Nanjing Forestry University Experience 南京林业大学经验 Gilda Naka June 6, 2013-July 3, 2013

Nanjing Forestry University Experience 南京林业大学经验 Gilda Naka June 6, 2013-July 3, 2013

Effects of water on Rhizopusoryzaewhole cell-catalyzed transesterification of bean oil for biodiesel production在以米根霉全细胞催化剂催化大豆油制备生物柴油的反应体系中，含水率对反应的影响 Gilda Naka Special Thanks to Dr. Wang Fei, Dr. LiawGoang, Dr. Zhang Yu，He Qiyang 特别感谢王飞教授、廖广信教授、张瑜副教授、何琦阳同学

What is Biodiesel Production?什么是生物柴油？ • The process of producing biofuel through the transesterfication of triglycerides. • This involves reacting vegetable or animal fats with short-chain alcohols (typically methanol or ethanol). • Advantages: • Domestically produced • Biodegradable • Renewable and Clean energy

Advantages ofRhizopusoryzaelipase (ROL)米根霉脂肪酶的优势 • Industries use chemical methanolysis with alkalicatalysis • Difficult removal of catalyst • Excessive Energy Requirements • Using lipase is better • Glycerol is by-product (easily recovered) • Simple purification of fatty methyl esters

Advantages ofR. oryzaelipase (cont.) • Whole-cell biocatalyst • No purification necessary • R. oryzaewhole cell with polyurethane foaming plastic Two Types of Lipase Extracellular Intracellular • Expensive (recovery, purification, immobilization process)

Transesterfication Reaction Mechanism转脂化反应的反应机理 • Reaction involves two steps: • Hydrolyzation（水解反应） • Rate Determining Step • Esterification（酯化反应）

Reaction with ROL: ROL

Why is Water Important? • Reacts bean oil to glycerol and fatty acids • Maximum methyl ester = 66.6% • Reaction #1: Acyl migration (酰基迁移) • Reaction #2: • Water affects concentration of methyl ester • Smaller concentration  Slower reaction • Excess H2O limits Reaction #2

Objective • Investigate the optimal amount of water needed to produce biodiesel via whole cell-catalyzed transesterfication of bean oil

Experiment Outline Breed Cultivation of R.oryzae Prepare Fermentation Medium Introduce R.oryzaeto Medium Retrieving Whole-cell biocatalyst (foam) Biodiesel Production with Varying Water Content

Fermentation Medium Preparation发酵培养基成分 • pH = 5.5

Fermentation Medium Preparation (cont.) • 4 beakers with 80 mL of medium

Fermentation Medium Preparation (cont.) • Medium was autoclaved • 20 minutes at 121° C

Breed Cultivation保种培养 • Ingredients: • D-(+)-Glucose 2% • Potato 20% • 50 mL of potato juice and 0.1 g D-(+)-Glucose • Autoclaved for 1 hour then inoculate and place in incubator at 28°C 180 rpm until mold appears • Agar (2%) was added to the petri dish as a storage medium

Introducing Rhizopusoryzaeto Fermentation Medium接种至发酵培养基 • Inoculate ROL (after 10 min UV sanitation of beakers)

Introducing Rhizopusoryzaeto Fermentation Medium (cont.) • Cultivation • 60 hours at 28° C at rotation • speed of 180 rpm

Retrieving whole cell biocatalyst for Biodiesel Production 全细胞催化剂的预处理 • Wash polyurethane foaming plastic 3-5 times with water and squeeze • Add 200mL of glutaraldehyde (1%) • Glutaraldehyde contains the lipase in the foam • Covered with gauze and incubated for 1 hour at 25°C, 180 rpm

Retrieving Foam for Biodiesel Production (cont.) • Add phosphate buffer (diluted 10 times) • 6.5 mL of KH2PO4 • 50 mL of Na2HPO4*12H2O • Put in ice water for 5 min • Put in watch glass and dry in multi-thermo incubator for 10 hours at 30°C

Rhizopusoryzaewhole cell in Polyurethane Foam Polyurethane Foam with no R. oryzae Polyurethane Foam with R. oryzae

Begin Water Variation • 6 beakers • Bean oil (15g) • Methanol (680mL) • 25 pieces of R. oryzae foaming plastic (not in control) • Varying percents of water (0%-20%) • In shaker for 12 hours at 37°C

Stepwise Sampling取样 • 150 μL into 6 test tubes every 12 hours • 680μL methanol added and shake for 12 hours • Methanol added stepwise because poisonous to the mould • Gas Chromatography done on each round of samples to measure methyl ester content

Gas Chromatography 气相色谱 • Use n-hexane as solvent • Sample is diluted 20 times • Nitrogen is the mobile phase • Samples were centrifuged before GC

Gas Chromatography (cont.) • 4 peaks: methyl palmitate, methyl linoleate, methyl oleate, and methyl linolenate • Methyl heptadecanoate used as interior label

Gas Chromatography (cont.)

Gas Chromatography (cont.) • Control had zero peaks throughout

Why the Results Matter! • Optimal Water Content was found to be 10% • Most methyl ester produced after 60 hours • One step closer to a most efficient biodiesel production process • Discovering optimal conditions with cheap techniques • Push towards cleaner, alternative energy • Worldwide adaption • Save Mother Earth!

Future Studies • Time stages in which methanol is added • Build apparatus • How the amount of catalyst effects biodiesel • Ideal amount of foam for mass transfer • Best type of foaming plastic • How functional groups play a role in mass transfer • Use Jatropha seed oil • Found in the Guangxi Province

THANK YOU! 谢谢! The skills I have gained will help me for years to come! 我所学到的技能在未来很多年都会对我有所帮助！ Special Thanks to Dr. Wang Fei, Dr. Wang Yong, Dr. Moss Elica Dr. LiawGoang, Dr. Zhang Yu，He Qiyang 特别感谢王飞教授、廖广信教授、张瑜副教授、何琦阳同学

Literature Cited • 1) Kazuhiro Ban, Masaru Kaieda, et. “Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopusoryzaecells immobilized within biomass support particles”. Biochemical Engineering Journal 8 (2001) 39-43 • 2) Jing Zeng, Wei Du, et. “Study on the effect of cultivation parameters and pretreatment on Rhizopusoryzaecell-catalyzed transesterification of vegetable oils for biodiesel production”. Journal of Molecular Catalysis B: Enzymatic 43 (2006) 15-16 • 3) Yunjun Yan, Li Xu, et. “A synergetic whole-cell biocatalyst for biodiesel production”. The Royal Society of Chemistry. 2012.

Questions???

Nanjing Forestry University Experience 南京林业大学经验 Gilda Naka June 6, 2013-July 3, 2013