Dr. Gary PeterProfessorUniversity of Florida

Southern Pines: The Bioenergy & Renewable Chemicals Star of the Southeastern US

Gary Peter University of Florida

gfpeter@ufl.edu

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

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

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

TimberMart-South

Operating & Proposed Wood Biomass to Electric Power & Wood Pellet Facilities

Approx. 30 actual or proposed plants

Approx. 40 actual or proposed plants

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 ?

Stable Cost & Large Supply

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

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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.

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

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

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

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:

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

• 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”

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

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

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

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

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

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

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

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

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

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

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

Phenotypic variation in oleoresin drymass is positively skewed

Oleoresin drymass by site

Xylem growth increment per year

Resin canal number per year

Associated SNPs accurately predict additive genetic variation in oleoresin drymass

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

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

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

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

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

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

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

Dr. Gary PeterProfessorUniversity of Florida