1. Dr. Gary Peter
Professor
University of Florida

2. Gary Peter University of Florida gfpeter@ufl.edu
Southern Pines: The Bioenergy & Renewable Chemicals Star of the Southeastern US
Gary Peter
University of Florida

3. 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

4. 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 & capacity
Johnson & Steppleton, 2011

5. Forest Products Supply Chain
Feedstock 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

6. Operating & Proposed Wood Biomass to Electric Power & Wood Pellet Facilities
Approx. 30 actual or proposed plants
Approx. 40 actual or proposed plants
TimberMart-South 6

7. 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 ?

8. Stable Cost & Large Supply

9. 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
Redrawn from: Fox, T.R., E.J. Jokela and H.L. Allen The development of pine plantation silviculture in the southern United States. J. Forestry 105:

10. Traditional Phenotypic Breeding with Recurrent Selection
Phenotype: Total Height
Quantitative Trait
BLUP
Variance Components
P= G + E
G= A + D + I
-Heritability (h2)
-G x E
-G x Age
Breeding Values
-Ranking genotypes
-Selection

11. CFGRP: Slash Pine Deployment Gains & Value
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 estate
Source: Greg Powell, Univ Florida

12. Pine Breeding Cycle Pine Breeding is a long multi-step process
>30 years 1st to 2nd Generation
Reduction of more than 10 years:
-Early Selection
-Smaller populations
-Top-grafting
White et al. 2008
Can be partitioned in three stages
Breeding
Testing
Propagation
Start Breeding
Commercial Production

13. Marker Assisted Selection
B. Indirect markers based on linkage disequilibrium:
I - QTL analysis
III – Genomic selection
II – Genetic association
Resolution
Low
Medium
High
Linkage Blocks
Large
Medium
Small

14. Genome-Wide Selection
Fit all SNPs in a prediction model
Y =  SNP + e
Training
population
Genotypes
Phenotypes
Define multi-loci models to predict phenotypes
Validation
Meuwissen et al. (2001) Genetics 157:

15. Genomic Selection “Current status in breeding”
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

17. GWS Accuracies in CCLONES
DBH & Height

18. GWS Incorporated into Pine Breeding B T P
10+ years
8+ years T
8+ years B
4 years P
<1yr B T P
4 years
1yr

19. 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

20. Why Terpenes? Terpenes as Biofuels Terpene Biosynthesis
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
Conserved biosynthetic pathways in microbes & plants
Large variety of natural terpenes with varying chemical properties
Mono, sesqui-, di- and triterpenes

21. Biofuels & Co-products
1st GENERATION BIOFUELS
2nd GENERATION BIOFUELS
Extraction
Deconstruction
Sugar
Ferment to EtOH
Starch
Amylase + ferment to EtOH
Oil, animal feed
Oil
Transesterification to biodiesel
Glycerin
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

22. Conversion of Biomass to Fuel
Extraction Based
Deconstruction Based
Compound highly concentrated in biomass that facilitates efficient recovery
Starting material has high chemical uniformity
High efficiency conversion with limited input costs
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

23. 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

24. Current Pine Terpene Industry
Biosynthesis
Pine
Extraction
Pulp Mill
Wood Rosin
Live Tree
Crude Products
Gum Turpentine & Rosin
CTO & CST
Wood Turpentine& Rosin
Final Products
Industrial Biofuels
Specialty Chemicals
Flavors & Fragrances

25. 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

26. Phenotypes 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)
Resin canals
Number of resin canals per year (averaged over triplicate samples)
543 cloned genotypes
3 sites
3 clonal replicates per site
2 years
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
302 93
204

27. Oleoresin traits are heritable
H2 resin canal number
single site: 0.15 – 0.21
across sites: 0.12
H2 oleoresin drymass
single site: 0.18 – 0.34
across sites: 0.18

28. Phenotypic variation in oleoresin drymass is positively skewed
Oleoresin drymass by site
Xylem growth increment per year
Resin canal number per year

29. Associated SNPs accurately predict additive genetic variation in oleoresin drymass

30. Fold-increase breed top 10% Fold-increase breed top 1%
Estimated F1 genetic gains in oleoresin drymass under varying selection intensities
site h2
Fold-increase breed top 10%
Fold-increase
breed 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
2.33
PAL
0.12
1.54
1.61
1.77
ALL
1.72
1.92
h2: narrow sense heritability

31. TE-Pine Can Exceed PETRO Metrics
Plants Engineered To Replace Oil Increase the mass of readily extractable hydrocarbons to meet technical targets at costs competitive with crude oil
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
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
1.5-Process cost
< $10 GJ-1
2.1- CO2 use
Atmospheric CO2
Ambient
2.2- 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

32. 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
Discovery phase
Candidate genes
RNAi mediated silencing
Overexpression
Wild-type v. mutant phenotypes
Validation phase

33. To increase terpene production 5 fold
Project Overview
To increase terpene production 5 fold
Three Synergistic Strategies for Increasing Pine Terpene Synthesis & Storage Will Be Used
Triple Resin Capacity
Activation
Constitutive Resinosis
25% Greater Flux
Upregulate Carbon Flux to Terpenes
Pathway
1.5X Faster Synthesis
Optimize Composition & Production of Terpenes
Enzyme

34. Terpenes & the Future Forest Biorefinery
Issue
Alignment
Land Use
Environmental Sustainability
Conversion Efficiency
Cost effective
Net positive energy relative to fossil fuels
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

35. Acknowledgements COLLABORATORS FUNDING DOE/ARPA-E USDA/NIFA
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
DOE/ARPA-E
USDA/NIFA
Forest Biology Research Cooperative
Plum Creek Timber, Rayonier, Weyerhaeuser, RMS, F & W

36. Project Summary Technoeconomic Modeling
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
Project Summary
Technoeconomic Modeling
Forest tree growth
Terpene recovery
Fuel production
Value Chain Analysis & Proposition
Germplasm providers
Landowners
Harvesting/transport
Wood processors
Fuel synthesis
Commercialization Partners
Pulp & paper Biofuel Producers
Wood products Bioenergy
Oleochemical Refiners
Flavor & Fragrances

37. Dr. Gary Peter
Professor
University of Florida