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The Faculty of Natural Resources and Agricultural Sciences A comparative study on four oleaginous yeasts on their lipid accumulating capacity

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The Faculty of Natural Resources and Agricultural Sciences A comparative study on four oleaginous yeasts on their lipid accumulating capacity Qier Sha Department of Microbiology Master s thesis 45 hec
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The Faculty of Natural Resources and Agricultural Sciences A comparative study on four oleaginous yeasts on their lipid accumulating capacity Qier Sha Department of Microbiology Master s thesis 45 hec Second cycle, A2E Biology Examensarbete/Sveriges lantbruksuniversitet, Institutionen för mikrobiologi: 213:7 ISSN Uppsala 213 A comparative study on four oleaginous yeasts on their lipid accumulating capacity Qier Sha Supervisor: Examiner: Johanna Blomqvist, Swedish University of Agricultural Sciences, Department of Microbiology Volkmar Passoth, Swedish University of Agricultural Sciences, Department of Microbiology Credits: 45 hec Level: Second cycle, A2E Course title: Independent project in Biology - Master's thesis Course code: EX596 Programme/education: Biotechnology Place of publication: Uppsala Year of publication: 213 Title of series: Examensarbete/Sveriges lantbruksuniversitet, Institutionen för mikrobiologi: no: 213:7 ISSN: Online publication: Key Words: Biodiesel; Yeast; Strains; Glucose; Xylose; Fermentation; Cell dry weight; Lipid content; Lipid yield; Optical density Sveriges lantbruksuniversitet Swedish University of Agricultural Sciences The Faculty of Natural Resources and Agricultural Sciences Uppsala BioCenter Department of Microbiology Abstract Under the premise of high energy price with estimated petroleum crisis, biodiesel extracted from plant oil, animal fat and microorganisms is considered as a promising replacement for fossil raw material. However, the increasing price of animal feeding and decreasing tillable land for oil plants have brought great difficulty to achieve biodiesel from feedstock plant oil and animal fat. In this study, four oleaginous yeasts including Lipomyces lipofer, Lipomyces starkeyi, Rhodotorula glutinis and Yarrowia lipolytica were compared for their lipid accumulating productivity in different carbon sources: glucose, xylose and a mixture of both glucose and xylose. According to the experimental results, Rhodotorula glutinis had the highest lipid yield in both glucose sole carbon source and mixed sugar in shake flask test. Lipomyces starkeyi was able to utilize xylose as sole carbon source to accumulate lipid with highest yield in shake flask test. In comparison with Lipomyces lipofer, Lipomyces starkeyi had higher lipid yield in both shake flask test and fermentation bioreactors. Yarrowia lipolytica could not utilize xylose sole carbon source and its lipid yield was the lowest one when compared to other three oleaginous yeasts. During fermentation in the bioreactor test, lipid content of Lipomyces lipofer showed a maximum, later the lipid content decreased. A similar peak of lipid content was observed in the fermentation test in bioreactor of Lipomyces starkeyi, but after a temporary decrease the lipid content increased again. Lipid content reached its highest amount at early stationary phase and decreased may be due to lipid remobilization. Table of Contents 1. Introduction Feedstock for biodiesel Oleaginous yeasts Fatty acid metabolism Influencing factors for lipid accumulation Nitrogen and sulfate limitation Minerals and other growth factors Temperature ph value Aeration Culturing time Study aim Material and Methods Media Pre-culture Main culture Yeasts used in the study Growth curve determination Inoculums preparation Shake flask culturing Freeze drying Lipid extraction Dry weight determination Bioreactor test HPLC analysis Lipid composition analysis Results Growth curve determination Shake flask batch test in different sugars Cell dry weight and lipid weight comparison Lipid content and lipid yield comparison Lipid yield comparison in different sugars Fermentation test Optical density, cell weight, lipid content and lipid weight Lipid content and sugar consumption during fermentation Fatty acid composition analysis Fatty acid composition from shake flask test Fatty acid composition change during fermentation Dry weight determination...23 4. Discussion Culturing condition was important Dry weight determination needed other solution Further study is required Conclusions and Future perspectives Acknowledgment References...27 Appendix...29 1. Introduction Because the global crude oil reserves are shrinking there is a globally increasing interest in developing substitutable renewable energy materials. Considering energy price and environmental concerns biodiesel had been regarded as one of the most promising potential substitute for traditional energy oil. The advantages of biodiesel is that it does not contain any polycyclic hydrocarbons which can be damaging to a wide variety of substances, so that biodiesel is less corrosive and thus easier to transport and store than conventional petroleum. Due to its ability to mix with petroleum diesel in any ratio and still to function, biodiesel can be considered as clean and renewable first alternative solution of oily energy resource (Sheedlo, 28). It has great features for example, higher energy density, lower S content than conventional petroleum with sufficient burning ability and lubricating property. It also has the characteristic that it is easily biodegradable, safe to transport and hardly to explode (Tyson et al., 1998). 1.1 Feedstock for biodiesel A variety of feedstock can be used to produce biodiesel. The most commonly used vegetable feedstock includes rapeseed and soybeans. The first amount of oil extracted from feedstock which was called virgin oil also could be obtained from other crops such as mustard, sunflower and coconut (Antolin et al., 22). Animal fats were also good choice for feedstock, which included tallow, lard and chicken fat etc (Lang et al., 21). Recently, microorganisms have been studied for their potential to be used for producing biodiesel with similar composition as oil from plants and animal fats. Microorganisms available for biodiesel production included bacteria, algae, filamentous fungi and yeasts. Some waste material such as sewage can be used as substrate for growing algae to produce biodiesel (Yusuf et al., 27). In the oleaginous fungus Mucor circinelloides lipid content up to 3 % was found when cultured in high carbon: nitrogen ratio and high temperature condition. The advantages of fungi fermentation was that very easy to harvest because of the formation of cell pellets (Xia et al., 211). Nowadays, efficient generation of lipids could be obtained from cheap and abundant material such as lignocellulosic materials. This kind of feedstock could be from agricultural residues such as corn stover, wood residues such as sawdust or paper mill residues, and energy crops such as willow and poplar. Lignocellulosic materials are composed of carbohydrate polymers (cellulose and hemicellulose), and lignin. The cellulose and hemicelluloses typically comprise up to two thirds of the lignocellulosic materials. Hemicelluloses consist of pentose (typically xylose) and hexose (typically glucose). Depending on the process and conditions used during pre-treatment, hemicelluloses can be hydrolyzed into monomeric and oligomeric forms which are the main substrates for biodiesel production and directly influence the lipid yield potential (Girio et al., 21). Some research reports have been published that are describing oleaginous yeasts converting lignocellulosic materials to biodiesel which has potential 7 developing prospect. Biodiesel production produced by various microorganisms is now called Single cell oils (Ratledge et al., 28). 1.2 Oleaginous yeasts Some oleaginous yeasts have been reported to accumulate lipids up to almost 7 % of their cell dry weight when cultured under nitrogen limited condition. These yeasts belong to genera like Rhodosporidium, Rhodotorula, Yarrowia, Candida, Cryptococcus, Trichosporon and Lipomyces (Ageitos et al., 211). Lipids synthesized by oleaginous yeasts mainly contained stearic (18:), oleic (18:1) and linoleic (18:2) acids together with palmitic acid (16:), which was almost similar to the oily product from plants, which means that yeast lipids could be used for the same purpose as biodiesel from plants (Xin et al., 29). Table 1: Common fatty acids of yeast accumulating lipids were summarized in Table 1 (Lin et al., 211). Fatty acid Palmitic (16:) Stearic (18:) Oleic (18:1)(n-9) Linoleic (18:2)(n-6) Linolenic (18:3)(n-3) 1.3 Fatty acid metabolism Chemical structure CH 3 (CH 2 ) 14 COOH CH 3 (CH 2 ) 16 COOH CH 3 (CH 2 ) 7 CH=CH(CH 2 ) 7 COOH CH 3 (CH 2 ) 4 CH=CHCH 2 CH=CH(CH 2 ) 7 COOH CH 3 CH 2 CH=CHCH 2 CH=CHCH 2 CH=CH(CH 2 ) 7 COOH Mainly, under nitrogen-limited condition, the first step of lipid accumulation is an increase in glucose catabolism through pentose phosphate pathway from glycolysis to pyruvate which is the basic substrate for fatty acid synthesis in both cytosol and mitochondria. Nitrogen limitation in the culture directly results in decreasing of the concentration of AMP: adenosine monophosphate because of de-amination of adenosine to inosine by AMP deaminase. Isocitrate dehydrogenase is an enzyme of the citrate cycle (TCC) which is highly dependent on AMP. Thus decreasing concentration of AMP leads to increasing of the concentration of isocitrate which is then converted to citrate (Ageitos et al., 211). Citrate is transported out of the mitochondria and in the cytoplasm converted to Acetyl-CoA and oxaloacetate, by the ATP: citrate lyase (ACL). In the transhydrogenase cycle pyruvate is carboxylated to oxaloacetate, which is then converted to malate (NADH-dependent). Malic enzyme converts malate to pyruvate, generating NADPH and releasing CO 2. NADPH is very important for fatty acid synthesis, because each step of the carbon chain elongation needs two molecules of NADPH (Beopoulos et al., 211). Acetyl-CoA is the initial biosynthetic unit for fatty acid synthesis which is converted to acyl-coa. Glycerol 3 phosphate (G3P) as the acyl acceptor which is followed by Kennedy pathway by using two acyltransferases to form lysophospatidic acid (LPA), and phosphatidic acid (PA) step by step. Then PA is 8 dephosphorylated to release diacylglycerol (DAG). Finally DAG is acylated to produce triacylglycerol (TAG).The final step in biodiesel production is the chemical conversion of these neutral lipids into fatty acid short-chain alcohol esters (Kosa et al., 21). 1.4 Influencing factors for lipid accumulation Nitrogen and sulfate limitation Lipid accumulation in oleaginous yeasts occurs at a surplus of carbon source with one limiting element usually nitrogen limitation (Wu et al., 211). Common nitrogen source for yeast accumulating lipids can be yeast extract, peptone, and ammonium sulfate. It has been reported that using yeast extract containing broth as a nitrogen source is the most common way to culture yeasts and this can contribute to produce high amount of cell biomass and lipid content for example around 24 % of lipid in Rhodotorula glutinis and 22 % of it in Lipomyces starkeyi (Kumar et al., 21). Sulfate limitation in the medium is another influencing factor which can affect the biosynthesis of some special amino acids and cellular cofactors such as cysteine, biotin, thiamine and iron-sulfur clusters. Limitation for synthesis of these cellular assemblies could influence cell growth greatly and induce lipid accumulation. (Scott et al., 27) It has been reported that sulfate limitation can directly influence fatty acid composition of the total lipid. Main fatty acids component- cocoa butter equivalent content was increased from 47 % to 63 % when Rhodosporidium toruloides was cultured under sulfate limitation (Wu et al., 211) Minerals and other growth factors Mineral elements such as Magnesium, Potassium, and Calcium etc. are normally essential for yeast growth or lipid accumulation at lower concentration (Spencer et al., 1997). Other compounds also affect lipid accumulation in the yeast cells for instance inositol, pantothenic acid and biotin omission results in a decrease of the total lipid amount (Rattray et al., 1975). It has been recorded that Vitamin B 6 absence of medium leaded to decreasing of total lipids level with dramatically reduction on Palmitoleic acid by Hanseniaspora valbyensis (Betty et al., 1965) Temperature Optimal temperature for high growth rate for each yeast strain is different but generally around 25 ºC. Too high or too low temperature affects the cell growth and lilpid accumulation. During yeast cells lipid accumulation, dropping temperature from optimal growth generally results in increasing of the lipid content and influence lipid composition. Usually, melting point of unsaturated fatty acids is lower than saturated fatty acids and for short chain fatty acids lower than long chain fatty acids. Thus, temperature decreasing results in increasing level of unsaturated and short chain fatty acids, for example increasing ratio of linoleic acid to oleic acid (Rattray et al., 1975). 9 1.4.4 ph value The optimal growth ph value for each yeast strain is different and varies from ph 3. to ph 7. (Rattray et al., 1975). However, it has been shown that the ph optimal for lipid accumulation is generally lower than that for optimal growth. For example, when using sewage sludge to accumulate lipids by Lipomyces starkeyi, highest lipid content was observed at ph 5. while the highest growth was at ph 6.5 (Angerbauer et al., 28). Another case is that highest lipid content occurred at ph 3.2 in Trichoderma reesei in glucose substrate while the highest growth was calculated at ph 4. (Brown et al., 199) Aeration Aeration is a very important factor for yeast cell growth as well as total lipid level. Dissolved oxygen amount in the culture could highly influence fatty acids composition in lipids. Under oxygen limited conditions, the glyceride fraction varies highly and the amounts of phospholipids and sterol decrease. These variations directly result in increasing amounts of saturated fatty acids, which become the main component of lipids. In an aerated culture, cellular viability increased and free fatty acids were oxidized to unsaturated fatty acids since yeast cells need unsaturated fatty acids for continued growth. For example after input of plenty of oxygen to a culture contents of linolenic acid was highest in Candida utilis (Babij et al., 1969) and palmitoleic acid was highest in Saccharomyces cerevisiae (Valero et al., 21) Culturing time Previous studies have shown that lipid accumulation in the yeast cell dramatically increased in the logarithmic growth period because lipids are used for membrane lipid synthesis to support cellular growth in size first. When cells reach their maximum size they turn to accumulate lipids as an oil bubble inside the cell. Cell lipids content reached highest value at stationary phase and upon nutrient depletion fatty acid accumulation rate decreased gradually until cells exit starvation, accumulated lipids will degraded to free fatty acid rapidly. Based on upon, harvesting cell time is better at early stationary phase and prevents lipids degradation (Beopoulos et al., 28). 1.5 Study aim Several oleaginous yeasts have been reported which could accumulate lipids under nitrogen limited condition. In this study, lipids accumulation potential of several oleaginous yeasts from glucose and xylose, and indentifying the influence of cultivation factors like temperature on lipid accumulation was tested in lab work. Quantitative work of cell dry weight determination was made as well. 1 2. Material and methods 2.1 Media Pre-culture YM medium was used as pre-culture to cultivate yeast cells. It contained glucose as carbon source 1 g/l, yeast extract 3 g/l, malt extract 3 g/l and peptone 5 g/l. To obtain solid medium, 16 g/l agar was added. High temperature moist heat sterilization method was used for medium sterilization, 12 ºC temperature for 2 minutes Main culture Semi-synthetic medium was prepared to grow oleaginous yeasts. Glucose, xylose or a mixture of both were used as carbon sources with concentration 7 g/l, nitrogen sources (yeast extract.75 g/l and NH 4 Cl.1 g/l), MgCl 2 6H 2 O 1 g/l, Na 2 SO 4.1 g/l, and phosphate buffer (KH 2 PO g/l and K 2 HPO 4 3H 2 O 3.7 g/l or K 2 HPO g/l). Mineral-element solution was a of: CaCl 2 2H 2 O 4 mg/l, FeSO 4 7H 2 O 5.5 mg/l, citric acid H 2 O 5.2 mg/l, ZnSO 4 7H 2 O 1. mg/l, MnSO 4 H 2 O.76 mg/l and 18 mol/l of H 2 SO 4 diluted by mg/l. Medium ph was measured as 5.8 by ph meter (based on high buffering properties of the medium itself, it is possibly no chance to adjust its ph value without influencing liquid volume) (Hu et al. 211). 2.2 Yeasts used in the study Lipomyces lipofer CBS 5842 and Lipomyces starkeyi CBS 187 were ordered from CBS. Rhodotorula glutinis J195, Yarrowia lipolytica J134 were from obtained from the strain collection of the Department of Microbiology. All yeasts were grown on solid YM medium and incubated in 25 C room. When yeast colonies became visible, then culture plates were stored in 2 C until use. 2.3 Growth curve determination All yeasts were cultured in YM medium and optical density (OD) was measured every 2 hours in an Ultrospec 11 Pro, Biochrom (Agilent, Germany) spectrophotometer at wave length 6 nm. Clear YM medium was used as blank for the OD measurement. For getting appropriate OD values which was between.1-.5 for accurate readings, the cell suspensions were diluted 1, 2 or 1 times if required. Yeasts were inoculated in two batches of pre-cultures, cultivated at different temperatures (25 and 3 ºC and samples were taken at different time points from the two cultures in order to get complementary data to complete the growth curve and identifying the impact of the growth temperature. 2.4 Inoculums preparation Cultures of yeasts were inoculated in liquid YM medium and incubated at 25 C before inoculation. Measuring OD of the culture and calculating the amount of cell suspension 11 needed in main culture to reach its OD to 1 were the next steps. An appropriate volume of cells was calculated and centrifuged at 8 g for 5 min and washed with normal saline (9 g/l NaCl) once and centrifuged again. The pellets were re-suspended in 1 ml normal saline and used as inoculums. 2.5 Shake flask culturing All four oleaginous yeasts studied during the project were inoculated in three different carbon source conditions (each experiment was performed in triplicates). 1 ml semi-synthetic medium was prepared in 5ml shake flasks. Inoculation cultures were shaking at 22 rpm and 25 C. Every 3-4 hours the OD values of the cultures were measured. At the same time, 1 ml sample was taken from each flask, and centrifuged at 13 g for 3 min. The supernatant was filter sterilized through a.2 μm filter to remove remaining particles. Samples were stored in 2 ml centrifuge tubes at -2 C until analyzed by HPLC. 2.6 Freeze drying 5 ml of yeast cells were harvested at the stationary phase. Collected samples were centrifuged at 4 g 1 min and washed with sterile water once, then centrifuged again. The pellets were transferred into pre-weighed vials and kept at -5 C until freeze drying. After 5 hours freeze drying, the vials were weighed again and the cell dry weight was determined. All determinations were performed in triplicates. 2.7 Lipid extraction Lipid extraction was performed using the chloroform- methanol method (Folch et al., 1957). Freeze-dried pellets were dissolved in 4 M HCl 3.2 ml and incubated at 55 C for two hours. Then 8 ml of chloroform: methanol mixture (1:1 as molarity) was added, transferred into glass tubes and shaken at 2 C for three hours. Then samples were centrifuged at 2 g for 15 min to obtain a clear chloroform phase (lower phase). The lower phase was carefully transferred into new tubes by using rubber head dropper. Then 4 ml chloroform was added into the chloroform phase and shaken vigorously. The new tubes were centrifuged once more. Clear chloroform was obtained and transferred into pre-weighted glass tubes. Then tubes were gassed with N 2 two hours to evaporate all
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