Growth and Lipid Production of L. Starkeyi Mutants

Growth and Lipid Production of L. Starkeyi Mutants CHAPTER 1 INTRODUCTION Diesel is one of the components in fossil fuel. However, the over-use of diesel is producing greenhouse gases such as carbon dioxide gases which are the major elements leading to global warming. Hence, due to increase in demand and source limitation, biodiesel is introduced as a substitute for diesel fuel (Wild et al., 2010). Biodiesel is a diesel fuel substitute that is extracted from renewable biomass. Biodiesel can be produced from plant oils, animal fats and microorganisms. Traditionally, biodiesel is produced from plant oils which were transesterify with methanol (Dai et al., 2007). However, production of biodiesel from plant oils is not suitable due to the quality of tillable land (Li et al., 2008) and competition with food production (Wahlen et al., 2012). Furthermore, the increase in animal fats prices due to the increase in animal feed makes it not suitable as biodiesel feedstock (Li et al., 2008). Hence, oleaginous microorganisms have been introduced as good candidates for biodiesel feedstock. Oleaginous microorganisms can accumulate lipid up to 20% of its cell dry weight (Ageitos et al., 2011). Oleaginous microorganisms have the ability to utilize different carbon source (Ageitos et al., 2011). In this study, Lipomyces starkeyi will be used. This type of yeast has the ability to produce lipid up to 70 % of its cell dry weight (Wild et al., 2010). L. starkeyi can utilize different types of carbon as its sole carbon and it is flexible in terms of culture conditions (Ageitos et al.,2011). However, L. starkeyi is still not economically practical because of the limitations in the wild-type strains (Ageitos et al., 2011). Therefore, in our research, we will be using L. starkeyi mutants in an attempt to produce more lipid more lipid in the fungal cells. The L. starkeyi mutants will be cultured in modified media consists of glucose, (NH4) SO4, yeast extract, Na2HPO4.7H20, KH2PO4, MgSO4. 7H20, CaCl2. 2H20, FeSO4, ZnSO4.H20 and CuSO4 supplied with 2.5% (w/v) and 5.0% (w/v) of glucose and sago effluents in separated schott bottles. pH 5 and pH 6 will also be used in order to optimize the production of lipid. The temperature that will be used is room temperature ( ± 27 °C). In this experiment, sago effluent and glucose would serve as carbon source for L. starkeyi. The total carbohydrate that would be consumed by L. starkeyi will be tested using phenol-sulphuric test. Our objectives in this research are: To optimize growth and lipid production of L. starkeyi mutants To measure the amount of lipid produced by L. starkeyi mutants cultured in 2.5 % and 5 % of glucose medium To measure the amount of lipid produced by L. starkeyi mutants cultured in sago effluent CHAPTER 2:  LITERATURE REVIEW 2.1 Biodiesel Biodiesel consists of alkly ester of fatty acids or triglycerides. Conventionally, triglyceride is produced from soybeans oil with the addition of alcohol and acid or base catalyst. This process is known as transesterifications which will produce Fatty Acid Methyl Ester (FAME) (Wahlen et al., 2012). Basically, biodiesel can be derived from 3 sources which are plants oil, animal fat and microorganisms (Meng et al., 2008). Plant oils that involve in the production of biodiesel are rapeseed, palm oil, soybeans, cottonseed, sunflower and many possible crops (Perritano, 2010). However, the practical used of plant oils raises critical issues on the decreasing in quality of land that is needed to plant the crops could affect the quality of the crops produced (Li et al., 2008). In addition, it also competes with the food production (Wahlen et al., 2012). Animal fat is also not a good biodiesel feedstock due to economical reasons (Meng et al., 2008). Hence, oleaginous microorganisms stand out as a potential feedstock provider. 2.2 Oleaginous microorganisms Oleginous yeasts (OY) are known producers of single cell oil (SCO). SCO produced from this organism are triacylglycerides (TAG) that have long-chain of fatty acids and have similar properties with plant oils. TAG acts as source of energy and it assist in phospholipid membrane formation. OY also utilizes various its carbon sources from waste substrate thus the cost to culture this microorganism is low (El-Fadaly et al., 2009). There are four groups of oleaginous microorganisms that capable of producing biodiesel which are bacteria, algae, filamentous fungi and yeast (Kitcha and Cheirsilp, 2011). The genera of oleaginous yeast are Yarrowia, Candida, Rhodotorula, Rhodosporium, Crytococcus, Trichosporon and Lipomyces (Ageitos et al., 2011). The specific name for the most preferable candidates for production of lipid are Cryptococcus albidus, Rhodosporidium toruloides, Rhodotorula glutinis, Lipomyces starkeyi and Yarrowia lipolytica. These microorganisms are capable of producing intracellular lipid more than 20% of its cell dry weight (Tapia et al., 2012). The duplication rate of yeast is lower than 1 hour and it is easy to culture compared to other microalgae. Other than that, certain oily yeast also has the ability to produce lipid up to 80% of their dry weight, while utilizing different carbon source including the lipid present in media (Ageitos et al., 2011). 2.3 Factors affecting lipid accumulations in Oleginous yeast Lipid accumulations occur when yeast is cultured under high amount of carbon source but in limited source of nitrogen. This is due to the nutrient imbalance that helps in triggering the accumulation of lipid because the remaining substrate would be assimilated by the yeast’s cells hence convert it into fat for storage (Ageitos et al., 2011). The fat that accumulated could be extracted to produce biodiesel. In addition, the accumulations of lipid also affected by other factors such as the present of microelements and inorganic salts in media. These elements help in ATP (AdenosineTriPhosphate) citrate lyse which important in lipid production (Ageitos et al., 2011). 2.4 Lipomyces starkeyi L. starkeyi is one of the members of Saccharomycetales and considered as true inhabitant of soil which have a worldwide distribution (Ansschau et al., 2014). L. starkeyi have the ability to accumulate lipid up to 70% of its dry weight (Wild et al., 2010). It also has a high flexibility in utilization of carbon source and culture environment. Other than that, fatty acid produced by L. starkeyi is almost similar to the vegetable oil (Tapia et al., 2012). According to Wild et al. (2010), L. starkeyi need a high ratio of carbon to nitrogen in order to optimize the production of lipid. The lipid bodies (LB) of L. starkeyi will receive the excess carbon source in the form of triglycerides (TAGs) (Ageitos et al., 2011) 2.5 Sago effluent Sago effluent is a form of sago liquid waste. In normal processes, this effluent would be channeled into the river, thus polluting the river and environment (Awang-Adeni et al., 2010). The releasing of sago effluent into the river can cause decreasing in water pH and increase in biochemical oxygen demand (BOD) and chemical oxygen demand (COD) (Ayyasamy et al., 2008) Sago effluent contains a high amount of organic materials and non-starch polysaccharide (NSP) (Awang-Adeni et al., 2010). NSP are made of cellulose, hemicellulose and lignin. In cellulose, the sub-components are 89% glucose and small amount of xylose, rhamnose, arabinose, mannose, fructose and galactose. In contrast to cellulose, hemicellulose main components are glucose and xylose accompanied with arabinose, galactose, rhmnose, fucose and uranic acid. Lignin functions in rigidity and stability of the wood. To sum up, sago effluent contains up to 66% of starch, 14 % fiber and 25 % lignin (Awang-Adeni et al., 2010). Sago effluents which flow from the sago mill usually have the ratio of carbon to nitrogen high which is 105: 0.12 (Awang-Adeni et al., 2010). As stated by Ageitos et al. (2011), L. starkeyi have the ability to utilize starch as its sole carbon. Hence, sago effluent is an excellent choice because it has a high amount of starch which can helps in optimizing the lipid production. 2.6 Phenol-sulphuric test Phenol-sulphuric test is the quantitative assays which often used in estimation of carbohydrate. This test could detect the presence of neutral sugar in oligosaccharides, proteoglycan, glycoproteins and glycolipids (Albalasmeh et al., 2013). When phenol-sulphuric is added, the glucose that presence in samples would dehydrate thus forms hydroxymethyl furfurax. It would yield a yellow-brown product and the OD could be checked at 490 nm (Albalasmeh et al., 2013). CHAPTER 3:  MATERIALS AND METHOD 3.1 Materials Modified media as suggested by Wild et al. (2010). Lipomyces Starkeyi mutants (LS R1 and LS R2) 2.5 % (w/v) and 5.0 % (w/v) of glucose (Ee Syn, Malaysia) 2.5 % (w/v) and 5.0 % (w/v) of sago effluent (Pusa, Malaysia) 80 % (w/v) of Glycerol stock (HmbG, Germany) 5 % Phenol (Nacalai Tesque, Japan) Hexane (Reagents, USA) Isopropanol (Amresco, USA) Microcentrifuge (Hettich EBA 21, England) Schotts bottles (Duran, Germany) 3.2 Glycerol stock A single colony of L. starkeyi mutants R3 will be inoculated into 100 ml of modified media. 800 ÃŽ ¼l of L. starkeyi mutants R3 that have grown will be transferred into vial that contained 1200 ÃŽ ¼l of glycerol stock. The glycerol stock steps of L. starkeyi will be repeated for L. starkeyi mutants R4. The solution will be stored in freezer at -20  °C. 3.3 Propagation of cell 1.5 L of modified media with pH 5 will be prepared into two Liter schott bottles and L. starkeyi mutants R3 and R4 will be inoculated in respective bottles (Wild et al., 2010). This step will be repeated for pH 6. For day 1 until day 6, three (3) falcon tubes will be autoclave and weight. After that, 50 ml of the cultured from first bottle will be transferred into each three (3) falcon tubes and it will be weighted again. The sample will be sent for centrifuge for 5 minutes at 5000 rpm. The supernatant will be discarded and the pellet with falcon tube will be weight again for its wet weight. The sample will be dry in the oven for 1 or 2 days. After that, the sample will be weight again for its dry weight. All experiments will be performed in duplications. 3.4 Standard curve for L. starkeyi 1 ml of culture which will be incubated for 3 days earlier will be added into 9 ml of modified media in test tube. Serial dilution will take place with the factors of 10-1 until 10-7. For factors of 10-1 until 10-7, their OD will be checked for 600 nm. For factors 10-5 until 10-7, 300 ÃŽ ¼l from each sample will be taken and poured onto plate count agar. The plate will be incubated overnight before colony counting will be performed. 3.5 Lipid accumulation stage for L. starkeyi mutants The L. starkeyi mutants culture will be incubated for 3 days (optimum growth) at room temperature. After 3 days, 750 ml of 10.0% (w/v) of glucose will be added into 750 ml modified media to achieve final concentration of 5% (w/v) in the schott bottle and it will be incubated further for 6 days. From day 1 to day 6, 150 ml of cultured will be harvested into each three (3) falcon tubes. This step will be repeated for pH 5 with 5.0% (w/v) of glucose and pH 6 with 10.0% (w/v) and 5.0% (w/v) of sago effluent. 3.6 Sampling biomass The samples will be weighted in wet condition before dry in the oven. After that, the samples will be dried in the oven for 3 days. The dried mass will be taken and weighted again for dry weight. 3.7 Lipid extraction Hexane: propanol in the ratio of 3:2 will be added into the falcon tubes consists of the dry mass. The mixture will be homogenized for 2 minutes. The homogenized sample will be incubated for 1 hour before centrifuge for 5 minutes. The supernatant will be taken and placed in an empty beaker and weight. The supernatant will be heated until the hexane and propanol solution have evaporated completely. The remaining oil will be weighted again. This step will be repeated for 5.0% (w/v) of glucose, 2.5% (w/v) of sago effluent and 5.0% (w/v) of sago effluent. 3.8 Phenol-sulphuric carbohydrate test Phenol test is used to detect the amount of carbohydrate that is not consumed by L. starkeyi. For each sample, phenol-sulphuric carbohydrate test will be performed by adding 0.2 ml of 5% (w/v) of phenol and 1 ml of 96% (w/v) of sulphuric acid. After that, 1 ml from each mixture will be placed into a clean cuvette and read at 490 nm in a spectrophotometer. EXPECTED OUTCOME By the end of this experiment, we expect to measure the amount of lipid produced by Lipomyces starkeyi mutants in 2.5% (w/v) and 5.0% (w/v) concentration of glucose and sago effluent at different pH. WORK SCHEDULE Project Activities 2014 2015 Aug Sept Oct Nov Dec Jan Feb March Apr May Data collection à ¢-  Proposal writing and presentation à ¢- º à ¢- º à ¢-  Bench work and sample processing à ¢- º à ¢- º à ¢- º à ¢-  Progress report à ¢- º à ¢- º à ¢- º à ¢-  Data analysis à ¢- º à ¢- º à ¢-  Data validation: Statistical analysis à ¢- º à ¢- º à ¢- º à ¢-  Report writing and presentation à ¢- º à ¢- º à ¢- º à ¢- º à ¢-  Legends à ¢- º: In progress à ¢-  : End of progress REFERENCES Ageitos, J.M., Vallejo, J.A., Veiga-Crespo, P., Villa, T.G. (2011). Oily yeast as oleaginouscell factories. Applied Microbiology and Biotechnoogy, 90(12), 1219-1227. Albalasmeh, A.A., Berhe, A.A., Ghezzehei, T.A. (2013). 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