dr. gary peter professor university of florida. southern pines: the bioenergy & renewable...
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Dr. Gary PeterProfessorUniversity of Florida
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Southern Pines: The Bioenergy & Renewable Chemicals Star of the Southeastern US
Gary Peter University of Florida
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Pine Forests of the Southeastern US
• Forests occupy over 200 million acres (60% of the land area), with a large fraction dominated by pines– 10 species, loblolly and slash economically important
• ~85% of all forestlands are privately owned• About half the pine forest is planted with genetically
improved seedlings– About 10 million ha / 25 million ac each
• Contains 12 Pg of C, 36% of the sequestered forest C in the contiguous United States
• Annually sequester 76 Tg C, equivalent to 13% of regional greenhouse gas emissions
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US South: Forestry & Forest Industry• Largest biomass industry in world
– Produces 16% of global industrial wood supply• More than any other country
– Supplies 60% of US & 25% of world pulp & paper markets• 43 million tons of annual capacity
• Generates ~2/3’s of all industrial bioenergy– Used on site
• Sustainability is a key focus for industry– >93% of stem is utilized
Southern pulp mill location & capacityJohnson & Steppleton, 2011
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Forest Products Supply ChainFeedstock production
Feedstock logistics Biomaterials Distribution
& use
• Scalable– Large land area– Large stable markets
• Sustainable– More volume growth than harvested
• Cost competitive for traditional products– Pulp, paper, wood
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TimberMart-South
Operating & Proposed Wood Biomass to Electric Power & Wood Pellet Facilities
Approx. 30 actual or proposed plants
Approx. 40 actual or proposed plants
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Biofuel Production in the Southeast
• 2010 USDA biofuels report estimates that ~50% of the advanced biofuel production capacity will be located in the southeast US– Most favorable growing
conditions & available land• Advanced biofuel facilities
that can use pine feedstock– KiOR (thermochem)– Bluefire (acid hydrolysis)– Ineos (thermochem)– Bluesugar ?
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Stable Cost & Large Supply
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Since 1940s, planted pine productivity has tripled, primarily due to improved genetic stock and silvicultural technology
developed and disseminated by University / Government / Industry Research Cooperatives
0
2000
4000
6000
8000
1940 1950 1960 1970 1980 1990 2000 2010
Establishment Decade
Vo
lum
e a
t H
arv
es
t (f
t3 /
ac
)
0
10
20
30
40
50
60
Ro
tati
on
ag
e (
ye
ars
)
Natural Stand Planting Site Prep FertilizationWeed Control Tree Improve Biotech/Clonal Rotation Age
Redrawn from: Fox, T.R., E.J. Jokela and H.L. Allen. 2007. The development of pine plantation silviculture in the southern United States. J. Forestry 105:337-347.
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Quantitative Trait
BLUP
Variance ComponentsP= G + EG= A + D + I
-Heritability (h2)-G x E-G x Age
Breeding Values-Ranking genotypes-Selection
Phenotype: Total Height
Traditional Phenotypic Breeding with Recurrent Selection
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CFGRP: Slash Pine Deployment Gains & Value
1.0 Unrogued 1.0 Rogued 1.5 Unrogued 2.0 Unrogued 2.0 Rogued 2.5 Unrogued 3.0 Unrogued0
1020304050
Genetic Gains in Harvest Yields (%)
Low Hazard
Conservative estimate of incremental increase in stumpage value (6% interest) due to increased yields from planting genetically improved stock in FL estateSource: Greg Powell, Univ Florida
1 2 30
100200300400500600
Cycle of Genetic Improvement
$ in
mill
ions
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Breeding Testing Propagation
Start Breeding Commercial Production
Pine Breeding Cycle
Pine Breeding is a long multi-step
process
Can be partitioned in three stages
>30 years 1st to 2nd Generation
Reduction of more than 10 years:
-Early Selection
-Smaller populations
-Top-grafting
White et al. 2008
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I - QTL analysis II – Genetic association
III – Genomic selection
Linkage Blocks
Large
Small
Medium
Resolution
Low HighMedium
Marker Assisted Selection
B. Indirect markers based on linkage disequilibrium:
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ValidationFit all SNPs in a prediction modelY = SNP + e
Training population
GenotypesPhenotypes
Define multi-loci models to predict phenotypes
Genome-Wide Selection
Meuwissen et al. (2001) Genetics 157: 1819-1829
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• Genomic Selection is operational in cattle breeding and evaluated in other animals, crops and trees
• Focus has been on development of methods (e.g. GBLUP, RR-BLUP, Bayes A, Bayes B, LASSO, RKHS, Machine learning, etc.)
• Everybody agrees that GS application depends on the accuracy of predicting phenotype with markers
• Theoretically accuracy depends on: – Linkage disequilibrium extent– Training population size– Heritability– Number of QTLs
• But also depends on the BV quality used to construct the GS model
Genomic Selection “Current status in breeding”
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DBH & HeightGWS Accuracies in CCLONES
B.F. Grant Cuthbert Nassau Palatka0
0.10.20.30.40.50.60.70.80.9
DBH
HT
Interval Generation
VariationIntensityAccuracyYr/BV
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1 2 3 4 5 6 7 8 9 10 12 13 14 15 16 17 18 19 20 21 22 23 24 25
B T P
10+ years 8+ years 8+ years
1 2 3 4 5 6 7 8 9 10 12 13 14
T
8+ years
B
4 years
P
<1yr
1 2 3 4 5 6 7 8 9 10 12 13 14
T
8+ years
B
4 years
P
<1yr
B T P
4 years 1yr
1 2 3 4 5
B T P
4 years 1yr
1 2 3 4 5
B T P
4 years 1yr
1 2 3 4 5
GWS Incorporated into Pine Breeding
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Conifer Oleoresin Canal System for Insect and Fungal Resistance
• The wood resin canals (vertical and horizontal) are organized into a 3D network for terpene synthesis and storage– Thin walled resin canal
epithelial cells line the canal and synthesize and secrete terpenes into the lumen of the canals or duct
• Resin flows out of stem after wounding, typically by boring insects– Constitutive resin under
positive pressure in resin canals
Resin canals
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Why Terpenes?
Terpene Biosynthesis • Conserved biosynthetic
pathways in microbes & plants
• Large variety of natural terpenes with varying chemical properties– Mono, sesqui-, di- and
triterpenes
Terpenes as Biofuels• High energy density - carbon and
hydrogen rich and low in oxygen• Simple & efficient chemical
methods for conversion of natural terpenes to drop-in fuels suitable for blending or replacement of petroleum are available– b pinene dimers as a jet fuel
replacement– Bisabolene to bisabolane a D2
diesel fuel replacement– Farensene as a diesel fuel
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Biofuels & Co-products
Extraction•Sugar – Ferment to EtOH– Sugar
•Starch– Amylase + ferment
to EtOH– Oil, animal feed
•Oil– Transesterification
to biodiesel– Glycerin
Deconstruction•Lignocellulose– Sugar Platform• Size reduction + degradation
+ fermentation• Power, lignin
– Gas Platform• Anaerobic digestion to biogas• Gasification + catalytic
synthesis to liquid fuel• Power
– Liquid Platform• Cracking / pyrolysis +
upgrading
1st GENERATION BIOFUELS 2nd GENERATION BIOFUELS
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Conversion of Biomass to FuelExtraction Based
• Compound highly concentrated in biomass that facilitates efficient recovery
• Starting material has high chemical uniformity
• High efficiency conversion with limited input costs
Deconstruction Based• Biomass is large &
heterogeneous• Starting material has
relatively low chemical uniformity
• Requires substantial energy and/or chemical inputs to reduce
Come from domesticated plants breed & selected for
concentration & yield of food
Non-edible parts of food plants & undomesticated
grasses & trees
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Pine Terpenes
• Naturally synthesize a large diversity of mono-, sesqui- and diterpenes as defense compounds against insects & fungi
• Terpenes accumulate in wood naturally to >20%– Constitutive synthesis– Inducible synthesis
• Genetic and environmental control of wood terpene content
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Current Pine Terpene Industry
Pine
Wood RosinPulp Mill
Biosynthesis
Extraction
Crude Products
Final Products
Specialty Chemicals
Gum Turpentine
& Rosin
CTO & CST
Industrial Biofuels
Flavors & Fragrances
Live Tree
Wood Turpentine
& Rosin
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Pine Terpenes: A $3 Billion Global Industry
• Pine Terpene collection > 1 billion tonne/yr– Turpentine (mono- & sesquiterpene) rosin (diterpenes)– Gum terpene (60%), crude sulfated turpentine & crude tall
oil (35%), wood naval stores (5%)• Gum terpenes collected by tapping living trees >
850,000 tonne/yr– China, Portugal, USSR, Brazil, Indonesia, Mexico, India– China >500,000 tonne/yr [60% of global supply but little is
exported]• Pulp & paper industry collects terpenes as a co-product
– Crude sulfated turpentine & Crude tall oil (CTO)– US south 450,000 tonne/yr of CTO
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Phenotypes
Resin canals• Number of resin canals per
year (averaged over triplicate samples)• 543 cloned genotypes• 3 sites• 3 clonal replicates per site• 2 years
Oleoresin drymass• Box-Cox transformed oleoresin
drymass exuded over 24 hours• 1002 cloned genotypes• 3 sites• 3 clonal replicates per site• 3 years (one site)
Wood terpene content• Diterpene content in dry
wood• 940 cloned genotypes• 2 sites• 2 clonal replicates per
site• Total, mono- & diterpene
content in wet wood• 750 cloned genotypes• 1 site• 4 clonal replicates per
site
30 66 10213817421024628231835439042602468
101214
m/zIn
tens
ity (a
rb u
nits
)
30293
204
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Oleoresin traits are heritableH2 resin canal number•single site: 0.15 – 0.21•across sites: 0.12H2 oleoresin drymass•single site: 0.18 – 0.34•across sites: 0.18
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Phenotypic variation in oleoresin drymass is positively skewed
Oleoresin drymass by site
Xylem growth increment per year
Resin canal number per year
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Associated SNPs accurately predict additive genetic variation in oleoresin drymass
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Estimated F1 genetic gains in oleoresin drymass under varying selection intensities
site h2 Fold-increase breed top
10%
Fold-increasebreed top 5%
Fold-increase breed top 1%
CUT 0.14 1.62 1.74 1.98
NAS yr 6
0.31 1.86 2.05 2.41
NAS yr 7
0.24 1.80 1.98 2.33
PAL 0.12 1.54 1.61 1.77
ALL 0.12 1.61 1.72 1.92h2: narrow sense heritability
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TE-Pine Can Exceed PETRO Metrics• Scalable
– 13 million+ h planted pine exist– Yield gains achievable
• Environmentally Sustainable– High harvest index– Strong positive net energy– Strong negative CO2eq
• Economically Sustainable– Lignocellulose & terpene co-product
synergy– Adds value across supply chain
• Adds Flexibility– No clear detrimental change in
current product mix– Strengthens possibility of pine as a
dedicated biofuel crop– Multiple routes to extraction
Plants Engineered To Replace Oil Increase the mass of readily extractable hydrocarbons to meet technical targets at costs competitive with crude oil
Technical Targets Value Required HT- Pine
1.1-Energy density > 26.5 MJ/L (LHV)
1.2–Melting point < -40oC <-63oC
1.3–Boiling point > 35oC >135oC
1.4-Energy > 160 GJ ha-1 y-1 > 160 GJ ha-1 y-1
1.5-Process cost < $10 GJ-1 < $10 GJ-1
2.1- CO2 use Atmospheric CO2 Ambient2.2- H2O requirement < 22 inch y-1 No irrigation
2.3- Fertilizer requirement
<201 kg ha-1 y-1 N, <77 kg ha-1 y-1 P, <56 kg ha-1 y-1 K
58.5 kg ha-1 y-1 N, 7.5 kg ha-1 y-1 P
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Genetic engineering to rapidly increase oleoresin production in pine stems
Association genetics
•Multi-site analysis of correlated oleoresin traits in a structured clonal population
Gene expression•Differential expression with chemical elicitors of resinosis•Tissue-specific expression in resin canals
Dis
covery
p
hase
Validation phase
Candidate genes
•RNAi mediated silencing•Overexpression•Wild-type v. mutant phenotypes
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Project Overview• To increase terpene production 5 fold
Triple Resin Capacity
25% Greater Flux
Activation
Pathway
1.5X Faster SynthesisEnzyme
Three Synergistic Strategies for Increasing Pine Terpene Synthesis
& Storage Will Be Used
Constitutive Resinosis
Upregulate Carbon Flux to Terpenes
Optimize Composition & Production of Terpenes
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Terpenes & the Future Forest Biorefinery
Issue• Land Use• Environmental
Sustainability• Conversion Efficiency• Cost effective• Net positive energy relative
to fossil fuels
Alignment• Dramatic increase in GJ/ha/y• Increased value to landowners
sustains forest land• Extracted as a co-product –
lignocellulose still useful for all traditional products or energy
• Existing capital• Flexible end product markets• Strongly positive to fossil fuels
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Acknowledgements
COLLABORATORS• University of Florida
– John Davis, Chris Dervinis, Matias Kirst, Patricio Munoz, Marcio Resende, Alejandro Riveros-Walker, Jared Westbrook
• ArborGen– Will Rottmann
• NREL– Mark Davis, Robert Sykes
• University of California, Berkeley– Jim Keasling, Jim Kirby, Pamela
Peralta-Yayha, Blake Simmons
FUNDING• DOE/ARPA-E• USDA/NIFA• Forest Biology Research
Cooperative – Plum Creek Timber, Rayonier,
Weyerhaeuser, RMS, F & W
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Project Summary
Combinatorial engineering 20% wood terpene
Increased Resin canal
#/volume
Increased terpene
synthesis
Resinosis
Improved enzymes
Increased carbon flux
Five fold increase in
wood terpene
Discovery
Technoeconomic ModelingForest tree growthTerpene recoveryFuel production
Value Chain Analysis & Proposition
Germplasm providersLandownersHarvesting/transportWood processorsFuel synthesis
Commercialization PartnersPulp & paper Biofuel ProducersWood products Bioenergy Oleochemical RefinersFlavor & Fragrances
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Dr. Gary PeterProfessorUniversity of Florida