Using Waste Cooking Oil as Feedstock and Candida Antarctica

Production of Biodiesel by Enzymatic Transesterification employ extravagance Cooking Oil as feedstock and Candida Antarctica Lipase B as Biocatalyst. CHAPTER 1 INTRODUCTON The game speak to of bio-diesel, comp ard to petroleum-based diesel, is a major barrier to its commercialization. It has been inform that 60-90% of bio-diesel constitute arises from the cost of the feedstock fossil fossil petroleum (C. C. Lai et al. , 2005). Studies showed the potential of waste-cooking oil (WCO) as a real for biodiesel occupation (Sulaiman Al-Zuhair, 2008).Therefore, the handling of WCO should greatly turn off the cost of bio-diesel. In addition to the cream of lipase occupied, factors which make the transesterification work let out feasible and ready for commercialization ar enzyme modification, the option of feedstock and alcoholic drink, rehearse of super acid results, pretreatment of the lipase , alcohol to oil molar ratio, water activeness/content and response temperat ure. Optimization of these parameters is needful in order to reduce the cost of biodiesel drudgery.Use of no/low cost waste materials such as the WCO will have double environmental benefits by reducing the environmental contaminant potential of the wastes and producing an environmentally friendly fuel. In addition, take of bio-diesel from WCO is considered an st roamgic dance step in reducing and recycling waste. A fresh veg oil and its waste differ signifi brooktly in water and unaffixed oily acids (FFAs) contents, which are around 2000 ppm and 10-15%, respective(prenominal)ly (C. C. Lai et al. , 2005 Y. Zhang et al. , 2003). Beca intent of this the traditional footne-catalyzed biodiesel production is inapposite (Zhang et al. 2003). The use of the enzyme lipase as a biocatalyst for the transesterification answer step in biodiesel production has been extensively investigated. Lipase is fetchd by all living organisms and can be apply intracellularly or extracellularly. In order to design an stintingally and environmentally sustainable biodiesel production process, a proper understanding of the factors scratching the process and their congenator importance of enzyme-catalyzed biodiesel production is necessary. A general equation for transesterification (where group R is a fatty acid, R is the ength of the acyl acceptor and R is the rest of the triglyercide molecule) is as follows Methanol is the some(prenominal) popular alcohol utilise in the transesterification process because of its relatively cheaper price compared to opposite alcohols. When methanol is use in the process, the answer is known as methanolysis as shown in the following equation Lipases from microorganisms (bacterial and fungal) are the most apply as biocatalysts in biotechnological applications and organic chemistry. Fungal beginning lipases have been found to produce high yields of lipases compare to the animal and plants.Because their spate production is easier, comme rcialization of microbial lipases and their involvement in enzymatic biodiesel production are more common than animal and plant angiotensin-converting enzymes (Hasan et al. , 2006Akoh et al. , 2007 Antczak et al. , 2009). The lipase to be employed as the biocatalyst is Candida Antarctica lipase B (Novozyme CABL L), one of the most common fungal lipase used for the production of biodiesel (Vasudevan and Briggs, 2008). Lipases are capable of converting all the triglycerides derive from the feed stocks into their respective fatty acids methyl esters (FAMEs).They act on the ester bonds of carboxylic acids allowing them to carry out their primary reaction of hydrolyzing fats (Joseph et al. , 2008). Enzyme immobilisation is an important approach that could be used as a tool to improve and optimize operation stability, performance and selectivity which allows the enzyme to study under harsher environmental condition and also provides their interval from the reaction multifariousness w ithout filtration in case of packed bed reactor (Fernandez-Lafuente et al. , 1998 Bhushan et al. , 2009 Gao et al. 2006) and, hence, could lead-in to more favorable economical benefits. The cost of lipase makes up 90% of the congeries cost of enzymatic biodiesel production. A significant portion of that is associated with the use of pricy carrier or support materials (Dizge et al. , 2009a). Search for cheaper support materials has been ongoing in order to reduce the overall cost of enzymatic biodiesel production (Robles et al. , 2009). indeed it is important to immobilize lipase, to be able to recover and reuse it repeatedly ( D. S. Clark,1994D.Cowan, 1996). Immobilization of lipase is the attachment of the enzyme onto a solid support or the elbow grease of the enzyme in a region of space (Jegannathan et al. , 2008). When proper strategy for the lipase immobilization technology is employed , it provides a number of important benefits including (a)enzyme reuse, (b) easy of brea kup of product from enzyme and (c) the potential to run continuous processes via packed-bed reactors (Peilow and Misbah, 2001). Methods of immobilization include chemic and physical means.Among these, the physical immobilization by way of entrapment is the most widely-used method, in which enzymes are entrapped within the sol- gel matrix reard by hydrolysis and polycondensation of precursors (Ko Woon Lee, et al. 2010). Tetramethylorthosilicate (TMOS) is a widely used precursor for sol-gel immobilization of the enzyme. However, CALB is unstable and shows low catalytic readiness in the reaction media contains high denseness of methanol and the lipase is also sub overdue by the by-product of glycerol.To over comply this, an amphiphilic matrix is developed to immobilize the lipase ((Ko Woon Lee, et al. 2010). The use of solvent in the transesterification process is also considered. Solvents are used to shelter the enzyme from denaturation by alcohol by increasing alcohol solubili ty (Kumari et al. , 2009). The solvent can also increase the solubility of glycerol which is beneficial since the spin-off can coat the enzyme and inhibit its performance (Royon etal. , 2007 ).The use of a common solvent for the reactants and products non only reduces enzyme inhibition but also determines a unvarying reaction classification, reduces the reaction mixture viscosity and stabilizes the immobilized enzyme (Ranganathan et al. , 2008Fjerbaek et al. , 2009). This is beneficial because homogeneous reaction mixture decreases problems associated with a multiple phase reaction mixture and a reduced viscosity reduces mass transfer problems around the enzyme (Fjerbaek et al. , 2009). The use of solvents significantly increases the reaction rate in comparison to solvent abandon systems (Vasudevan and Briggs, 2008).Some study also showed that methanolysis conversion using Candida antarctica was increased when tert-butanol was added to the system (Royon et al. , 2007). This i nspection and repair as the basis for the choice of tert-butyl to be the solvent use in the system, in order to reduce the inhibition cause of the use of a lower image alcohol, in this case, the methanol. OBJECTIVES This study aims to produce economical extension of feedstocks such as waste-cooking oil for the production of biodiesel and the use of enzyme Candida Antarctica Lipase B, to catalyze to transterification reaction.To be able to determine the yield biodiesel through vaunt Chromatographic abbreviation (Chrompack CP 9001, Holland). SIGNIFICANCE OF THE STUDY Oil is one of the most commonly reported types of water pollution, causing nearly a quarter of all pollution incidents. Careless disposal of oil into drainage systems, onto land or to watercourses is an offense. It can harm river birds, fish and other wildlife. Because of the way oil spreads, even a small quantity can cause a lot of harm.It is estimated that UK caterers produce between 50 90 million litres of waste cooking oil individually year. If this is not disposed of correctly the effects of oil pollution on the environment could be quite devastating. According to the Environmental shield Agency (EPA), estimates that over 200 million gallons of used oil ends up in the trash, and poured into the water each year. This study aims to promote conventional and economic source for the production of biodiesel by using home waste material such as waste cooking oil.Thus, resolving high cost of biodiesel production making it commercially producible and reduce devastation of environment due to high consumption of crude oils from fossil sources. This study will be a significant endeavour in promoting the social needs and to solve the high prices of the gasoline which is the major economical crisis face in the innovate society. The advantages of using lipases in biodiesel production are (a) ability to work in very different media which include biphasic systems, monophasic system (in the presence of hydrophilic or hydrophobic (Am.J. Biochem. & Biotech. , 6 (2) 54-76, 2010), (b) they are robust and versatile enzymes that can be produce in bulk because of their extracellular nature in most producing system, (c) many lipases show considerable activity to catalyze transesterification with long or branched chain alcohols, which can hardly be converted to fatty acid esters in the presence of conventional alkaline catalysts, (d) products and byproduct separation in downstream process are xtremely easier, (e) the immobilization of lipases on a carrier has facilitated the repeated use of enzymes after removal from the reaction mixture and when the lipase is in a packed bed reactor, no separation is necessary after transesterification and (f) higher(prenominal) thermostability and short-chain alcohol-tolerant capabilities of lipase make it very convenient for use in biodiesel production (Bacovsky et al. , 2007 Kato et al. , 2007 Robles et al. ,2009). SCOPE AND LIMITATION Like any method for enzymatic biodiesel production, the cost of the lipase to be used is one of great consideration .The limitations of using lipases in biodiesel production include (a) initial activity may be lost because of volume of the oil molecule (Marchetti et al. , 2008 Robles et al. , 2009), (b) the use of solvent does not guarantee the wind up protection of enzyme from the inhibitory effect of low chain alcohol, methanol (c) Although lipase is not affected by the high content of FFAs in WCO, the high water content remains a problem (d) the lipase in the biodiesel production is limited on a specific feedstock to be used because of the regioselectivity of the enzyme lipase.CHAPTER 2 REVIEW OF RELATED LITERATURE Biodiesel has shown its ability tomeet the energy penury of the instauration in the transportation, agriculture, commercial and industrial sectors of the economy (Akoh et al. , 2007 Basha et al. , 2009 Shafiee and Topal, 2009 Robles et al. , 2009). The annual world consumption of diesel is approximately 934 million tons, of which Canada and the United States consume 2. 14 and 19. 06%, respectively (Marchetti et al. , 2008).As a green renewable and potentially unlimited, biodiesel has recently come out as the superlative alternative fuel which can be used in compression ignition engines with minor or no modifications (Xu and Wu, 2003 Vasudevan and Briggs, 2008 Robles et al. , 2009 Leung et al. , 2010). The concept of biofuel is not new. Rudolph Diesel was the firstly to use a vegetable oil (peanut oil) in a diesel engine in 1911 (Akoh et al. , 2007 Antczak et al. ,2009). The use of biofuels in place of conventional fuels would slow the rogression of global warming by reducing sulfur and carbon oxides and hydrocarbon emissions (Fjerbaek et al. , 2009). Because of economic benefits and more power output, biodiesel is very much blended with diesel fuel in ratios of 2, 5 and 20% (Vasudevan and Briggs, 2008). The higher the ratio of biodiesel to diesel the lower the carbon dioxide emission (Fukuda et al. , 2001 Harding et al. , 2007). Using a mixture containing 20% biodiesel reduces carbon dioxide net emissions by 15. 66% (Fukuda et al. 2001) turn using pure biodiesel makes the net emission of carbon dioxide zero (Vasudevan and Briggs, 2008). The simplest and most efficient route for biodiesel production in large quantities, against less ecofriendly, pricey and eventual low yield methods is transesterification. One of the classic organic reactions (transesterification) is the step wise reversible reactions of a triglyceride (fat/oil) with an alcohol to form esters and glycerol. Little inordinateness of alcohol is used to shift the equilibrium towards the formation of esters.Transesterification using an alcohol is a sequence of three reversible consecutive steps. In the first step, triglycerides are converted to diglycerides. In thesecond step, diglycerides are converted to monoglycerides. In the third step, monoglycerides are converted to glycerin molecules (Freedman et al. , 1984 Noureddini and Zhu, 1997 Marchetti et al. , 2008). Each conversion step yields one FAAE molecule, giving a total of three FAAEs per triglyceride molecule as describe by the following equations (Murugesan et al. , 2009). 1. Conversion of triglycerides to diglycerides . Conversion of diglycerides to monoglycerides 3. Conversion of monoglycerides tto glycerin molecules In order for the transesterification reaction to be applicable for biodiesel production, the process must be accelerated by the use of catalyst which may be alkaline, acids or enzymes (Bacovsky et al. , 2007 Murugesan et al. ,2009 et al. , 2010). The catalyst employed directly effects the purity of the feedstock required, the reaction rate and the extent of post reaction processing needed (McNeff et al. , 2008). To speed up the reaction, heat is also applied.However, this process is very energy intensive and incompetent since FAAE yield below 350C is very low and temperatur es higher up four hundredC degrade the ester bonds (Ranganathan et al. , 2008). Generally, the reaction mix is kept just above the boiling point of the alcohol (71-72C) to speed up the reaction. The variables known to affect the reaction are temperature, alcohol to oil molar ratio, catalyst concentration and mixing intensity (Marchetti et al. ,2007). Transesterification catalysts The transesterification process is catalyzed by alkalis, acids or enzymes.However, the use of alkali catalysts is 100% in commercial sector. The most common alkaline catalysts are sodium hydroxide (NaOH) METHODOLOGY * LIPASE CABL ( Novozyme CABL L) can be purchased from Novozyme (Denmark). All other chemicals can be purchased from Sigma- Aldrich (St. Louis, MO, USA). Grown in thelaboratory,Candidaappears as large, round, white or cream (albicansis fromLatinmeaning whitish) colonies with a yeasty odor onagar platesat populate temperature. IMMOBILIZATION OF LIPASE Sol gel immobilization in an amphiphilic m atrix was shown in figure below mL of CABL (8. 2 mg/ml) is to be placed in a 50-ml Falcon thermionic tube with 1-mL of 0. 2 M phosphate buffer (pH 7). As a catalyst, 50 microliter of 1M sodium fluoride is to be added and the mixture is to be shaken with a twisting mixer. Then, TMOS (2 mM) and the following hydrophobic alkylsilanes (8 mM) is added methyltrimethoxysilanes (MTMS), ethyltrimethoxysilane (ETMS), propyltrimethoxysilanes (PTMS), and iso-butyltrimethoxysilane (iso-BTMS). Gelation is usually observed within a few minutes while a reaction vessel is gently shaken.Following complete polymerization for 12 hours in a closed Falcon tube, the gel was dried for 24 hours in an open Falcon tube. The gel is to wash with 10 mL of distilled water, 10 mL of 99. 8% iso-propanol, and 10 mL of 95% n-hexane respectively. The immobilized CALB is to be filtered using filter paper, dried at 30 for oC for 36 hours and then ground with mortar and pestle. The particles were sorted using choleca lciferol and 300 micrometer sieves and stored at 4 oC until use. ENZYME SOLUTION Immobilized P. cepacia lipase solution is prepare by adding 0. g of lipase to 1 ml of distilled water and soak in water for 30 minutes, prior to being used. This step is found experimentally essential to activate the enzymes. WASTE-COOKING OIL PREPARATION In order to ensure consistency, waste cooking oil is simulated from the commercially available ornamentation oil by heating 1 L of palm oil on a hot plate (Stuart, U. K. ), set at its maximum heating power for ii hours. The oil is then allowed to cool to room temperature and then 5 ml of water (around 5000 ppm) is to be added. The sample is shelved for two weeks before being used.Fresh WCO samples were prepared every two weeks. Bio-Diesel Production in tert-butyl Solvent System Using C. Antarctica Lipase The experiment will be conducted in a specially designed 150 ml capacity jacketed reactor cell. The cell will be kept on a magnetic stirrer (Velp Scientifica, Italy) to facilitate the agitation of the mixture. Water from a temperature controlled water john (Grant Instruments, UK) circulated through the jacket and will be set to maintain the temperature of the reaction media constant at 45 oC.The temperature used was that presented in the literature to be the optimum(M. M. Soumanou,et al, 2003 H. Fukuda,et al, 2001 ) and an agitation speed was chosen to provide suitable mixing without modify the activity of the enzyme. In this part, the working volume at the beginning of each experiment was 50 ml, consisting of 5 g of WCO, different volumes of methanol, in the background of 0. 4 to 0. 8 ml (correspond to 0. 57 to 1. 14 molar equivalents of ester bonds in the triglyceride chain), and tert-butyl solution to make up the total volume.The cell is to be cover tightly throughout the progress of the experiments to prevent evaporation. After thermal equilibrium is ensured, 1 ml of enzyme solution containing 0. 4% g of C. Antarctica lipase per g oil is added to initiate the reaction. At suitable intervals, 1. 5 ml samples are secluded into a capped vial, immediately immersed in boiling water for at least 5 minutes to denature the enzyme and stop the reaction, and then displace for analysis. The amounts of FAMEs in the samples are to be determine by using Gas Chromatograph (Chrompack CP 9001, Holland).