Production of Butanol From Lower Termite Protists and E. Coli Fermentation

1. Production of Butanol From Lower Termite Protists and E. Coli Fermentation Team Gold All On My Polypeptide Chain: Derek Weidlein Ricky Rodriguez Michelle Empleo Michael McClurg Jerrell Ross

2. PURPOSE • There is an increase demand for alternative biofuel. • Utilize otherwise useless plant material for energy • Produce a higher efficiency product

3. “In 2011, the United States consumed about 134 billion gallons of gasoline.”

4. One of the cleanest sources of energy • Established method of production • Takes away from human food sources • Low energy density • Absorbs moisture in the air Ethanol as a fuel source

5. Clean source of energy • Would mix well with gasoline • Not hygroscopic • Higher energy density than ethanol Our idea: Butanol

6. Plan Overview • High cellulose feedstock • Cellulose breakdown with termite hindgut protists and bacteria • Extract simple sugars • Use genetically altered E. Coli to ferment sugars into alcohol product • Distill product to isolate butanol for use as biofuel

7. Hardwood • Cotton • Flax • Hemp • Henequen • Jute • Ramie • Sisal Our feedstock? Useful or Not? Useful Not Useful Potentially Potentially Not Useful In U.S. Useful Not Useful Not Useful % Cellulose 50 95 63 70 78 71 76 73

8. Breaking down cellulose • Endocellulase-breaks crystal structure of complex cellulose (breaks H-bonds) • Exocellulase-cleaves 1 glucose off of 3-ringed cellulose • Cellobiase-splits 2-ringed cellobiose into 2 glucose molecules

9. TermiteS (Coptotermesformosanus) • Invasive species found in Southeast Asia (China, Japan, and Taiwan), but can also be found in the continental United States, South Africa, and Hawaii • Grouped in the Lower Termite class which means it relies on a symbiotic relationship with protists and bacteria to complete its metabolic functions (cellulose breakdown and glucose fermentation)

10. Digestion Overview

11. Protistsin hindgut • Some cellulose activity in the salivary glands and midgut of termite, but most activity is done in the hindgut by the protozoan fauna (full decomposition of cellulose to glucose), which feed by phagocytosis. • Pseudotrichonymphagrassi- largest protozoan that decomposes highly polymerized cellulose • Holomastigoteshartmannii- breaks down low molecular weight cellulose • Spirotrichonymphaleidyi- smallest protozoan breaks down low molecular weight cellulose • Trichomitopsistermopsidis- ferments the glucose into acetate, carbon dioxide, and hydrogen

12. In more detail • Cellulose-containing product would first be chipped and then ground as small as possible with machinery • This sawdust would be mixed with water into a cellulose soup • This soup would sit in a large pool where it will be aerated to maintain an oxygen-rich environment, and inoculated in many separate locations with about one total gram of p. grassii, h. hartmannii, and s. leidyi • This mixture would be maintained at a pH of about 6.5, and kept slightly above room temperature

13. Protist assumptions • All assumptions based on typical protists, since we could not find specific information on our protists • 1 colony consumes .3685kg wood per day, there are 1,000,000 termites per colony, each termite weighs about 2mg, and “termites can be up to 50% digestive protists and bacteria”

14. Consumption Rate vs time (under ideal conditions)

15. Expected yield • In order to make about 10,000kg of glucose , we would need about 18.7 metric tons of hardwood feedstock. This translates to about 7 large trees, about 2/3 the capacity of one semi-truck • With a 1g inoculum and an assumed doubling time of about 2 hours, the protists would be capable of consuming 6200kg of cellulose per day after the first two days. • Total estimated time to consume cellulose without lag phase or death phase is about 47.2 hours

16. Removal of Glucose from the batch • Rotating drum filtration to remove the solid non-cellulose wood components • Precipitate acetate via a sodium salt 3. Filter out the salt product

17. E. coli • Naturally converts glucose to valine via a pyruvate and 2-Keto-isovalerate intermediate • We know a lot about E. Coli’s DNA, so we can add in new genetic material to utilize this 2-Keto-isovalerate intermediate

18. Pathway Glucose Pyruvate 2-Keto-isovalerate Valine 2-keto-acid decarboxylase (KDC) Isobutanol Isobutyraldehyde alcohol dehydrogenase (ADH)

19. Adding new dna • KDC gene comes from Lactococcuslactisbacterium • ADH gene comes from Saccharomyces cerevisiaebacterium • Cut new DNA and E. Coli plasmid with restriction enzyme with selectible marker, creating new recombinant DNA in a single bacterium when treated with DNA ligase

20. Bioreactor conditions • A glucose-water solution would be continuously fed into bioreactor inoculated with 1g of genetically altered E. Coli • The bioreactor would be heated to maintain a constant 37 degrees Celsius • The bioreactor will be aerated for maximum E. Coli function

21. Centrifugation and Distillation Procedure: Boiling points of different components: Valine-melts at 295°C Isobutanol-107.89°C Isobutyraldehyde-63°C Ketoisovalerate-170.5°C Pyruvate-165°C • Drum centrifugation will remove any solids (“excess glucose, cell metabolites, biomass”) • Any valine will be removed prior to distillation (solid) • The isobutanol can be collected in the second fraction. It has a significant boiling point difference from other main components

22. E. Coli Assumptions

23. Expected Yield • Taking the 10,000kg of glucose as a basis for this process: • Ideally, .41g of isobutanol would be formed per gram of glucose • Assuming about 75% efficiency due to byproducts and lost product during seperation, the 10,000kg of glucose would yield about 1,015 gallons of isobutanol. Mixed at 15% per gallon of gasoline, this is 6,762 gallons of mixed fuel gasoline.

24. Schematic part 1 Covered Cellulose Fermenter Wood Processing • Covered Cellulose Fermenter Rotating Drum • Covered Cellulose Fermenter Boiler Schematic Part 2

25. Schematic Part 2 Centrifuge Bioreactor Incoming Glucose Solution Waste Isobutanol Boom. Distillation Water and waste liquids

26. Feasibility • Using only one cellulose fermenter would not make a big difference • With multiple cellulose fermenters, the plan becomes more and more reasonable. The E. Coli functions faster than the protists, and could easily handle an increase in feed glucose. With a significant plant size, a plant could produce tens of thousands of gallons of mixed biofuel per day.

27. Thanks for listening

28. Questions?