1. N OT Y OUR O RDINARY G RASS : S WITCHGRASS AS A C ANDIDATE FOR C ELLULOSIC E THANOL Avery Tucker December 6, 2012

2. C ELLULOSE  E THANOL Primarily produced from starch, but recent emphasis on cellulose derived ethanol so as not to compete with food sources (From Stricklen 2008)

3. S WITCHGRASS AS A C ANDIDATE Panicum virgatum L. (monocot) Fast growth on marginal lands, low water and nutrient requirements Energy analysis show: Switchgrass produced more renewable than nonrenewable energy consumed by 540% Increased biomass yield when grown in monoculture by 93% Lower emissions from switchgrass derived ethanol compared with gasoline by 94% http://en.wikipedia.org/wiki/File:Panicum_virgatum.jpg

4. W HAT IS L IGNIN Lignin is a complex secondary cell wall component found in plants Adds mechanical strength to a plant http://en.wikipedia.org/wiki/Lignin

5. W HAT IS L IGNIN Monolignols(H, S, and G) polymerize to form lignin Used to measure lignin content Similar biochemical pathways COMT http://www.plantphysiol.org/content/153/3/895/F1.large.jpg

6. R EDUCING L IGNIN The removal of lignin as a key step in cellulosic ethanol production Reduces pretreatment costs Down-regulation of lignin related genes can alter the concentration of lignin with several effects Increased ethanol yield Decreased pretreatment costs (From Stricklen 2008)

7. G ENETIC MANIPULATION OF LIGNIN REDUCES RECALCITRANCE AND IMPROVES ETHANOL PRODUCTION FROM SWITCHGRASS (C HENG ET AL. 2011) Down-regulation of the switchgrass O- methyltransferease (COMT) Cellulosic ethanol yields showed: 38% increase in biomass 300-400% lower treatment requirement of cellulase for equivalent yield Higher yields of ethanol after fermentation with Clostridium thermocellum without the addition of enzymes.

8. COMT D OWN - REGULATION 1. Create cDNA library 2. Identification of fragment based on expressed sequence tag information 3. Amplification of fragment yielded cDNA 4. Construction of RNAi binary vector based on pANDA gateway vector 5. COMT fragment placed in the sense and anti- sense orientation 6. Construct inserted via Agrobacterium tumefacians

9. Fig. 1 Structure of the pANDA vector and procedure for RNAi vector construction. Miki D, Shimamoto K Plant Cell Physiol 2004;45:490-495 ©2004 by Oxford University Press

10. RT-PCR A NALYSIS Shows expression levels of COMT in T0 generation plants Figure A. housekeeping gene ELFIA vs. COMT Figure B. expression of COMT transcripts Figure C. COMT activity levels with substrates 5-OH coniferaldehyde immediate precursor Caffeyl aldehyde precursor of 5-OH coniferaldehyde (Fu et. al 2011)

11. E FFECTS OF COMT S UPPRESSION T0-2, T0-3, and T0-12 selected for further analysis due to high down-regulation AcBr lignin content reduced in T0 and T1 T0-2: -12.2% T0-3: -14.7% T0-12: -6.4% Plants maintained low levels even after outcrossing S/G ratio shows a greater reduction in S content. As COMT is part of the S biosynthesis pathway this would be expected Minor impact on cellulose content (~3% to -%5 difference)

12. E FFECTS OF COMT S UPPRESSION (Fu et. al 2011)

13. E FFECTS OF COMT S UPPRESSION (Fu et. al 2011) Figure A Switchgrass: Control, T1-2, T1-3, T1-12 Normal growth and development Figure B showing biomass of tiller and stem Figure C internode cross section of mature Switchgrass stems showing brown coloration (C, E are control and D, F are transgenic) No explanation of brown coloring, useful as an phenotypic indicator of transgenic plants

14. I MPACT OF D OWN - REGULATION ON H YDROLYSIS AND F ERMENTATION Saccharification assay used to evaluate acceptibility of plant material for bioconversion Cellulases and other enzymes used to hydrolyze cell wall carbohydrates Increased digestibility of pretreated and non- pretreated plant material (Fu et. al 2011)

15. I MPACT OF D OWN - REGULATION ON H YDROLYSIS AND F ERMENTATION Enzymatic digestibility does not necessarily reflect true bioconversion potential Pretreatment followed by fermentation shows true potential Ethanol yield per gram from whole tillers: T1-2: 38% T1-3: 30% Stem material produced more ethanol (~59%) than the tillers using equivalent weights

16. I MPACT OF D OWN - REGULATION ON H YDROLYSIS AND F ERMENTATION (Fu et. al 2011) Figure A. showing T1 tiller and T0, T1 Stem ethanol conversion Figure B. Showing weight loss vs. ferment ation

17. C ONCLUSIONS Recalcitrance is the inherent resistance of plants to microbial and enzymatic deconstruction. Shown to be reduced in COMT lines 300-400% less enzyme needed Improved ethanol yield Improved forage quality, or digestibility, would allow transgenic Switchgrass to serve as both a fuel source and feed for livestock

18. P USHING B IOFUELS Renewable Fuels Standard (2007) By 2022 require 20 billion gallons of cellulosic biofuels Oil companies required to blend fuel stocks with ethanol, creating a market Alternative transportation fuels should meet the following criteria to be a substitute for conventional gasoline 1. have superior environmental benefits 2. be economically competitive 3. have meaningful supplies to meet energy demands 4. have a positive net energy value (NEV)

19. S OURCES Bulls, Kevin. "What's Holding Biofuels Back?" MIT Technology Review. N.p., 06 Aug. 2010. Web. 04 Dec. 2012.. Fu, C., J. R. Mielenz, X. Xiao, Y. Ge, C. Y. Hamilton, M. Rodriguez, F. Chen, M. Foston, A. Ragauskas, J. Bouton, R. A. Dixon, and Z.-Y. Wang. "Genetic Manipulation of Lignin Reduces Recalcitrance and Improves Ethanol Production from Switchgrass." Proceedings of the National Academy of Sciences 108.9 (2011): 3803-808. Glick, Bernard R., Jack J. Pasternak, and Cheryl L. Patten. Molecular Biotechnology. Washington: ASM, 2010. Print. Schmer MR, Vogel KP, Mitchell RB, Perrin RK. Net energy of cellulosic ethanol from switchgrass. Proceedings of the National Academy of Sciences of the USA. 2008;105:464–469. Stricklen, Miriam B. "Plant Genetic Engineering for Biofuel Production: Towards Affordable Cellulosic Ethanol." Nature.com. Nature Publishing Group, June 2008. Web. 05 Dec. 2012.