Growth and Lipid Production of L. Starkeyi Mutants

Growth and Lipid Production of L. Starkeyi MutantsCHAPTER 1INTRODUCTION diesel motor is whizz of the components in fossil fuel. However, the over-use of diesel is producing greenhouse gases such as degree Celsius dioxide gases which be the major elements leading to global warming. Hence, due to add-on in bring and ascendent limitation, biodiesel is introduced as a substitute for diesel fuel ( brainsick et al., 2010).Biodiesel is a diesel fuel substitute that is extracted from renewable biomass. Biodiesel notify be produced from go under oils, animal fill outs and microorganisms. Traditionally, biodiesel is produced from plant oils which were transesterify with methanol (Dai et al., 2007). However, occupation of biodiesel from plant oils is not suitable due to the quality of tillable land (Li et al., 2008) and competitor with food production (Wahlen et al., 2012). Further much, the growing in animal fertiles prices due to the increase in animal feed makes it not suitable as biodiesel feedstock (Li et al., 2008). Hence, buttery microorganisms flummox been introduced as good candidates for biodiesel feedstock.Oleaginous microorganisms can accumulate lipid up to 20% of its cell alter angle (Ageitos et al., 2011). Oleaginous microorganisms need the ability to utilize different snow source (Ageitos et al., 2011). In this study, Lipomyces starkeyi exit be employ. 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 wrong 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 depart be using L. starkeyi mutants in an attempt to produce more lipid more lipid in the fungal cells.The L. starkeyi mutants depart be polished 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 wastewaters in separated schott bottles. pH 5 and pH 6 testament overly be apply in order to optimize the production of lipid. The temperature that forget be apply is room temperature ( 27C). 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 depart be testinged using carbolic acid-sulphuric test.Our objectives in this research areTo optimize increment and lipid production of L. starkeyi mutantsTo measure the nub of lipid produced by L. starkeyi mutants well-bred in 2.5 % and 5 % of glucose mediumTo measure the amount of lipid produced by L. starkeyi mutants obliging in sago effluentCHAPTER 2LITERATURE REVIEW2.1 BiodieselBiodiesel consists of alkly ester of dipper acids or triglycerides. Conventionall y, triglyceride is produced from soybeans oil with the addition of alcohol and acid or abode catalyst. This process is known as transesterifications which give produce Fatty social disease 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, sunf imprinter 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 disturb 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, insincere microorganisms stand out as a potential feedstock provider.2.2 Oleaginous microorga nismsOleginous yeasts (OY) are known producers of single cell oil (SCO). SCO produced from this organism are triacylglycerides (TAG) that charter 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 and so the cost to culture this microorganism is low (El-Fadaly et al., 2009).There are four groups of oleaginous microorganisms that confident of producing biodiesel which are bacteria, algae, threadlike 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 surname 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 docile to culture compared to early(a) microalgae. Other than that, certain oily yeast also has the ability to produce lipid up to 80% of their dry weight, magic spell utilizing different carbon source including the lipid present in media (Ageitos et al., 2011).2.3 Factors affect lipid accumulations in Oleginous yeastLipid accumulations occur when yeast is cultured under heights 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 rest substrate would be assimilated by the yeasts 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 ino rganic salts in media. These elements help in ATP (AdenosineTriPhosphate) citrate lyse which important in lipid production (Ageitos et al., 2011).2.4 Lipomyces starkeyiL. starkeyi is one of the members of Saccharomycetales and considered as true inhabitant of soil which have a cosmopolitan 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 go away receive the overabundance carbon source in the form of triglycerides (TAGs) (Ageitos et al., 2011)2.5 Sago effluentSago effluent is a form of sago liquid waste. In normal processes, this ef fluent 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- stiffen 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 lurk usually have the ratio of carbon to nitrogen high which is cv 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 testPhenol-sulphuric test is the quantitative assays which often used in estimation of carbohydrate. This test could come across 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 3MATERIALS AND METHOD3.1 MaterialsModified 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 stockA single dependency of L. starkeyi mutants R3 will be inoculated into 100 ml of modified media. 800 l of L. starkeyi mutants R3 that have grown will be transferred into vial that contained 1200 l of glycerin 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 cell1.5 L of modified media with pH 5 will be prepared into 2 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, common chord (3) falcon tubes will be autoclave and weight. After that, 50 ml of the cultured from first bottle will be transferred into each ternary (3) falcon tubes and it will be burthen again. The sample will be sent for centrifuge for 5 minutes at 5000 rpm. The supported 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. starkeyi1 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 say 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 l from each sample will be taken and poured onto plate take care agar. The plate will be incubated overnight in front colony tally will be performed.3.5 Lipid accumulation stage for L. starkeyi mutantsThe 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 utmost concentration of 5% (w/v) in the schott bottle and it will be incubated raise 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 take biomassThe 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 extractionHexane propanol in the ratio of 32 will be added into the falcon tubes consists of the dry mass. The salmagundi 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 s upernatant 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 testPhenol 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 dissipated cuvette and read at 490 nm in a spectrophotometer.EXPECTED subjectBy 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 documentProject Activities20142015AugSeptOctNovDecJanFebMarchAprMayData gathering-Proposal writing and presentation---Bench work and sample touch on----Progress report----Data analysis---Data validation statistical analysis----Report writing and presentation-----Legends- In progress- barricade of progressREFERENCESAgeitos, J.M., Vallejo, J.A., Veiga-Crespo, P., Villa, T.G. (2011). oil coloury 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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