153L

• Chem 153L is the intro biochem laboratory class of the Chemistry and Biochemistry department, with the main mission to learn how to make biofuel in bacterial cells.
• Biofuels are fuel molecules that can be generated by treating fresh biomass, with the energy stored as chemical energy in the main storage forms starch or sucrose and was previously converted from light energy by plants or microcorganisms through photosynthesis.
• The advantage of biofuels in comparison to fossil fuels is that they are easy to regenerate as long as the sun is still shining and the earth has a climate that allows plants to grow.
• Fossil fuels have a finite storage and will take millions of years to replenish.
• The energy for biofuels comes from sunlight, that the plants convert to carbohydrates via photosynthesis.
• The various material, either high in carbohydrates or high in lipids, can be used to produce biofuels.
• New strategies can also harness energy stored in cellulose or material high in proteins.
• Raw material can be fed to microorganisms, containing enzymes to produce the specific fuel.
• Algae can be engineered directly to produce specific fuels, since they can perform photosynthesis and produce the fuels themselves.
• Algae can be grown in tanks/bioreactors and do not compete with land that can also be used for food production.
• The lab work in Chem 153L can be scaled up in industrial plants where microorganisms are cultivated in bioreactors.
• Glyceraldehyde can be phosphorylated also by triose kinase to yield glyceraldehyde-3-phosphate to continue in the glycolytic pathway.
• Another form of carbohydrate for energy storage is starch.
• In the absence of oxygen, the NADH will build up and needs to be oxidized to NAD+, to keep glycolysis running.
• Phosphoglucomutase then switches the phosphate group from the first to the 6th position.
• Sucrose is digested by the enzyme sucrase to fructose and glucose.
• In C, the gasoline phase decreases to 8 mL, whereas the water phase increases to 2 mL.
• In order to re-utilize the carbon skeletons from these storage forms for biofuels, they have to be channeled through glycolysis.
• In B, the ethanol that was mixed with the gasoline switches to the water phase during shaking.
• Fructose-1-phosphate is then cleaved by fructose 1-phosphate aldolase to glyceraldehyde and dihydroxyacetone phosphate.
• Starch is a large polymer made out of glucose units that are linked via alpha 1,4 glycosidic bonds to amylose chains, that are cross-linked through alpha 1,6 branchpoints in amylopectin.
• The fructose part can either be also phosphorylated by hexokinase to fructose-6-phosphate, or it can be phosphorylated to fructose-1-phosphate by the enzyme fructokinase.
• Starch enters the glycolytic pathway via alpha amylase, that randomly cleaves alpha 1,4 glucosidic bonds.
• Phosphorylase is able to cleave off glucose-1-P units from the non-reducing end.
• The starch molecules are very similar to glycogen molecules (animal carbohydrate storage form), which has more alpha 1,6 glycosidic branch points to connect the linear chains.
• Cellulose is hard to digest because degradation is slow, due to the fact that enzymes (collectively named cellulases, that are only found in few species) cannot access polymer chains easily, as they are tightly packed and mixed with hemicellulose and lignin polymers.
• Pathways to produce lipids directly in the algal cells can be engineered.
• Isobutanol has a higher energy density and is less miscible with water, offering a logistic advantage for transport as addition to fuel.
• One of the carbohydrates that store energy in cells is the disaccharide sucrose, which consists of a glucose pyranose (= 6-membered ring) and a fructose furanose (5-membered ring) hexoses and is a major source for energy during biofuel production.
• Glucose-6-P then continues through the regular glycolysis pathway.
• Algae can be grown in illuminated bioreactors and no feedstock has to be provided as they convert the CO2 from the air directly to carbohydrates via photosynthesis.
• Glucose can enter the glycolysis pathway via hexokinase, which phosphorylates D-glucose to glucose-6-phosphate.
• At the same time the keto =O is reduced to a hydroxylgroup at the 3 rd C.
• NADH is oxidized to NAD+.
• Two pyruvate molecules are fused together, while one carbon dioxide is eliminated to yield the C5- molecule 2-aceto-lactate.
• Isobutanol stays in the hydrophobic phase after mixing with the water, while ethanol is in the hydrophilic phase.
• In our pathway here, we pull the 2-ketoisovalerate by overexpressing the 2-ketoacid decarbox
• The enzyme pyruvate decarboxylase decarboxylates pyruvate to acetaldehyde, which is then reduced to ethanol.
• Ethanol used as biofuel has to be transported separately and only added to the fuel at the gas station.
• In the center, 9 mL gasoline- isobutanol blend was mixed with 1 mL water and time was allowed for phase separation.