pongamia presentation uoq
TRANSCRIPT
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
osyn
thes
is o
f Alg
as
Aqua
tic L
ife B
reat
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
The REAL BIO-FUEL Cycle for CORN/Wheat/Sugarcane
NITROGEN Fertiliser
Crop Plant Biomass
The REAL BIO-FUEL Cycle for CORN/Wheat/Sugarcane
NITROGEN Fertiliser
HABER-BOSCH process
Transport/storage/application
Crop Plant Biomass
Run-off/pollution
The REAL BIO-FUEL Cycle for CORN/Wheat/Sugarcane
NITROGEN Fertiliser
FossilFuel
HABER-BOSCH process
CO2
Transport/storage/application
Crop Plant Biomass
CO2Run-off/pollution
Nitrous oxides/250 x GHG
The REAL BIO-FUEL Cycle for CORN/Wheat/Sugarcane
NITROGEN Fertiliser
FossilFuel
HABER-BOSCH process
CO2
Transport/storage/application
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
LIFE CYCLE ANALYSISIs CRITICAL
Hill et al, 2006, PNAS
LIFE CYCLE ANALYSISIs CRITICAL
LIFE CYCLE ANALYSISIs CRITICAL
Hill et al, 2006, PNAS
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