Production of Microbial Lipids by Yarrowia Lipolytica

Romanian Biotechnological Letters Vol. 2, No. 6, 215 Copyright 215 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER Production of Microbial Lipids by Yarrowia Lipolytica Received for publication, May 1, 215 Accepted, October, 215 DIANA GROPOȘILĂ-CONSTANTINESCU, OVIDIU POPA, Department of Industrial Biotechnologies, University of Agronomical Sciences and Veterinary Medicine, 59 Marasti Blvd., 116, Bucharest, Romania *Corresponding author: diana_gconst@yahoo.com Abstract Experiments performed in this work have aimed to establish conditions that allow fermentation directing with a Yarowia lipolytica strain to the biosynthesis of lipids at a high and reproducible level. Evaluation of lipid accumulation was achieved by Soxhlet method. The effects of several culture conditions on growth and lipid production of Yarrowia lipolytica were studied in this research. Lipid accumulation during fermentation was significantly influenced by the medium composition, the most important factor proving to be the C/N ratio. Results showed that maximum productivity, of 7.5 g/l lipid, corresponding to 5.3% of lipid in dry biomass (g/g), was accomplished in the presence of glucose g/l, (NH ) 2 SO as nitrogen source and a 5:1 C/N ratio. This study allowed the identification of parameters affecting the production of lipids by Yarrowia lipolytica. Optimum conditions for biosynthesis of lipids were obtained using the following parameters: inoculum 1% (v/v), temperature 2 C, initial ph 6, agitation rate 2 rpm, intense aeration (1. l air/l medium/minute) and duration 96 hours. Results showed that Yarowia lipolytica exhibited impressive cell growth and lipid production, accumulating up to 5% of lipid in dry biomass. Keywords: fermentation, lipid, C/N ratio, Yarrowia lipolytica 1. Introduction Biotechnology of lipids covers the microbial production and the biotechnological transformation of lipid and lipid-soluble compounds. Storage lipid in the form of triacylglycerols and their different fatty acid types are the main targets for biotechnological product development. Biotechnological processes have recently started entering the oleochemical business [1]. Positive examples for biotechnological developments are found mainly in the field of specialty products for the cosmetic, health food and pharmaceutical industry, with microbial steroid transformation representing a prominent example for the production of active pharmaceutical ingredients [2]. The aim of all biotechnological processes is to obtain products that are either cheaper than can be obtained from other sources. Within the field of lipids, the opportunities to produce triacylglycerol lipids are limited to the highest valued materials [3]. Not all microorganisms can be considered as abundant sources of oils and fats, though, like all living cells, microorganisms always contain lipids for the normal functioning of membranes and membranous structures. Those microorganisms that do produce a high 1936 Romanian Biotechnological Letters, Vol. 2, No. 6, 215

Production of Microbial Lipids by Yarrowia Lipolytica content of lipids may be termed oleaginous in parallel with the designation given to oilbearing plant seeds. Of about 6 different yeast species, only 25 or so are able to accumulate more than 2% lipids; of 6, fungal species, fewer than 5 accumulate more than 25% lipid []. The lipids which accumulate in oleaginous microorganisms are mainly triacylglycerols. Of current interest are oils containing the polyunsaturated fatty acids: g- linolenic acid (1:3), arachidonic acid (2:), eicosapentaenoic acid (2:5) and docosahexaenoic acid (22:6). With few exceptions, oleaginous microorganisms are eukaryotes and thus representative species include algae, yeasts, and molds. Bacteria do not usually accumulate high amounts of triacylglycerols [1]. The key to lipid accumulation lies in allowing the amount of nitrogen supplied to the culture to become exhausted within about 2- h. Exhaustion of nutrients other than nitrogen can also lead to the onset of lipid accumulation [1] but, in practice, cell proliferation is effected by using a limiting amount of N (usually NH or urea) in the medium. The excess carbon which is available to the culture after N exhaustion continues to be assimilated by the cells and, by virtue of the oleaginous organism possessing the requisite enzymes, it is converted directly into lipids [2]. The essential mechanism which operates is that the organism is unable to synthesize essential cell materials because of nutrient deprivation and thus cannot continue to produce new cells. Because of the continued uptake of carbon and its conversion to lipids, the cells can then be seen to become engorged with lipid droplets [2]. The medium has to be formulated with a high carbon-to-nitrogen ratio, higher than 2:1. The culture must be grown at a rate which is about 25-3% of the maximum. Under this condition, the concentration of nitrogen in the medium is virtually nil and then the organism has sufficient residence time within the chemostat to assimilate the excess carbon and convert it into lipids [5]. At the end of the lipid accumulation phase, it is essential that the cells are promptly harvested and processed. If glucose, or other substrate, has become exhausted at the end of the fermentation, then the organism will begin to utilize the lipids, as the role of the accumulated material is to act as a reserve storage of carbon, energy, and possibly even water [2]. For this research, oleaginous yeast Yarrowia lipolytica was chosen because it is one of the most extensively studied nonconventional yeasts. This yeast is able to accumulate large amounts of lipids (in some cases, more than 5% of its dry weight [7, ]. Several technologies including different fermentation configurations have been applied for single-cell oil production by strains of Yarrowia lipolytica grown on various agro-industrial by-products or wastes (i.e., industrial fats, crude glycerol, etc.) [7, 9]. 2. Materials and methods Microorganism and maintenance The laboratory strain (wild type) Yarrowia lipolytica used in this study was provided by the Collection of Microorganisms of the Faculty of Biotechnology University of Agronomical Sciences and Veterinary Medicine, Bucharest. The strain was maintained by periodic transfer on YPD agar slants containing (g/l): glucose 2., bactopeptone 2., yeast extract 1., agar 15., ph 6. Yarrowia lipolytica was incubated for hours at 3 C and maintained at C for maximum three months [1, 11]. Romanian Biotechnological Letters, Vol. 2, No. 6, 215 1937

DIANA GROPOȘILĂ-CONSTANTINESCU, OVIDIU POPA, Inoculum, growth medium From a YPD agar slant, a first preinoculum was inoculated into YPD medium (2 ml in 5 ml Erlenmeyer flasks) and incubated in a rotary shaker for h at 3 C and 22 rpm. Cells from the preculture were used to inoculate (1%) the fermentation medium (5-2 ml in 5 ml Erlenmeyer flasks). Fermentation was carried out in different conditions, in order to determine the optimum conditions for lipid biosynthesis [1, 11]. Fermentation medium has the following basic composition (g/l): glucose, lactose, sucrose, fructose, maltose (as carbon sources) 1-5; KH 2 PO, 5.; Na 2 HPO, 2.5; MgSO, 1.5; organic nitrogen sources (peptone, yeast extract) and inorganic sources (NH NO 3, NaNO 3 and (NH) 2 SO ) 1.; CaCl 2,.1 [1, 11]. Fermentation conditions Fermentations were carried out batch wise in 5 ml Erlenmeyer flasks, containing 5, 1, 15, 2 ml of fermentation medium []. The sterilized medium was inoculated with 1 % (v/v) preculture and incubated at 25, 3, 35 C, initial ph 5., 5.5, 6., 6.5 and 7., for 96 hours, in a rotary shaker, at 15, 1, 21, 2 rpm [13, 1]. Determination of yeast growth and biomass Biomass concentration was determined measuring the optical density (OD) of the fermentation medium by visible spectrophotometry (Helios γ-thermo Electron Corporation- USA) at 57 nm and hour intervals [15, 16]. For absorbance readings,.5 ml of culture medium were diluted in water (1:25) and measured with a spectrophotometer, at 57 nm. Calibration curves were performed using absorbance readings [16]. The quantity of yeast cells was assessed by dry weight determination [16]. Biomass separation At the end of fermentation process, the cultivation medium was centrifuged for 2 minutes at rpm. The sediment was washed with ethanol (95%) and hexane, in order to remove extracellular fat from the cell surface. The wet biomass was dried at a temperature of 6 C to constant weight [7]. Lipid extraction and determination Lipids were extracted by treating the dried biomass with chloroform: methanol 2:1 in a Soxhlet extraction apparatus and estimated gravimetrically [7, 17]. Quantitative determination of lipids was done by solvent evaporation and weighing the remaining product [3, 7]. Other analytical methods Residual sugar content was determined by 3.5-dinitro-salicylic acid method, in order to monitor the glucose content at the end of the fermentation process [16]. 3. Results and discussions The effects of several culture conditions on growth and lipid production of Yarrowia lipolytica is given below. 193 Romanian Biotechnological Letters, Vol. 2, No. 6, 215

Production of Microbial Lipids by Yarrowia Lipolytica Influence of carbon sources on biomass and lipid production To study the effects of carbon sources on lipid production and biomass, various available substrates were used: glucose, lactose, sucrose, fructose and maltose. Cells were cultivated in a rotary shaker incubator at 3 C, 3 g/l carbon source, ph 6 and 22 rpm, for 96 hours. The results show that all tested substances were suitable carbon sources for the growth and lipid accumulation of the strain, among which glucose was the most suitable. The lipid production was.9 g/l, followed by sucrose with a yield of.1 g/l. Therefore, glucose was chosen as carbon source in the following experiments. Figure 1 shows the effect of carbon sources on lipid production and biomass. Maltose Fructose Sucrose Lactose Glucose 3,7 3,2,1 3,6,9 1,9 11,9,5 Biomass (g/l) 11,1 13,1 2 6 1 1 Figure 1. Effect of carbon sources on biomass and lipid production To study the effects of glucose concentration on lipid production and biomass, various concentrations were used: 1, 2, 3, and 5 (g/l). Both the biomass and lipid productivity enhanced with an increase in initial glucose concentration. Noticeable growth (biomass=.9-13. g/l) and lipid accumulation (35.-1.1% of its dry weight) were observed in all cases. The highest amount of lipid was observed in media with g/l glucose (biomass yield=13.3 g/l, lipid yield=5. g/l). Figure 2 shows the effect of glucose concentration on biomass and lipid production. 1 1 6 2 13 13,2 13, 13,3,9,6, 5,1 5, 5,3 1 2 3 5 Glucose (g/l) Biomass (g/l) Figure 2. Effect of glucose concentration on biomass and lipid production Influence of nitrogen sources on biomass and lipid production The effect of nitrogen sources on lipid production and biomass is shown in Table 1. Compared with organic nitrogen sources (peptone, yeast extract), when inorganic ones (NH NO 3, NaNO 3 and (NH) 2 SO ) were used, the cells grew more quickly and reached a higher lipid productivity. Among the inorganic nitrogen sources, (NH) 2 SO was the most suitable and the lipid productivity and biomass was 5.6 g/l and 13.5 g/l, respectively. It is reasonable that Romanian Biotechnological Letters, Vol. 2, No. 6, 215 1939

DIANA GROPOȘILĂ-CONSTANTINESCU, OVIDIU POPA, (NH) 2 SO was chosen as nitrogen source in following experiments. Experiments were carried out at 3 C, glucose g/l, nitrogen source 1 g/l, ph 6 and 22 rpm, for 96 hours. Table 1. Effect of nitrogen source on cell growth and lipid production Nitrogen source Biomass (g/l) Lipid production (g/l) Peptone 11. 3. Yeast extract 11.3 3.6 NH NO 3.5.9 NaNO 3.3. (NH) 2 SO 13.5 5.6 Influence of C/N ratio on lipid production Experiments were conducted covering a large range of C/N ratios from 1:1 to 5:1. The effect of the C/N ratio on biomass at different sugar concentrations is shown in Figure 3. As the C/N ratio increases, the biomass increases rapidly, to reach a maximum value at a C/N ratio of 5:1. At C/N ratios below this, the biomass declines. The effect of the C/N ratio on lipid productivity is shown in Figure 3. As the C/N ratio increases, the accumulation of lipid increases to reach a maximum, giving an optimum C/N ratio for lipid production of 5:1. The highest value of lipid reached 6.1 g/l. From Figure, the effect of the C/N ratio on lipid productivity appears to be more pronounced at high values. At low C/N ratio, nitrogen limiting conditions necessary for lipid overproduction are not attained, resulting in low quantity of lipids, even though the biomass may be high. 1 1 6 2,9 13,3 13, 13,6 13,7 5, 5,6 6,1,2,7 1:1 2:1 3:1 :1 5:1 C/N ratio Biomass (g/l) Figure 3. Effect of C/N ratio on biomass and lipid production Influence of temperature on biomass and lipid production In order to investigate the effects of incubation temperature on biomass and lipid production, experiments were carried out at varying temperature, 25, 2, 31 and 35 C. Experiments were carried out at 22 rpm, ph 6, for 96 hours. The result (Figure ) shows that both the biomass and lipid production enhanced with an increase in culture temperature at 2-31 C. The highest lipid production was obtained at 2 C (biomass yield=13.6 g/l, lipid production=6.2 g/l, corresponding to 5.6% of its dry weight), whereas at 25 C and 35 C, lower amounts of lipids were accumulated. Therefore, for further experiments, a temperature of 2 C was selected as optimum parameter for lipid production. 19 Romanian Biotechnological Letters, Vol. 2, No. 6, 215

Production of Microbial Lipids by Yarrowia Lipolytica 1 1 6 2 5,5 13,6 13,5 6,2 5,9,1 5, 25⁰C 2⁰C 31⁰C 35⁰C Temperature Biomass (g/l) Figure. Effect of temperature on biomass and lipid production Influence of initial ph on biomass and lipid production To study the effects of initial ph on biomass and lipid production, Yarrowia lipolytica was incubated at 2 C, 22 rpm and varying initial values of ph (5., 5.5, 6., 6.5 and 7.). Substantial growth was observed (Figure 5) at ph 6 and 6.5 (13.5 and.6 g/l, respectively), whereas lipid (L) accumulation was favoured at ph 6 (Y L =6.3 g/l). At ph 5 and 7, restricted microbial growth was observed. The ph decreased slightly during growth, as low amounts of organic acids were produced. The result suggests that initial ph had a significant effect on the lipid accumulation and the optimum initial ph value was 6.. 1 1 6 2 1,1 3,9 11,9 5,3 13,5,6 6,3 5,6 11,1 5, 5,5 6, 6,5 7, ph value,6 Biomass (g/l) Figure 5. Effect of initial ph on biomass and lipids production Influence of culture volume on biomass and lipid production The study of this parameter was performed in fermentation experiments carried out in 5-mL Erlenmeyer flasks, using 5, 1, 15, 2 ml medium, in the following conditions: temperature 2 C, ph 6, for 96 hours, 2 rpm. The effects of medium volume on biomass and lipid production are shown in Figure 6. Both the biomass and lipid yield decreased with an increase in culture volume. This suggests that the strain is an aerobic strain because the less culture volume the better aeration provided in the flask. When culture volume was 5 and 1 ml/5 ml, the biomass yield was 1.5 and 1.3 g/l, respectively. Considering economic benefits, small volumes of medium is not advantageous. Therefore, 1 ml (in a 5 ml flask) was selected as the suitable culture volume for lipid biosynthesis. Romanian Biotechnological Letters, Vol. 2, No. 6, 215 191

DIANA GROPOȘILĂ-CONSTANTINESCU, OVIDIU POPA, 16 1,5 1,3 7,2 7,,9 5,9 11,2,6 Biomass (g/l) 5 1 15 2 Volume of medium/flask (ml/5 ml) Figure 6. Effect of culture volume on biomass and lipids production Influence of agitation speed on biomass and lipid production In order to investigate the effects of agitation speed on biomass and lipid biosynthesis, the yeast Yarrowia lipolytica was incubated at 2 C, ph 6 and varying agitation speed (15, 1, 21, 2, 2 rpm). The results (Figure 7) show a gradual and significant increase in biomass and lipid content with the increase of agitation speed during the bioprocess, up to 2 rpm. At 2 rpm, the biomass and lipid production were 1.7 and 7.3 g/l, respectively, corresponding to 9.7% (g/g) lipids of dry biomass. Higher agitation speed was advantageous to oxygen supply, resulting only a higher biomass yield. Therefore, further experiments will be conducted at 2 rpm. 16 9,7,6 11, 5,7 13,1 6, 1,7 1,9 7,3 6,9 Biomass (g/l) 15 1 21 2 2 Agitation (rpm) Figure 7. Effect of agitation speed on biomass and lipids production Verification of the optimum conditions for lipid production in a stirred-tank bioreactor Based on the results obtained from the previous experiments, further investigations aimed at the verification of the reproducibility of optimum conditions for biosynthesis of lipids in a stirred-tank bioreactor. Figure shows the time courses of cell growth and lipid production under the optimized culture conditions. The concentration of residual sugars in the fermentation medium decreased to 15.2 g/l at h, and.5 g/l at 96 h. The amount of lipids increased from 2 h to 96 h, when it reached the maximum productivity (7.5 g/l). The quantity of biomass kept rising from h to 96 h, and reached the maximum weight (at 96 h, 1.9 g/l). It was obvious that the accumulation of biomass and lipids were achieved during the stationary growth phase. 192 Romanian Biotechnological Letters, Vol. 2, No. 6, 215

Production of Microbial Lipids by Yarrowia Lipolytica 16, 2,1,1, 9,6 1,9 1,9 1,7 7,5 7,2 2 h h 72 h 96 h 1 h Time (hours) Biomass (g/l) Figure. Biomass and lipids production in stirred-tank bioreactor. Conclusions The experiments performed in order to establish the influence of the medium composition. On the biosynthesis of lipids by Yarowia lipolytica, pointed out that maximum production (7.5 g/l) was accomplished in the presence of glucose g/l, (NH ) 2 SO as nitrogen source and a 5:1 C/N ratio. This study also allowed the identification of parameters affecting the production of lipids by Yarrowia lipolytica. Optimum conditions for biosynthesis of lipid were obtained using the following parameters: inoculum 1% (v/v), temperature 2 C, initial ph 6, agitation rate 2 rpm, aeration corresponding to a medium/flasks ratio of 1:5 (v/v) at laboratory level and 1. l air/l medium/minute in bioreactor level, fermentation time 96 hours. Yarowia lipolytica exhibited impressive cell growth and lipid production, accumulating up to 5% of lipids in dry biomass. 5. Acknowledgements This research was carried out with the support of the program Invest in people!, project co-financed by the European Social Fund POSDRU 27-213, POSDRU/159/1.5/S/132765 6. References 1. U. SCHÖRKEN, P. KEMPERS, Lipid biotechnology: Industrially relevant production processes, European Journal of Lipid Science and Technology, 111(7), pp. 627-65, 29. 2. C. RATLEDGE, Microbial Lipids, in Biotechnology: Products of Secondary Metabolism, 7, Second Edition, Ed. H.-J. Rehm and G. Reed, Wiley-VCH Verlag GmbH, Weinheim, Germany, 1997, pp. 133-197. 3. C. RATLEDGE, Yeasts, moulds, algae and bacteria as sources of lipids, in: Kamel BS, Kakuda Y (eds) Technological advances in improved and alternative sources of lipids, Blackie, London, 199, pp. 235-291.. C. RATLEDGE, Single cell oils for the 21 st century, in Z. Cohen & C. Ratledge (Eds.), Single cell oils, Illinois, US: AOCS Press, 25, pp. 1-2. 5. J.B. RATTRAY, A. SCHIBECI, D.K. KIDBY, Lipids of yeasts, Bacteriol. Rev., 39(3), pp. 197, 1975. 6. A. BEOPOULOS, Z. MROZOVA, F. THEVENIEAU, M.T. LE DALL, I. HAPALA, S. PAPANIKOLAOU, T. CHARDOT, Control of Lipid Accumulation in the Yeast Yarrowia lipolytica, Applied and Environmental Microbiology, 7(2), pp. 7779-779, 2. 7. S. PAPANIKOLAOU, I. CHEVALOT, M. KOMAITIS, I. MARC, G. AGGELIS, Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures, Appl. Microbiol. Biotechnol, 5, pp. 3-3, 22.. S. PAPANIKOLAOU, G. AGGELIS, Lipids of oleaginous yeasts. Part I: Biochemistry of single cell oil production, European Journal of Lipid Science and Technology, 113(), pp. 131-151, 211. 9. A. BEOPOULOS, J. CESCUT, R. HADDOUCHE, J.L. URIBELARREA, C. MOLINA-JOUVE, J.M. NICAUD, Yarrowia lipolytica as a model for bio-oil production, Progress in Lipid Research,, 375-37, 29. Romanian Biotechnological Letters, Vol. 2, No. 6, 215 193

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