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Home , Miscanthus x giganteus

The Energy Biosciences Institute – Realizing Cellulosic and

Benefiting the Environment.http://energybiosciencesinstitute.org

Steve Long, Clive Beale, Emily Heaton & Frank Dohleman, Biology, Crop Sciences, Institute for Genomic Biology, National Center for Supercomputer Applications, University of Illinois. CSU 11/11/08 What are second generation feedstocks? In this context: sustainable crops beyond food grains - mainly perennials. ozone

control ROADMAP

Why fuels from crops?

What is the ideal crop? Performance of and switchgrass.

Food vs. Fuel ROADMAP

Why fuels from crops?

What is the ideal biomass crop?

Performance of Miscanthus and switchgrass.

Food vs. Fuel DRIVERS FOR RENEWABLE TRANSPORTATION FUELS? http://www.informify.com/top-stories/46-natural-world/274-north-pole-ice- may-melt-by-september-accelerate-global-warming FUEL PER UNIT AREA OF Yield of various species varies widelyLAND

2000

1600

1200

Sugar beet Sugar Castor Sunflower Jatropha

Rapeseed Corn Sugar Soy Palm

800 Gallons per Acre per Gallons

400

0 Cellulosic Worldwatch 2006 (Miscanthus) THE ENERGY BIOSCIENCES INSTITUTE

RESEARCH & DEVELOPMENT

Environmentally and economically sustainable second generation feedstock and business

$500M investment over 10 years by BP

DIRECTOR: Chris Somerville (Berkeley) DEPUTY DIRECTOR: Steve Long (Illinois) ASSOCIATE DIRECTOR: Paul Willems (BP Group) WHATINSTITUTE IS THE EBI? R&D AREAS

Thermal Treatment Methanogens/Others Syn Gas

Consolidated Process

Sunlight Biomass Monomers Fuels FEEDSTOCK BIOMASS BIOFUELS DEVELOPMENT DEPOLYMERIZATION PRODUCTION Fossil Fuels Bio-processing & MEOR Photosynthetic Microbes

Coal/Oil Economic, Environmental and Social Impacts Carbon & Energy Balance, Carbon Sequestration, Water use, Commercial Systems, Business Ecosystem. EDUCATION & EXTENSION ebi program leaders EBI PROGRAMS

FEEDSTOCK PRODUCTION/ AGRONOMY PROGRAM Tom Voigt (NRES) ENGINEERING SOLUTIONS FOR BIOMASS FEEDSTOCK PRODUCTION K.C. Ting (ABE) BIOFUEL ECONOMIC AND ENVIRONMENTAL IMPACTS Madhu Khanna (ACE) ENVIRONMENTAL IMPACT AND SUSTAINABILITY OF FEEDSTOCK PRODUCTION Evan DeLucia (Plant Bio) ASSESSING IMPACT OF INSECT PESTS AND PLANT PATHOGENS ON BIOMASS PRODUCTION Michael Gray (Crop Sci) GENOMIC-DIRECTED IMPROVEMENT OF FEEDSTOCKS DISCOVERY OF ENZYMES TO Stephen Moose (Crop Sci) DEGRADE PLANT CELL WALL BIOFUELS LAW AND REGULATION LIGNOCELLULOSE Jay Kesan (Law) Isaac Cann (Animal Sci.) ENGINEERING ENZYMES AND METABOLIC PATHWAYS TO ENGINEERING SUGAR DEGRADE PLANT CELL WALL FERMENTATION LIGNOCELLULOSE Huimin Zhao (Chem Eng) John Gerlt (Biochemistry) WHATINTEGRATING IS THE EBI? PROGRAMS

Under One Roof

Programs Projects Feedstock Development

Feedstock Deconstruction

Fuel Synthesis

Environment, Economics & Policy WHATON CAMPUS IS THE EBI? ENERGY FARM CELLULOSIC BIOFUELS – 1.3 BILLION TON POTENTIAL FEEDSTOCK IN USA.

Wheat Corn 6.1% ~ 2 t/ha 19.9% Soy 6.2% Crop residues 7.6%

Grains 5.2%

Manure 4.1% Urban waste Perennial crops 2.9% 35.2% Forest 12.8%

From: Billion ton Study, DOE & USDA 2005 ROADMAP

Why fuels from crops?

What is the ideal biomass crop?

Performance of Miscanthus and switchgrass. Basis of differences

Miscanthus and potential biofuel production. ebi program leaders WHICH FEEDSTOCKS? POOR INFORMATION BASE DETERMINANTSebi program OF YIELDleaders

Total solar Conversion energy efficiency

Wh = S i c

Harvested yield Interception efficiency THEORETICAL EFFICIENCY OF

LOSSES AT DIFFERENT STAGES IN ENERGY TRANSDUCTION 100% 51% outside usable spectrum 49% 5% reflected and transmitted 44% 37% 7% photochemical inefficiency

C C 28.5% carbohydrate 25.5% 3 4 11.6% 8.5% synthesis 5.1% 0% photorespiration 6.5% 8.5% 1.9% 2.5% respiration

From Zhu, Long & Ort (2008) 4.6% 6.0% Current Opinion in 2008, 19:153–159 INTERCEPTING SOLAR ebi program leaders

RADIATION

1 - 20

10 Solar radiation Solarradiation d MJ 0 J F M A M J J A S O N D ebi program leaders Why is low input a critical factor?

•Better energy and carbon balance. •Lower or no environmental impact. •Marginal lands NITROGEN USE EFFICIENCY THEORY

SPRING/

SUMMER FALL WINTER

Mineralnutrients Mineralnutrients

Translocation Translocation Lignocellulose from to as dry shoots to growing shoot harvested, shoot senesces stay in rhizomes Prairie, Steppe & Savannaebi program Life leaders-form

Prairie, Steppe and Savanna perennial grasses are sustainable if cropped or burned annually, and accumulate carbon in the soil. C4 PERENNIALebi GRASSES program leaders

The Best of Both Worlds? USA – Switchgrass (Panicum Europe – Miscanthus (Miscanthus virgatum L.) x giganteus Greef et Deu.) THE IDEAL BIOMASS CROP?

C4 photosynthesis Long canopy duration Recycles nutrients to roots Low input High water use efficiency Sterile – non-invasive Can store harvest in field Easily removed No known pests/diseases Uses existing farm equipment THE IDEAL BIOMASS CROP?

CROP

C4 photosynthesis  Long canopy duration Recycles nutrients to roots Low input High water use efficiency Sterile – non-invasive n/a Can store harvest in field Easily removed  No known pests/diseases Uses existing farm equipment  THE IDEAL BIOMASS CROP?

CROP Corn SRC

C4 photosynthesis  Long canopy duration  Recycles nutrients to roots  Low input  High water use efficiency Sterile – non-invasive n/a  Can store harvest in field  Easily removed  No known pests/diseases Uses existing farm equipment  THE IDEAL BIOMASS CROP?

CROP Corn SRC C4PG

C4 photosynthesis   Long canopy duration   Recycles nutrients to roots   Low input   High water use efficiency   Sterile – non-invasive n/a   Can store harvest in field   Easily removed   No known pests/diseases  Uses existing farm equipment   ROADMAP

Why fuels from crops?

What is the ideal biomass crop?

Performance of Miscanthus and switchgrass.

Food vs. Fuel Switchgrass and Miscanthus C4 Perennial Grasses

USA – Switchgrass (Panicum Europe – Miscanthus (Miscanthus virgatum L.) x giganteus Greef et Deu.) InterAn Inter-specific-specific triploidhybrid

Miscanthus Miscanthus sacchariflorus x giganteus

+ =

“Diploid” “Tetraploid” “Triploid” 2n=2x=38 2n=4x=76 2n=3x=57 STERILE

Planting in 2002 Randomized plot trials March Pre-Emergence & Post-Harvest

Miscanthus and Switchgrass Early Spring (April) before maize is planted Efficient Solar Radiation Capture 4th July Early August Late October January February Harvest

Winter cutting (followed by baling) of demonstration plot of at the South Farms, University of Illinois, Urbana, early 2007. Harvested Bales Miscanthus – Traditionalebi program thatching leaders material in Japan

FEEDSTOCK PRODUCTION/ AGRONOMY PROGRAM Tom Voigt ENGINEERING SOLUTIONS FOR BIOMASS FEEDSTOCK PRODUCTION K.C. Ting BIOFUEL ECONOMIC AND ENVIRONMENTAL IMPACTS Madhu Khanna ENVIRONMENTAL IMPACT AND SUSTAINABILITY OF FEEDSTOCK PRODUCTION Evan DeLucia ASSESSING IMPACT OF INSECT PESTS AND PLANT PATHOGENS ON BIOMASS PRODUCTION Michael Gray GENOMIC-DIRECTED IMPROVEMENT OF FEEDSTOCKS Stephen Moose BIOFUELS LAW AND REGULATION Jay Kesan ENGINEERING ENZYMES AND METABOLIC PATHWAYS TO DISCOVERY OF ENZYMES TO DEGRADE PLANT CELL WALL DEGRADE PLANT CELL WALL LIGNOCELLULOSE LIGNOCELLULOSE John Gerlt Isaac Cann Miscanthus and cold-ebitolerance program leaders of WesternC4 photosynthesis. Blot Analysis M. x giganteus Z. mays 25 14 25 14

Wang, Moose, Portis & Long (2008) Plant Physiology (current issue) PPDK

PEPc

LS Rubisco Light interception by Miscanthus and corn Champaign, IL. BELOW GROUND BIOMASS – MISCANTHUS AND SWITCHGRASS 5 YEARS AFTER PLANTING THE BOTTOM LINE

Harvestable % 2006 Dry Mha needed for harvested Biomass Ethanol 133 billion US Feedstock (t/ha) (liters/ha) liters of ethanol cropland Maize grain 10.1 3,830 35 24.4 Maize stover 6.71 2,554 52 37.2 Maize Total 17.5 6,640 20 14.8 Prairie mix 3.8 1,447 92 72.5 Switchgrass 12.5 4,767 28 22.0 Miscanthus 29.1 11,066 12 9.3

Source: Heaton, Dohleman & Long (2008) Global Change Biology 14, 2000–2014. ROADMAP

Why fuels from crops?

What is the ideal biomass crop?

Performance of Miscanthus and switchgrass.

Food vs. Fuel Spatial Variability in Yields SPATIAL VARIABILITY IN YIELD MiscanthusMISCANTHUScan be productive IS PRODUCTIVE on marginal land ON MARGINAL LAND

The Miscanthus x giganteus in Cashel Co. Tipperary, Ireland photographed in September 1996 with 72HP tractor for scale. How WATERmuch water?REQUIREMENT

• 10 g kPa-1 kg-1 = 0.1 t ha-1 kPa-1 mm-1 Beale, Morison & Long (1999) Agric For Met 96, 103-115

• 1000 mm at 1.0 kPa would allow 100 t ha-1 – (assumes all water available)

• 500 mm at 4.0 kPa would allow 12.5 t ha-1 Enough land/enough water? US AVERAGE PRECIPITATION Enough land/enough water? PREDICTED MISCANTHUS YIELDS

t/ha

Data of Dr. Fernando Miguez, EBI, Univ. Illinois. US ARABLE CROPPING 1900

From: US Geological Survey - http://biology.usgs.gov/luhna/chap2.html US ARABLE CROPPING 1992

From: US Geological Survey - http://biology.usgs.gov/luhna/chap2.html WHAT IS POSSIBLE WITH THIS RETIRED LAND? • Miscanthus yields: 40 dry tons/ha feasible • 400 liters of ethanol / dry ton 16000 liters/ha • 40 M out of 200 M ha ~640 B liters / year of ethanol • US consumption (2004) = 780 B liters ethanol equivalent (excludes diesel) OVER 1 BILLION ACRES OF RECENTLY ABANDONED AGRICULTURAL LAND >>A billion acres of agricultural land have been abandoned

Campbell et al., Env. Sci. Technol. (2008) ASAP Article, 10.1021/es800052w CAN THESE CROPS BE SCALED UP? IRELAND 50 ha/day

Next generation Miscanthus planter (50 acres per day). MISCANTHUS FOR DRAX B Looking beyondLOOKING crop BEYOND CURRENT CROPS TRANSFORMATION SYSTEMS Feedstock Genomics Program

The primary aim of the EBI Feedstocks Genomics Program is to generate resources that will enable genomics-directed improvement of Saccharum and Miscanthus species as biofuels feedstocks.

1. Deep sequencing of the Miscanthus x giganteus transcriptome 2. Miscanthus and Saccharum genome sequencing 3. Assess genetic diversity within Miscanthus and Saccharum 4. Identify molecular markers for high-density genetic mapping in Miscanthus and Saccharum, and associate marker genotypes with phenotypes that contribute to biomass yield and composition in structured genetic populations. 5. Develop genome-scale expression profiling platforms for Miscanthus and Saccharum. 6. Develop an integrated bioinformatics system for functional genomics. Miscanthus EST sequencing

PolyA+ cDNA for Tissue Collected Total RNA RNA Library

Yes Yes Mature Leaf Yes No [1000+/1000ug] [5+/5ug] Post-Flowering Mature Leaf Yes No No No Yes Yes Emerging Shoot, Above Ground (Purple) Yes No [1000+/1000ug] [5+/5ug] Yes Yes Emerging Shoot, Above Ground (Green) Yes No [1000+/1000ug] [5/5ug] Yes Yes Emerging Shoot, Still Underground Yes No [1000+/1000ug] [5/5ug] No Apical Meristem (Midseason) Yes No No [889/1000ug] Apical Meristem (Pre-Flowering) Yes No No No Apical Meristem (During Flowering) Yes No No No

Apical Meristem (Post-Flowering) Yes No No No No Second Node Down (Midseason) Yes No No [552/1000ug] Yes Yes Leaves Beginning to Open Yes No [1000+/1000ug] [5+/5ug] • Sanger sequencing Yes Yes Rhizome Lateral Buds Yes No [1000+/1000ug] [5+/5ug] approach to identify No Rhizomes that had Emerging Shoots Yes No No [348/1000ug] allelic variants among Post-Flowering Rhizomes Yes No No No polyploid homeologs. Yes No Juvenile Roots Yes No [1000+/1000ug] [3+/5ug] • Verbal commitment Pollen Yes No No No from JGI to sequence Immature Inflorescence Yes No No No 150K M. x g ESTs. Mature Inflorescence Yes No No No 454 Survey Sequence of M. x g genome

Used flow cytometry to estimate M. x g genome at 7.5Gbp, or ~2.5 Gbp per haploid genome.

Genome Shotgun Genome skim

84 Mbp of DNA in 366,448 reads average read length was 229bp 44% GC content 1.2% coverage of the Mxg nuclear subgenomes

Whole chromosome sequences 100kb average chunks <1kb chunks Done clone by clone Need physical map Better than nothing? Agave mapisaga and A. saimiana 40 t/ha in semi-desert, Mexico DF.

Nobel, Garcia- Moya & Quero (1992) Plant Cell & Environment 15, 329-335. Spartina alterniflora 22 t/ha, Georgia USA, in seawater

Wiegert, Chalmers & Randerson (1983) Oikos 41, 1-6 ALTERATION % Increase in c Speculated relative to current time horizon value. (years) Improved canopy 10% (0-40%) 0-10 architecture

Rubisco with decreased 30% (5-60%) >20 oxygenase activity

Bypassing glycine 20% (2-35%) 0-10 decarboxylation.

Increased rate of recovery 15% (6-40%) 5 from photoprotection of photosynthesis

Introduction of higher 22% (17-30%) 5-10 catalytic rate foreign forms of Rubisco

Altered allocation of 40% (0-60%) 0-5 resources within the photosynthetic apparatus Long (2006) Plant Cell Env. , Evolutionary algorithm at work 400

300

200 % beginning of % 100

0

PRK

SPS SPP

GDC

GSAT

GGAT

FBPase

SBPase

UDPGP

Rubisco

ADPGPP

GAPDH Aldolase cFBPase

F26BPase

PGCAPase

PGA Kinase PGA

GCEA Kinase GCEA

FBP aldolase FBP

GOA Oxidase GOA

cFBP aldolase cFBP

Transketolase

HPR reductase HPR Photosynthesis

Zhu et al (2007) Plant Physiology 145: 513-526 Agronomists as seen by Plant Molecular Biologists

© Daily Illini 9/15/1907 Value of Plant Molecular Biology as viewed by Agronomists WHATINTEGRATING IS THE EBI? PROGRAMS

Under One Roof

Programs Projects Feedstock Development

Feedstock Deconstruction

Fuel Synthesis

Environment, Economics & Policy Meeting Global Productivity Needs or A Perfect Storm for Plant Sciences. •Global Feed and Food Shortage. •Replace Use of Fossil Fuels. •Increased productivity solution to feed, food & fuel. •„Omics coming of age. •Crop sequences completed & annotation advancing. •Transformation technologies •Cell wall, oil and carbohydrate synthetic pathways being unraveled. •Plant systems biology coupled with computational biology tools emerging. •Plant synthetic biology underway. WHICH FEEDSTOCK

Poplars Spartina Eucalypts Sugar Black Phylostachys cane Mangrove

Willows Miscanthus Sorghum Haloxylon

Sitka Spruce Switchgrass Echinochloa p. Casuarina

Jatropha Eucalypts Poplars Crambe Crambe Big Bluestem Willows