five different COD/TKN ratios of 75, 100, 125, 150 and 175 were selected. Carbon sources were added to the medium in early stationary phase to increase carbon concentration of FFW and obtain favorable COD/TKN ratios. In order to identify the appropriate fermentation time to collect the biomass to evaluate its lipid content, biomass samples were collected at both early and late stationary phase in YPD (the optimum growth medium for yeast growth) and FFW medium. The results indicated lipid content of 21.54 ± 1.4 and 14.97 ± 0.51% lipid along with biomass concentration of 9.63 ± 0.24 and 8.34 ± 0.82 g/L in early and late stationary phase respectively, for YPD medium. These values were 19.5 ± 0.5 and 15.52 ± 0.31% lipid content along with 8.31 ± 0.51 and 7.10 ± 0.34 g/L biomass generation for Y. lipolytica cultivated on FFW medium. Results illustrated the early stationary phase as the optimum fermentation point to obtain highest lipid content and microbial cell because the biodegradation of yeast cell and intracellular lipid is occurred by time during stationary phase. This biodegradation caused drop of intracellular lipid content and yeast cell quantity at the end of stationary phase. In COD/TKN 75, lipid content of biomass was 26.7 ± 0.5, 34.2 ± 1.12 and 33.2 ± 1.59% with biomass concentration of 9.50 ± 0.37, 8.40 ± 0.72 and 9.32 ± 0.12 g/L for mediums contain glucose, glycerol, and potassium acetate, respectively. By increasing the ratio to 100, lipid content of medium with glucose increased to 29.2 ± 0.28, in medium contains glycerol lipid content was 34.3 ± 2.16 and in medium with potassium acetate the lipid content was slightly decreased to 31.2 ± 1.60%. In COD/TKN ratio of 125, the significant lipid content of 42.2 ± 1.72% in the medium supplemented with glycerol was measured. The other mediums contain glucose and potassium acetate have intracellular lipid content of 38.7 ± 0.35 and 34.7 ± 3.1%, respectively. Biomass concentration was measured as 18.52 ± 1.97, 12.95 ± 1.95 and 17.60 ± 0.75 g/L in culture sets supplemented with glucose, glycerol, and potassium acetate, respectively. In COD/TKN ratios 150 and 175, the amount of accumulated lipid and generated biomass were decreased that demonstrated the adverse effect of high concentration of carbon source on intracellular lipid accumulation of the cell. In these two ratios the amount of lipid content and cell concentration was 36.7 ± 1.3 and 9.77 ± 0.97; and 38.1 ± 3.0% and 11.57 ± 0.77 g/L, respectively. Highest lipid concentration of 7.61 ± 0.17 g/L was observed in medium with potassium acetate in COD/TKN 100 that is related to high biomass concentration in this experimental set. Lipid concentration started to decrease in higher COD/TKN ratios and dropped to 3.59 ± 0.13 g/L in COD/TKN ratio 150 with glycerol. Although the final pH value of the mediums with COD/TKN 125 and lower was always over 8, in ratios over 125 the pH was dropped to 4.3. This pH indicated formation of secondary metabolites such as organic acids in higher ratios that was due to high glycerol concentration in the mediums. This metabolic shift from lipid accumulation to organic acid generation led to the pH drop of the batch cultures. In next step of the thesis, Yeast Extract (YE), Iron Sulphate (IS) and Trace elements Solution (TS) was supplied to FFW along with glycerol as second carbon source to boost the lipid content further. Firstly, various amount of mentioned components were added to FFW to investigate the optimum concentration of each one to enhance lipid content of the cell. The concentrations of 1000 mg/L YE, 150 mg/L IS, and 5 ml/L TS were identified as the optimum. Different dual and triple combinations of these components along with glycerol were added to the mediums. In COD/TKN 150, accumulated lipid was reached to 44.72 ± 0.31% in the medium contains YE+IS+TS. Accumulated lipid reached to its maximum in ratio 175 i.e., 45.94 ± 0.21% in experimental set supplemented with YE+IS+TS. This value was the highest lipid content obtained in this thesis and by increasing the COD/TKN ratio over 175, the lipid accumulation dropped significantly. The highest concentration of lipid was 7.12 ± 0.12 g/L obtained in ratio 175 in medium supplied with YE+TS. The highest cell production was measured in the same culture with biomass concentration of 16.67 ± 0.27 g/L. Investigation of metabolic behavior of Y. lipolytica in this thesis revealed that in general, by increasing concentration of organic carbon in the medium, the carbon consumption is enhanced as well. In ratio 75, the highest COD consumption was measured in culture contains glycerol as 30.11 g/L COD. In ratio 100, the COD consumption of medium contain glycerol was increased slightly to 32.54 mg/L COD. In ratio 125, the COD consumption of 38.84 g/L was observed that caused the lipid storage of 42.2 ± 1.72% w/w in the cell. By increasing the COD/TKN ratios to 150 and 175, the microbial lipid content decreased although COD consumption was increased. This demonstrated the metabolic shift of Y. lipolytica from lipid generation to secondary metabolite production which caused drop of lipid biosynthesis. By adding YE, IS and TS to the medium, in ratio 150 the lipid content enhanced to 44.72 ± 0.31% with COD consumption of 48.37 g/L and in ratio 175, highest lipid content of 45.94 ± 0.21% was obtained by COD consumption of 49.63 g/L. However, decreasing pH value in experimental sets with COD/TKN over 125 indicated the generation of secondary metabolites in the medium. Additionally, in ratio 200 and 225 despite increase in COD consumption in the culture, the amount of stored lipid was reduced. Gas chromatography analyses of accumulated lipid revealed the fatty acid (FA) composition of stearic acid (C18:0), linoleic acid (C18:2), pentadecanoic acid (C15:1), palmitic acid (C16:0) and heptadecenoic acid (C17:1), similar to the FA profile of plant oil and appropriate for biodiesel generation. This thesis presented the efficient conversion of FFW and glycerol to considerable amount of microbial lipid. It indicated that FW and glycerol as two available and economic carbon sources can be evaluated as feedstock for intracellular lipid accumulation of Y. lipolytica. Additionally, this study demonstrated that the two-stage batch cultivation method by using FFW as initial carbon source and glycerol as the second one, can improve the lipid content of the microbial cell in significant amount. This thesis provides not only an economic waste treatment strategy, but also a sustainable and profitable method of microbial oil production by using carbon rich wastes as substrate for oleaginous yeast.