7What is Biodiesel? Biodiesel FAME (Fatty acid methyl ester) Alkyl ester of long chain fatty acid (C16-C18)FAME (Fatty acid methyl ester)FAEE (Fatty acid ethyl ester)(TAG)(Biodiesel)Catalysts: Two of the most commonly used catalysts for transesterification are NaOH and KOH.R1, R2, and R3 are long chains of carbons and hydrogen atoms, sometimes called fatty acid chains.
8Sources of Renewable Oils and Fats Plant-derived oils (TAG)Green plants grow through the photosynthesis process with CO2 as a carbon sourceThe combustion of plant-derived oils will release CO2 which has previously been fixed through photosynthesisAdvantagesRenewableInexhaustibleNontoxicBiodegradableSimilar energy content to fossil diesel fuel(Source: http:
9Fuel Ingredients Comparison Fuel ingredients Fossil diesel BiodieselFuel Standard ASTM D ASTM PS121Fuel Composition C10-C21 HC C16-C18 FAMELower Heating Value, Btu/gal , ,093Kin. Viscosity, at 40oCWater, ppm by wt % max.Carbon, wt %Hydrogen, wt %Oxygen, wt %Sulfur, wt % maxBoiling Point, oCFlash Point, oCPour Point, oC to – to 10Cetane NumberBOCLE Scuff, grams , ,000HFRR, microns(Source: National Renewable Energy Laboratory, Sept. 2001)-閃火點(FLASH POINT) 並非燃點, 它是燃料在自然揮發下其油氣得以點燃卻不足以維持燃料燃燒的最低溫度-流動點(Pour Point)為測定潤滑油在規定條件下，將油料冷卻至不可流動之最低溫度。-凝結的溫度則叫做「雲點(cloud point)」
10Sources of Renewable Oils and Fats Waste oils and fatsFrying oils, lard, beef tallow, yellow grease, and other hard stock fatsAdvantageCheapDisadvantagesHigh polymerization productsHigh free fatty acid contentsSusceptibility to oxidationHigh viscosityPoor-quality oils may inactivate the basic or even enzyme catalystsSolve strategyPreliminary treatmentSuch as the use of adsorbent materials (magnesium silicates)Reduce free fatty acid content and polar containminants
11Sources of Renewable Oils and Fats Microbial oils—Algal oilsLargely produced through substrate feeding and heterotrophic fermentationAnother cheap source of renewable new materialsStrainLipids content(%)EnzymeBiodiesel yieldReaction conditionsChlorella protothecoids44-48%Immobilized Candida sp. lipase98% of FAEEMolar ratio 3:1Reaction time 12 h–Pseudomonas fluorescens lipase (1,3-specific)92%20 ºC in 12 h(Source: Biotechnol. Bioeng. 2007, DOI: /bit In press; Appl. Microbiol. Biotechnol. 2006, 73, )
12Source of acyl acceptors Different alcohol and different fatty acid produce different biodiesel of different propertiesSource of acyl acceptorsMain purpose of alcoholysis (transesterification)Reduce the viscosity of the fatIncrease volatility and FA ester combustion in a diesel engineAcyl acceptorsProductsAdvantagesDisadvantagesMethanolFAME1. Cheaper than ethanol2. More reactive3. More volatile1.Toxic2.Mostly nonrenewableEthanolFAEE1. More renewable2. Environmental friendly1. More expensive2. Less reactiveOthers: propanol, isopropanol, butanol, branched-chain alcohols, t-butanol,octanol, methyl and ethyl acetate(Source: Akoh et al., J. Agric. Food Chem., 2007, 55, )
13Different alcohol and different fatty acid produce different biodiesel of different properties 杏核仁油月桂葉油玻璃苣油椰子油玉米油棉花子油海甘藍油落花生油榛果油麻瘋樹水黃皮籽油亞麻子油橄欖油棕櫚油花生油罌粟籽油葡萄籽油米糠油葵花籽油芝麻油大豆油葵花油烏桕核桃仁油小麥油(Source: Akoh et al., J. Agric. Food Chem., 2007, 55, )
14Fatty Acid Characterizations TABLE 1 Some naturally occuring fatty acids: structure, properties, and nomenclature[Source: Nelson and Cox 2005, “Lehninger Principles of Biochemistry, 4th ed.]
15Fatty Acids Extended Conformations Saturated Fatty Acid (SFA) Unsaturated Fatty Acid (UFA)[Source: Nelson and Cox 2005, “Lehninger Principles of Biochemistry, 4th ed.]
22Chemical Catalyst Transesterification Other unfavorable reactions
23Chemical Catalyst Transesterification Reaction condition(Sources: NoureddiniH et al.75(12), , 1998; DarnokoD. and CheryanM. JAOCS, 77, 1269–1272, 2000.;He et al. Transactions of the ASAE, 48, , 2005; He et al. Transactions of the ASABE, 49, , 2006)
24Biodiesel Production Two available approaches Chemical catalyst Enzyme catalystApproachParametersChemicalEnzymeCatalystSodium methoxideLipaseTemperatureHighMildSeparationDifficultEasyReusablePuritySide reactionNature(Source: Bioresour. Technol. 1999, 79, ; J. Mol. Catal. B: Enzymatic 2002, 17, )
26Enzymatic Biodiesel Reaction mechanism Materials Oil resources: soy bean, rapeseed, palm, and jatropha etc.Alcohol types: Ethanol, Methanol, and Isopropanol etc.Solvent systems: n-hexane, t-butanol, and solvent-free etc.COOR1RCOOR1COOR1OHOHLipaseROH+COOR2RCOOR2+COOR2+OH+OHCOOR3OHCOOR3OHRCOOR3Alcohol Oil Alkyl ester DG MG Glycerol(Triacylglycerol) (Biodiesel)R1, R2, and R3 are long chains of carbons and hydrogen atoms, sometimes called fatty acid chains.DG: Diacylglycerol; MG: Monoacylglycerol
27Lipases Advantages Mild reaction conditions Specificity Reuse Enzymes or whole cells can be immobilizedCan be genetically engineered to improveTheir efficiencyAccept new substratesMore thermostableThe reactions they catalyze are considered as “natural” and “green”
28CRL isozymes Similarity High-identity gene family (lip1 to lip7) Consisting of 534 amino acidsAn evident MW of 60 kDaConserved at a catalytic triadSer-209, His-449 and Glu-341Disulphide bond formation sitesCys-60/Cys-97Cys-268/Cys-277
30Related Publications Recombinant LIP1 Recombinant LIP2 and LIP4 Chang, S. W., C. J. Shieh, G. C. Lee and J. F. Shaw*, Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris. Appl. Microbiol. Biotechnol. 67: (SCI)Chang, S. W., G. C. Lee, J. F. Shaw*, Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase. J. Agric. Food Chem. 54, (SCI)Chang, S. W., G. C. Lee, C. C. Akoh, J. F. Shaw*, Optimized growth kinetics of Pichia pastoris and recombinant Candida rugosa LIP1 production by RSM, J. Mol. Microbiol. Biotechnol. 11, (SCI)Recombinant LIP2 and LIP4Lee, L. C., Y. T. Chen, C. C. Yen, T. C. Y. Chiang, S. J. Tang, G. C. Lee*, and J. F. Shaw*, Altering the substrate specificity of Candida rugosa LIP4 by engineering the substrate-binding sites. J. Agric. Food Chem. 55: 5103−5108. (SCI)Lee, G. C., L. C. Lee, and J. F. Shaw*, Multiple mutagenesis of nonuniversal serine codons of Candida rugosa LIP2 gene and its functional expression in Pichia pastoris. Biochem. J. 366: (SCI)Tang, S.J., J.F. Shaw, K.H. Sun, G.H. Sun, T.Y. Chang, C.K. Lin, Y.C. Lo and G.C. Lee Recombinant expression and characterization of the Candida Rugosa Lip4 lipase in Pichia pastoris：Comparison of glycosylation, activity and stability. Archives Biochem. Biophys. 387: (SCI)Recombinant LIP3Chang, S. W., G. C. Lee, J. F. Shaw*, Efficient production of active recombinant Candida rugosa LIP3 Lipase in Pichia pastoris and biochemical characterization of the purified enzyme. J. Agric. Food Chem. 54: (SCI)ReviewAkoh, C. C., G. C. Lee, and J. F. Shaw*, Protein Engineering and Applications of Candida rugosa Lipase Isoforms. Lipids 39 (6): (SCI)Akoh, C. C., Chang, S. W., G. C. Lee, and J. F. Shaw*, 2007.Enzymatic Approach to Biodiesel Production. J. Agric. Food Chem. 55:
31Recombinant CRL Isoform for Biodiesel Production Figure Effect of methanol addition times on biodiesel conversion catalyzed by LIP2. The reaction condition was subject to a loading of 0.5 g soybean oil, oil/methanol molar ratio = 1/4, 20% water content, and 12-h reaction time at 35 ºC. The enzyme solution (LIP2) used in this work was 70 μL. The time interval between the two methanol additions was 1 h.
33Protein Engineering Technology Table Important amino acid changes producing structural differences among C. rugosa LIP 1, LIP 2, LIP 3 and LIP 4Residue LIP LIP LIP LIP4Tyr Phe Phe TrpVal Leu Ile ValThr Leu Ile LeuPhe Val Phe AlaPhe Leu Ile ValSer Gly Ala Ala( Source: Mancheno, J.M. et al )
34Protein Engineering Technology (kb)MLane 1: Represent the WT lip4Lane 2: Represent the A296ILane 3: Represent the V344QLane 4: Represent the V344HLane 5: Represent the H448S3.01.52.0Figure PCR analysis of P. pastoris transformants. Using the genomic DNA as templates, and rector specific 5’ α-factor primer and 3’ AOX1 primer.
35wild-type Candida rugosa lip4 A296I（B）wild-type Candida rugosa lip4296V344QV344H（C）wild-type Candida rugosa lip4344H448S448Figure Genomic DNA-sequence comparisons between wild-type Candida rugosa lip4（CRL4） and A296（A）,CRL4, V344Q and V344H（B）CRL4, and H448S（C）. The difference locations between mutated codons and wild-type are cased in red squre.
36Protein Engineering Technology Wild-typeA296IV344QV344HH448SNegative control(P. pastoris KM 71)Figure Lipase plate(C4) assay. The wild-type, A296I, V344Q, V344H, H448S and negative control ( P. pastoris KM 71) were transferred on the YPD agar plate containing 100 μg/ml Zeocin and 1% tributyrin, and cultured for 48 hours at 30 ℃.
37Protein Engineering Technology M(kDa)97.484.066.055.4Figure 13 SDS-PAGE of the wild-type, A296I, V344Q, V344H, H448S and Negative control .Lane 1: Represent the wild-type; Lane 2: Represent the A296ILane 3: Represent the V344Q ; Lane 4: Represent the V344HLane 5: Represent the H448S ; Lane 6: Represent the Negative control（KM 71）
38Protein Engineering Technology Specific activity (U/mg)Figure The substrate specific activity of C. rugosa LIP4 wild-type , A296I, V344Q, V344H and a H448S with p-nitrophenyl (p-NP) esters of various chain-length fatty acids. The lipase sample was added to a reaction mixture containing 5 mM p-nitrophenyl ester（such as acetate、butyrate、caproate and caprylate）and 2.5 mM p-nitrophenyl ester（such as caprate、laurate、myristate、palmitate and stearate）at pH 7.0.
39Protein Engineering Technology Specific activity (U/mg)Figure The substrate specific activity of C. rugosa LIP4 wild-type , A296I, V344Q, V344H and a H448S with triglyceride of various chain-length fatty acid. The lipase sample was added to a reaction mixture containing 50 mM triglyceride (such as tributyrin、tricaprin) and 10 mM triglyceride (such as trilaurin、ripalmitin) and the activity was measured by pH stat at pH 7.0.
40Enzyme Immobilization AdvantagesEasy to control enzyme concentrationEasy separation of the immobilized enzymeEasy to control micro-environmentEasy separation of enzyme from productReuse of the enzyme
41Enzyme Immobilization Principal MethodsAdsorptionCovalent bindingEncapsulationEntrapmentCross-linking(Source: Gordon F. Bickerstaff ,1997)
42Lipolytic Activities of a Lipase Immobilized on Six Selected Supporting Materials (Source: Biotechnology and Bioengineering, 1990, 35, )
43Lipolytic Activities of a Lipase Immobilized on Six Selected Supporting Materials (Source: Biotechnology and Bioengineering, 1990, 35, )
44Continuous Bioreactor Systems TypesStirred-tank bioreactorMembrane bioreactorFluidized bed reactorPacked-bed bioreactorAdvantagesEasily usedContinuous process with automatic controlLong term reaction timeHigh product concentration(Source: Lipid biotechnology p. 387–398.; 生物固定化技術與產業應用。2000。 第121–155頁 )
46How to increase oil content of plants? Fatty acid biosynthesis pathwayLocationsER: Polyunsaturated fatty acids (PUFAs)Plastid: primary saturated and monounsaturated fatty acidsOil body: triglycerideFirst step: Acetyl-CoA + CO2 Malonyl-CoAFirst enzyme: Acetyl-CoA carboxylase (ACCase)
47How to increase oil content of plants? Fatty acid biosynthesis pathway in plantAcetyl-CoA carboxylase (ACCase)KASs=3-ketoacyl-ACP synthasesTEs=thioesterasesDEs=DesaturasesATs=Acyltransferases(Source:
48How to increase oil content of plants? Genetic modified technology for ACCase in plantsFunctional promoter region constructFunctional gene expression[Source: Ohlrogge and Browse (1995) The Plant Cell 7, ]
49Conclusion Advantages of Biodiesel from vegetable oils or their blends RenewableBiodegradableoxygenatedLess or nontoxicLow sulfur content and higher cetane numbersProduces less smoke and particulatesProduces lower carbon monoxide and hydrocarbon emissionsLow aromatic contentHigher heat content of about 88% of number 2 diesel fuelReadily available
50Conclusion Future trend for fuel production—Biotechnology Protein (enzyme) engineeringCatalytic efficiency improvementHigh specific activityNovel substrate specificityDifferent regio-selectivityEnantioselectivity improvementHigh stabilitiespHTemperatureOrganic solvents etc.
51Conclusion Future trend for fuel production—Biotechnology Genetic modified technologyIncrease oil content in various plantsFunctional promoter developmentHigh level expression of key enzymeMicrobial engineering--MicrodieselRecombinant E. coli host for ethanol productionCoexpression of the ethanol production genesPyruvate decarboxylase (pdc gene product)Alcohol dehydrogenase (adhB gene product)Unspecific acyltransferase WS/DGAT geneFrom Zymomonas mobilisFrom Acinetobacter baylyi strain ADP1(Source: Microbiology, 2006, 152, )
52Conclusion Microbial engineering--Microdiesel Figure Pathway of FAEE biosynthesis in recombinant E. coli. FAEE formation was achieved byCoexpression of the ethanolic enzymes pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB)From Z. mobilis and the unspecific acyltransferase WS/DGAT from A. baylyi strain ADP1.(Source: Microbiology, 2006, 152, )
53ReferencesLi, X.; Xu, H.; Wu, Q. Large-scale biodiesel production from microalga Chlorella protothecoids through heterotrophic cultivation in bioreactors. Biotechnol. Bioeng. 2007, DOI: /bit (in press).Luo, Y.; Zheng, Y.; Jiang, Z.; Ma, Y.; Wei, D. A novel psychrophilic lipase from Pseudomonas fluorescens with unique property in chiral resolution and biodiesel production via transesterification. Appl. Microbiol. Biotechnol. 2006, 73,Kalscheuer, R.; Stölting, T.; Steinbüchel, A. Microdiesel: Escherichia coli engineered for fuel production. Microbiology 2006, 152,Stowe, K. Exploring Ocean Science, 2nd Ed., Wiley, New York, 1996.Nelson, D. L; Cox, M. M. “Lehninger Principles of Biochemistry, 4th ed. W.H. Freeman and Company, New York, 2005.Kuo, T. M. and Gardner, H. W. Lipid biotechnology. p. 387–398. Marcel dekker. New York. USA, 2002.Ma, F.; Hanna M. A. Biodiesel production: a review. Bioresour. Technol. 1999, 70, 1–15.Shimada, Y.; Watanable, Y.; Sugihara, A.; Tominaga, Y. Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing. J. Mol. Catal. B: Enzymatic 2002, 17, 133–142.
54Contact InformationISBB Symposium Secretariat250, KuoKuang Rd., Taichung, 40227, TaiwanTel: ext304Fax:Welcome to Taichung, Taiwan. The main theme of the symposium is Agricultural Biotechnology. The following lists the main areas that will be focused in the meeting: 1). Functional Food and Industry Products2). Improvement of Agronomic and Microbial Traits3). Biofuel4). Nanobiotechnology
55Acknowledgement Shu-Wei Chang, Ph. D. Prof. Casimir C. Akoh Department of Nutrition and Health ScienceChung-Chou University of TechnologyProf. Casimir C. AkohDepartment of Food Science and TechnologyThe University of GeorgiaProf. Chwen-Jen ShiehDepartment of Bioindustry TechnologyDayeh UniversityMr. Chih-Chung YenInstitute of Agricultural BiotechnologyNational Chiayi University