Not Your Ordinary Grass: Switchgrass as a
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Transcript Not Your Ordinary Grass: Switchgrass as a
NOT YOUR ORDINARY GRASS:
SWITCHGRASS AS A
CANDIDATE FOR CELLULOSIC
ETHANOL
Avery Tucker
December 6, 2012
CELLULOSE ETHANOL
Primarily produced from starch, but recent
emphasis on cellulose derived ethanol so as not to
compete with food sources
(From Stricklen 2008)
SWITCHGRASS AS A CANDIDATE
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
WHAT IS LIGNIN
Lignin is a complex
secondary cell wall
component found in
plants
Adds mechanical
strength to a plant
http://en.wikipedia.org/wiki/Lignin
WHAT IS LIGNIN
• 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
REDUCING LIGNIN
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)
GENETIC MANIPULATION
OF LIGNIN REDUCES
RECALCITRANCE AND IMPROVES ETHANOL PRODUCTION
FROM SWITCHGRASS
(CHENG ET AL. 2011)
Down-regulation of the switchgrass Omethyltransferease (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.
COMT DOWN-REGULATION
1.
2.
3.
4.
5.
6.
Create cDNA library
Identification of fragment based on expressed
sequence tag information
Amplification of fragment yielded cDNA
Construction of RNAi binary vector based on
pANDA gateway vector
COMT fragment placed in the sense and antisense orientation
Construct inserted via Agrobacterium
tumefacians
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
RT-PCR ANALYSIS
• 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)
EFFECTS OF COMT SUPPRESSION
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)
EFFECTS OF COMT SUPPRESSION
(Fu et. al 2011)
EFFECTS OF COMT SUPPRESSION
• 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
(Fu et. al 2011)
IMPACT OF DOWN-REGULATION ON
HYDROLYSIS AND FERMENTATION
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 nonpretreated plant
material
(Fu et. al 2011)
IMPACT OF DOWN-REGULATION ON
HYDROLYSIS AND FERMENTATION
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
IMPACT OF DOWN-REGULATION ON
HYDROLYSIS AND FERMENTATION
• Figure A.
showing T1
tiller and T0,
T1 Stem
ethanol
conversion
• Figure
B.
Showing
weight
loss vs.
ferment
ation
(Fu et. al 2011)
CONCLUSIONS
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
PUSHING BIOFUELS
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.
2.
3.
4.
have superior environmental benefits
be economically competitive
have meaningful supplies to meet energy demands
have a positive net energy value (NEV)
SOURCES
Bulls, Kevin. "What's Holding Biofuels Back?" MIT Technology
Review. N.p., 06 Aug. 2010. Web. 04 Dec. 2012.
<http://www.technologyreview.com/news/420136/whats-holdingbiofuels-back/page/2/>.
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.