pongamia presentation uoq

Legumes: Vital for LifeCILRCILR

Global Problems, Legume Biotechnology and Ethical Biofuels

Peter M. GresshoffAustralian Research Council Centre of Excellencefor Integrative Legume [email protected]://www.cilr.uq.edu.au

Melbourne, March 2009

“The Team” (past and present)

UQ Industry Partners/FundingProf. Peter GresshoffDr Paul Scott Australian Research CouncilDr Qunyi Jiang Bioenergy Solutions, COMET Dr Bandana Biswas Pacific Renewable Energy, BER Mikiko Miyagi UQ Strategic Funds, UniQuestDr Ning Chen Origin Energy, Brisbane City CouncilJohanna Hadler Sunshine Coast Council, SEQC, C4OCHamid SolamaniDr Alvin van NiekerkLisette PregeljDong-Xue LiCherie WilkinsonStephen KazakoffJohn O’TooleANUDr Michael DjordjevicDr Charles Hocart

Global Problems:• population increase• land availability• human conflict/bioterrorism • water quality and availability • emerging diseases• dietary changes• environmental sensitivity• consumer expectations• governmental regulations• global warming/climate change

Global Problems:• population increase• land availability• human conflict/bioterrorism • water quality and availability • emerging diseases• dietary changes• environmental sensitivity• consumer expectations• governmental regulations• global warming/climate change

Terra nostra (NASA)‘Our spaceship’

#1: Social Change

#2: Education

#3. Science/Gene revolution

#1: Social Change

#2: Education

#3. Science/Gene revolution

DNA sequencing (ABI)

SOLUTIONS ???#3. Gene revolution:

• globalism and transferability• plant and animal genomes• bacterial genomes (over 300)• bioinformatics/robotics/computing• global information flow (Internet)• transcriptome/proteome/metabolome• genome/phenome research• transgenics/knock-outs/RNAi/mutants• computational biology• animal and plant cloning • stem cell research

#3. Gene revolution:• globalism and transferability• plant and animal genomes• bacterial genomes (over 300)• bioinformatics/robotics/computing• global information flow (Internet)• transcriptome/proteome/metabolome• genome/phenome research• transgenics/knock-outs/RNAi/mutants• computational biology• animal and plant cloning • stem cell research

SOLUTIONS ???

Sturt Desert Pea

The BIO-FUEL Concept

Key Issues:

1) Difference of FUEL and ENERGY

2) Energy input for NITROGEN

3) Competition with FOOD crops

4) Competition for ARABLE land

5) Competition for WATER

Key Issues:

1) Difference of FUEL and ENERGY

2) Energy input for NITROGEN

3) Competition with FOOD crops

4) Competition for ARABLE land

5) Competition for WATER

Food vs. Fuel

Different types of food

Different types of fuel

Food vs. Fuel

Different types of food

Different types of fuel

ISSUES

Energy: Electricity and fuel (33% vs 67% in Australia)

What will you learn?

• FOSSIL FUEL is LIMITED• biofuel is essential• sustainability is ESSENTIAL • not all biofuels are sustainable• legumes fix nitrogen• Pongamia is a suitable legume• biotechnology and marginal lands

Global Problems:• Food (especially protein) Security

• Fuel Security

161 trillion litres crude oil reserves3.8 trillion litres per year usageor 120,000 liters per second

PEAK OIL !!!

Global Problems:• Food (especially protein) Security

• Fuel Security

161 trillion litres crude oil reserves3.8 trillion litres per year usageor 120,000 liters per second

PEAK OIL !!!

Golden Rice(Prof. Ingo Potrykus)

The Fossil Fuel CycleCarbon Dioxide at Atmosphere

Oceans,lakes

RootsBreathing

Fossil Fuels:Coal, Natural Gas, Oil

VegetableGarbageSo

il an

d O

rgan

isms

Brea

thin

g

Anim

al B

reat

hing

Plan

ts B

reat

hing

Plan

tsPh

otos

ynth

esis

Phot

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is o

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as

Aqua

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ife B

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hing

Biofuels:fuels made from biological material- CRUDE OIL/COAL: but CO2 absorbed 150 Mya- OLD technology. WOOD, straw, oils, wax, candles- Carbon neutral (???)- Ecological- Sustainable- Renewable

- Australia legislation: 20% renewable energy by 2020

Biofuels:fuels made from biological material- CRUDE OIL/COAL: but CO2 absorbed 150 Mya- OLD technology. WOOD, straw, oils, wax, candles- Carbon neutral (???)- Ecological- Sustainable- Renewable

- Australia legislation: 20% renewable energy by 2020

The Assumed Carbon-neutral BIO-FUEL Cycle:

• proposed balance of input and output

• biomass or plant productfor biofuel production

FUEL

AIR

PLANTS

Potential Biofuels

First Generation: Ethanol ---- E10, E100 in Brazil

Second Generation: Biodiesel; Cellulytic Fermentation

Third Generation: Hydrogen gas (H2)

Modern Biofuels:• Ethanol from sugars• Ethanol from cellulytic digestion• Biohydrogen from algae

• Diesel from plant oils- Palm oil (12 tons/ha/year!!)- Canola (1.5 tons/ha/year)- Soybean (0.8 tons/ha/year)- Pongamia (5 tons/ha/year)

Modern Biofuels:• Ethanol from sugars• Ethanol from cellulytic digestion• Biohydrogen from algae

• Diesel from plant oils- Palm oil (12 tons/ha/year!!)- Canola (1.5 tons/ha/year)- Soybean (0.8 tons/ha/year)- Pongamia (5 tons/ha/year)

Switchgrassafter one year growth

Potential Biofuels-Issues to Consider

• land use and availability• fertiliser use• food crop competition• water use and requirements• mechanical planting, maintenance and harvesting• high yield• energy inputs vs. outputs (LIFE CYCLE ANALYSIS)

Potential Biofuels-Issues to Consider

Ethanol: low energy content, volatile, hydroscopic; low flash

Biodiesel: non-food feed stock needed; ecology; high flash

Cellulytic Fermentation: high unproven technology, lignin

Hydrogen gas (H2): non-polluting, small molecule, explosive

Biofuels and History:• Rudolf Diesel used peanut oilto power his first engine 100years ago; Crude oil was cheaper and easier then.

• Biofuels are renewable and sustainable; zero-sulphur

• Biofuel production can be sustainable IF…

Biofuels and History:• Rudolf Diesel used peanut oilto power his first engine 100years ago; Crude oil was cheaper and easier then.

• Biofuels are renewable and sustainable; zero-sulphur

• Biofuel production can be sustainable IF…

The Assumed BIO-FUEL Cycle:

FLAWS:• industrial inputs• land and water• fertiliser costs• fertiliser impact (NOx)

The REAL BIO-FUEL Cycle for non-legume crops

NITROGEN INPUT

SugarcaneCanolaCorn

Oil palmJatrophaPoplar

Switchgrass

SugarcaneCanolaCorn

Oil palmJatrophaPoplar

Switchgrass

The REAL BIO-FUEL Cycle for non-legume crops

NITROGEN INPUT

FossilFuel

EmissionsCO2, N20, NOx

The REAL BIO-FUEL Cycle for CORN/Wheat/Sugarcane

Crop Plant Biomass


NITROGEN Fertiliser

Crop Plant Biomass


NITROGEN Fertiliser

HABER-BOSCH process

Transport/storage/application

Crop Plant Biomass

Run-off/pollution


NITROGEN Fertiliser

FossilFuel

HABER-BOSCH process

CO2


Crop Plant Biomass

CO2Run-off/pollution

Nitrous oxides/250 x GHG


NITROGEN Fertiliser

FossilFuel

HABER-BOSCH process

CO2


CO2Run-off/pollution

Nitrous oxides/250 x GHG

THE NEGATIVESTHE NEGATIVES

Conclusions

Biofuel capture of CO2 and solar energy is limited bynitrogen supply

The C cycle needs a strong N cycle

Nitrogen supply has an energy and environmental cost

The BETTER BIO-FUEL Cycle with Legumes

NITROGEN INPUT

Nitrogen Fixation

Sugar

What is Nitrogen Fixation?

Nitrogen Gas

Ammonia

Conversion of NITROGEN gasfrom the atmosphere to AMMONIA,

used to make proteins.

Nitrogenase enzyme inan invading bacterium

(called Rhizobium) harboured within legume nodules

PROTEINS

Nodules and lateral roots share parts

Nodules are organised

Nodules harbour good bacteria

Nitrogen Fixation in Pongamia nodules removes N-fertiliser need

Nodules

Sucrose 1 mm

Nitrogen Gas

Ammonium Amino acidsP. Crutzen has the Nobel Prize in Chemistry

P. Crutzen has the Nobel Prize in Chemistry– but overlooks LEGUMES!!

How is diesel fuel made?• fuel is based on trans-esterification

• methanol plus oil = ESTER

• esters are volatile (e.g., perfume, methyl-jasmonate, fresh bread)

How is diesel fuel made?• fuel is based on trans-esterification

• methanol plus oil = ESTER

• esters are volatile (e.g., perfume, methyl-jasmonate, fresh bread)

Biodiesel Synthesis: the Industry Perspective

CRITICAL: the feed stockCRITICAL: the feed stock

Real Applications

Real Factories

Two KEY papers

Hill et al, 2006, PNAS

LIFE CYCLE ANALYSISIs CRITICAL






GainGain

Conclusions:

Input costs need to be reduced

Nitrogen fertiliser is major input

N fertiliser also yields NOx

NOx is 250 x GHG of CO2

(from Zemanek and Reinhardt, 1999)

Nitrogen fertiliser

Life Cycle Analysis (LCA)reveals that fertiliser cost

is a major component of plant oil production

For canola oil; a non-legume

Bio-diesel advantage over bioethanol:• Hill et all (2006) PNAS (LCA= life cycle analysis)Bio-diesel advantage over bioethanol:• Hill et all (2006) PNAS (LCA= life cycle analysis)

Bioethanol BiodieselEnergy gain

%23 96

N % run-off 100 3

P % run-off 100 7

Pesticide load 100 13

C sequestered 100 340

The CILRConcept of Plant

System Biology inCrop Improvement and

Understanding ofBiological Processes

The CILRConcept of Plant

System Biology inCrop Improvement and

Understanding ofBiological Processes

Feedstock

Biopolymers

GRAINGRAINIndustrial

ApplicationsFOOD

FEED

Embryo

Flower

Leaf

Root

Nitrogen Acquisition

Water Acquisition

Phosphorus Acquisition

BiofuelsNutriceuticals

SymbioticMineral

SymbioticMineral

SymbioticMineral

CarbonSequestration

CarbonSequestration

Feedstock

Biopolymers

GRAINGRAINIndustrial

ApplicationsFOODFOOD

FEEDFEED

Embryo

Flower

Leaf

Root

Nitrogen Acquisition

Water Acquisition

Phosphorus Acquisition

BiofuelsNutriceuticalsNutriceuticals

SymbioticMineral

SymbioticMineral

SymbioticMineral

SymbioticMineral

SymbioticMineral

SymbioticMineral

CarbonSequestration

CarbonSequestration

CarbonSequestration

Molecular Physiology Analysis

Genome and Transcriptome Analysis

Proteome and Metabolome Analysis

Interactive data bases and com

putational modeling

What is BIOTECHNOLOGY?

Application of biological knowledge for the production of a more valued product

Desirable

Etr1-1

Animal Feed

Bioethanol(starch/cellulose)

SolarEnergy

Input

WaterResource

Input

MineralNutrition

Input Pongamia pinnata

Biodiesel

Green Manure

CarbonCredits

SoilProtection

The Pongamia Biodiesel Biotechnology ProjectPBBP

Pongamia is officially termed Millettia pinnata

Typical legume flowersPongamia pinnata chromosomes

-Meiotic chromosomes (2n=22) x1,800.-Chromosome size is similar to that of soybean (2n=4x=40)-Soybean has a genome size of 1,100 megabases (1/3 of humans)-Presumed genome size of Pongamia pinnata : 600 Mb

At UQ we are using Solexa deep sequencing. 15 million runs (35 bp)

1 2

6 4

11

5 3

109

8 7

pachytene anaphasemetaphase

BioEnergy Research (2008) vol. 1, pg. 1-10.

Pongamia fatty acid/oil:

Questions:

How does the seed control the distribution of energy toprotein, starch and oil?

How does the seed controlfatty acid composition?

Sucrose

SEED Cotyledon

Protein Starch

Fatty Acids

C20:0

C22:0

Waxes

C16:1

C18:3

C18:1

Oleic acid

C18:0

Stearic acid

C16:0

Palmitic acid

C18:2

Linoleic acid

?

?

3 weeks !

CountryCity

Laboratory

Pongamia grows fast and in stressed conditions

Vegetative ‘Clonal’ Propagation

Rooted Cuttings Tissue culture

The Pongamia Biodiesel Biotechnology ProjectPBBP

A B

250 bp

500 bp

750 bp

1000 bp

1 2 3 MStorage proteins

DNAfingerprinting

Nodules

Growth

The Pongamia Biodiesel Biotechnology Project

DNA fingerprinting

M1 M2 K2.1 K1.1 K1.2 K1.3 K1.4 K2.2 Marker K2.3 K2.4 K2.5 K2.6 K2.7

01100100

0110

1110111001100110

1110

00010111111010010000

Fig. 6: demonstration of binary scoring of DNA profiling of Pongamia DNA. Leaf DNA was DAF amplified and separated by PAGE, and stained by silver

(see Bassam and Gresshoff, 2007). Band presence was scored as ‘0’ or ‘1’ depending on absence or presence. The figure shows a portion of a DAF gel with the binary codingfor band presence shown below.

Pongamia genomic DNA

CTAB method DNeasy Plant mini kit

Pongamia mRNA isolation

M Actin PpNARK-KD

RT-PCR of NARK and Actin

DNA isolation RNA isolation RNA quantification

undet.39.31:100000

undet.36.81:10000

37.733.61:1000

32.830.11:100

29.726.91:10

NARK-KDActinCTDilution

qRT-PCR

CCTTTCATAGAAGGCGGCGGTGGAATCGAAATCTCGTGATGGCAGGTTGGGCGTCGCTTGGTCGGTCATTTCGATAAGCTCAGATCTGTTAACATTAACGTTTACAATTTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGTCGACTCATTATTAATTCAGATCCTCTTCTGAGATGAGTTTTTGTTCCCGTTTGATCTCGAGCTTGGTGCCTCCACCGAACGTCCACGGGTATTCATTATGTTGTTGACAGAAATACATTGCAAAATCTTCAGGCTCCAGGCTACTGATGGTGAGAGTGAAATCTGTACCAGATCCACTGCCA

CTGAACCTTGATGGAATTCCAGATTGCAAAGTGGATCCAGAGTAGATAAGAAGTTTATTAGTTTTCCCAGGTTTCTCTTGAAACCAGGCTAAATACTTGCCAATGTTTTTACTTGCCCTGCAATTAATAGTAATGGTTTCTCCAGGAGATGCAGCAAGATAAGATGGAGACTGAGTGAGCTCGATGTCCGATCCGCCACCGCCAGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAGCAGTCCATAGCATAGCCGTAGTAACCCGATCTTGCACAGTAATAGACGGCAGAGTCCTCAGATGTCAGGCTGCTGAGTTGCATGTAGGCTGTGTTGGAGGATGTATCTGCAGTGAATGCGGCCTTGCCCTTGAACTTCTCATTGTAGTTAGTATTACCACTTCCAGGTAAAATCTCTCCAATCCACTCAAGGCCATGTCCAGGCCTCTGCTTTACCCACTCTATCCAGTAGCTACTGAGTGTGTAGCCAGTGGCCTTGCAGGATATCTTCACTGAGGCCCCAGGCTTCATCAGCTCAGCTCCTGACTCCTGCAGCTGCACACTAGTTCGTCGGTTCTGTAACTATCATCATCATCATAGACACACGAAATAAAGTAATCAGATTATCAGTTAAAGCTATGTAATATTTACACCATAACCAATCAATTAAAAAATAGATCAGTTTAAAGAAAGATCAAAGCTCAAAAAAATAAAAAGAGAAAAGGGTCCTAACCAAGAAAATGAAGGAGAAAAACTAGAAATTTACCCTCAGATCTAGGAGGGTTCTTGCACTGGTGGTCATACCAATGCTAAGGAGGACAAGGATGGCAAGAGCCAAATGCTTAGTAGTAGCCATGGTCAAGAGTCCCCCGTGTTCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGA

TGCTCC

PpNFR5-kinase

Pongamia gene discovery and characterisation

G. max NFR5b: 88% identical (703/793)

L. japonicus NFR5: 83% identical (650/779)

Fatty acid and triglyceride biosynthesis in plants

TAG=tri-acyl-glycerides

SUCROSESUCROSE

BIODIESELBIODIESEL

LEAFLEAF

SolarEnergySolar

Energy

ChemicalEnergy

ChemicalEnergy

Pongamia produces ‘good’ oilRT: 6.00 - 14.50

6 7 8 9 10 11 12 13 14Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

RT: 11.43AA: 10804270

RT: 11.90AA: 4950132

RT: 10.38AA: 3909312

RT: 9.59AA: 1836957 RT: 11.14

AA: 1811354

RT: 12.46AA: 638958 RT: 13.94

AA: 408022RT: 8.68AA: 69480

RT: 6.12AA: 84472

RT: 7.38AA: 70831

RT: 9.85AA: 12048

RT: 10.67AA: 9193

NL:5.63E6TIC F: MS ICIS pod_1_+_IS_c

Time (min)

Rel

ativ

e ab

unda

nce

C18:1

C18:0C16:0

C18:2

C18:3

Low saturated FAstability

Low cloud point

(Dr Charles Hocart, RSBS)

30% of Australia's land is affected by salinity

Costs ~ AU$270 Million/year in production losses due to salinity.

“There is an urgent need to utilize and reclaim these saline soils, and this may be possible by planting salt-tolerant tree

legumes”(Zou et al. 1995, – UQ and CSIRO)

High salinity has minimal effect on Pongamia growth

sowing.

ParameterEC

(dS/m)

12 weeksUn-Nodulated Nodulated

Shoot Height (cm) 0 14.9 ± 1.1 a 20.0 ± 0.8 a

4 15.7 ± 1.0 a * 20.5 ± 1.0 a *

10 15.7 ± 1.2 a 18.5 ± 1.4 a

20 17.5 ± 1.1 a 18.4 ± 0.8 a

EC (dS/m) Nitrogen Concentration (% of d. wt)

Un-Nodulated Nodulated0 2.52 ± 0.16 a 2.42 ± 0.05 a

4 1.99 ± 0.08 a 2.41 ± 0.10 ab

10 2.27 ± 0.10 a 2.16 ± 0.05 bc20 2.25 ± 0.11 a 2.04 ± 0.02 c

High salinity has minimal effect on Pongamia N gain

Rain-fedOpportunities with

BioEnergy Solutions

Caboolture, QLD April 2008RobertKane

GeorgeMuirhead

Coal Seam Gas-WaterOpportunities withORIGIN Energy

Coal Seam Gas-WaterOpportunities withORIGIN Energy

ORIGIN Energy CSG Phase I2.1 ha planted May 2008

Coal Seam Gas-Water

With ORIGIN Energy

Pongamia, despite its sub-tropical origins, grows in cold winter/hot

summer environments680 trees trial

Biodiesel Yields

0

500

1000

1500

2000

2500

3000

3500

Soybean Rape-seed Jatropha Pongamia

Feedstock

Litr

es p

er H

ecta

re

Biodiesel Yields – of Pongamia pinnata compared to other biodiesel crops

One hectare of Pongamia

40 tons of Pongamia

pods/seedsPods/seeds –

50/50

CRUSH

12 tons of Pongamia pulp 5 tons of oil

5,500 liters of biodiesel

Diesel at pump = $1.50/L

Maintenance costs:$100/ha

Seeds = 35% oil content65% starch & protein – 20%

protein

1 t oil = 1,136 l of biodiesel

350 trees5 m apart

Removal of toxic components

Oil cake suitable as fertiliser or

animal feed

Establishment/Production costs:$ 1,000/ha;$ 3,000/ha irrigated

Oil extraction and related

costs:$80/ton

Income/ha/yr:Oil: $ 3,000C-credit: $ 300Honey: $ 100Cattle feed: $ 500

Cost Estimates for Pongamia Biodiesel

Some broad calculations from broad assumptions:1) 1 ton of plant oil makes 1,100 liters of biodiesel2) 1 hectare =350 Pongamia trees3) 1 Pongamia tree (6-10 years old) makes 20,000* seeds4) 1 seed = 1.8 g* at 40%* oil content (*conservative estimates)

Therefore 1 hectare produces about 5 tons oil per year

Some broad calculations from broad assumptions:1) 1 ton of plant oil makes 1,100 liters of biodiesel2) 1 hectare =350 Pongamia trees3) 1 Pongamia tree (6-10 years old) makes 20,000* seeds4) 1 seed = 1.8 g* at 40%* oil content (*conservative estimates)

Therefore 1 hectare produces about 5 tons oil per year

1 square kilometer = 100 hectares = 500 t oil = 550,000 l1 square kilometer = 100 hectares = 500 t oil = 550,000 l

Australia needs 1.8 x 1010 liters of diesel (2007 figures)Assuming B20 (short term) = 3.6 x 109 liters= 7,000 square kilometers = 200 plantations of 6 km x 6 km

Australia needs 1.8 x 1010 liters of diesel (2007 figures)Assuming B20 (short term) = 3.6 x 109 liters= 7,000 square kilometers = 200 plantations of 6 km x 6 kmAustralia has 1-2 million square kilometers of marginal landsAustralia has 1-2 million square kilometers of marginal lands

Conclusions:1) Sustainable biofuel production is essential2) A Nitrogen cycle drives the Carbon cycle3) Nitrogen input is negative (Cost + Environment)4) Biological nitrogen fixation is a sustainable alternative5) Biofuels from LEGUMES needs to take advantage

of marginal land to avoid crop competition6) Pongamia pinnata offers a possible solution7) Biotechnology and plant improvement are in progress

Conclusions:1) Sustainable biofuel production is essential2) A Nitrogen cycle drives the Carbon cycle3) Nitrogen input is negative (Cost + Environment)4) Biological nitrogen fixation is a sustainable alternative5) Biofuels from LEGUMES needs to take advantage

of marginal land to avoid crop competition6) Pongamia pinnata offers a possible solution7) Biotechnology and plant improvement are in progress

pongamia presentation uoq

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