British Society of Sugar Technologists · Genetically Modified Crops: the devil’s spawn, saviours...
Transcript of British Society of Sugar Technologists · Genetically Modified Crops: the devil’s spawn, saviours...
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Cellulosic ethanol: facing the challenges
October 2014
British Society of Sugar Technologists
Leonardo Gomez
Center for Novel Agricultural Products
University of York
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Why cellulosic biofuels?
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• Environmental concerns about the impact
of human greenhouse gases on climate
change
• Dwindling petroleum reserves
• Security of oil supply
DRIVERS FOR THE EXPANSION OF BIOFUELS
(2004)
Shale gas
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• Environmental concerns about the impact
of human greenhouse gases on climate
change
DRIVERS FOR THE EXPANSION OF BIOFUELS
(present)
• Biofuels should avoid competing with
food production
• Biofuels should be sustainable
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Liquid biofuels
First generation = produced from food crops such as oils and starch
(corn ethanol, biodiesel)
Second generation = made from lignocellulosic
plant biomass
Third generation biofuels = designer fuels made from engineered microorganisms
Advanced
Biofuels
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Different procedures to produce liquid biofuels from biomass
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EU Renewable Energy Directive (RED, 2009) - 10% of renewable transport
fuels by 2020
Food crop derived biofuels limited to 7% (July 2014)
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Lignocellulosic Biomass = Cell wall
CELLULOSE
40%
MATRIX
20%
LIGNIN
25%
ASHES
5%
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Cell walls have evolved to develop recalcitrance
Cellulose
molecules
Cellulose
microfibrils
Hemicelluloses
Plasma
membrane
Lignins
Glucose
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Cell walls have evolved to develop recalcitrance
- Cellulose is a B-1-4 linked glucan that is grouped
in a crystalline status in microfilbrils which are very
difficult to hydrolise to release glucose
- Matrix polysaccharides are complex in
composition and structure. They are largely
composed by pentoses.
- Lignins are polyphenolics of quasi-random structure
that creates a hydrophobic coating for the
polysaccharides
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Size
reduction Pretreatment
Feedstock
Quality
Hydrolysis
Fermentation
Enzyme production
Co
nso
lida
ted
bio
pro
ce
ssin
g
SS
F
Bio
log
ica
l s
tep
s
Product
recovery
Residue
processing
Heat and
electricity
General biomass processing for cellulosic biofuels
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The success of cellulosic biofuels depends on their cost-competitiveness and sustainability
Cellu
losic
Eth
an
ol price (
cents
/gal)
Enzyme Feedstock Conversion
0
100
200
300
400
500
600
700
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
20
11
2012
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Commercial Cellulosic Ethanol Plants
- GranBio (Alagoas, Brazil) Operational since
October 2014. 82 million litres per year.
Investment $208M. Sugar cane bagasse.
- POET-DSM (Iowa, US) Operational since September
2014. 95 million litres per year.
Investment $250M. Corn stover.
- Beta Renewables (Crescentino, Italy)
Operational since July 2013. 76 million
litres per year. Investment $190M.
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Develop novel pretreatments
Identify the molecular basis of recalcitrance to
saccharification
Discover new enzymes for more efficient hydrolysis
and tailored fractionation
Improving biomass for industrial applications
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HTBP
Species involved: Arabidopsis, Brachypodium, Poplar, Barley, Maize, Tobacco, Sugar cane, Sorghum, Willow,
Rice, Wheat, etc.
Biomass Analytical Facility:
Developing tools for tailoring
biomass for industrial processes
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Developing novel chemical
pretreatments
Biomass pretreatment can be
considered biomass fractionation to
add value into different fractions
LIGNOCELLULOSIC MATERIAL
Supercritical extraction
Microwave assisted Thermochemical
pretreatment
Fermentation
Anaerobic Digestion
Pyrolysis
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-400
-300
-200
-100
0
100
200
300
400
-400 -300 -200 -100 0 100 200 300 400 500
t[2
]
t[1]
Scores Comp[1] vs. Comp[2] colored by File Text
123
1_180
1_180
1_180
1_20
1_201_20
2_180A2_180A2_180A2_180B
2_180B
2_180B
2_180C
2_180C2_180C
2_20A2_20A
2_20A
2_20B2_20B2_20B
2_20C
2_20C2_20C
3_180A3_180A
3_180A
3_180B3_180B3_180B
3_180C
3_180C
3_180C
3_20A3_20A3_20A3_20B3_20B3_20B3_20C3_20C3_20C
EZinf o 2 - bagasse3 (M2: PCA-X) - 2012-08-30 18:17:00 (UTC+1)
H2SO4
NaOH
WATER
Sugar cane bagasse
Biomass derivatives in the pretreatment liquor
Gomez et al. Bioenergy Research 2014
Developing novel chemical pretreatments
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Control
Maize
Antifoam Test at Ecover
Supercritical CO2 Extraction and
Fractionation of Maize wax
Andy Hunt-Tom Attard-
BDC
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Identify the molecular basis of recalcitrance to saccharification
Fermentation
Chemical
catalysis
Synthetic biology
Consolidated
bioprocessing
Alc
oh
ols
/Hyd
rocarb
on
s
The process of breaking a complex carbohydrate
into its monosaccharide components
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High Throughput Saccharification Analysis:
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“Hypothesis free” approaches to understand the main determinants
of the saccharification potential
- Screening of mutagenised populations Brachypodium
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Mutagen M1
M2 M3
Saccharification analysis of M2 plants
Characterisation and mapping of mutants with
altered saccharification
Forward genetic screening of Brachypodium for variability in saccharification
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0
20
40
60
80
100
120
140
160
180
200
40
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55
60
65
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80
85
90
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0
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5
16
0
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5
17
0
Mo
re
No
. of
mu
tan
t p
lan
ts
Saccharification Potential (nmoles reducing sugars hour-1 mg DW-1)
WT mean overall mean
Distribution of saccharification potential
Highest saccharification
Lowest saccharification
INRA
+72 %
-51 %
USDA
+74 %
-46 %
John Innes Centre
+65 %
-33 %
% = % higher/lower than WT for plate
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Characterising the sac1 mutant
Allele frequency plot for the five chromosomes of sac1
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Association genetics of saccharification in barley populations
BSBEC Cell Wall Lignin Programme
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Association Genetics experiment
• 640 elite 2-row spring barley genotypes
• Grown in polytunnel; 5 reps, watered, collared
• Various phenotypes measured (including straw biomass)
• Samples collected and SNP genotyped
• 3240 samples of 2nd internode base collected;
powdered
• Evaluated for saccharification at York
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0
1
2
3
4
5
6
-lo
g10 (
p-v
alu
e)
Saccharification
1H 2H 3H 4H 5H 6H 7H
GWAS Saccharification
2H 3H 7H5H4H3H 6H 7H5H2H 3H 7H5H4H3H 6H 7H5H
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Improving Enzymes
3 mm
Enzyme discovery in
Limnoria
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Teredinid bivalves (shipworm)
Isopod crustacean Limnoria (gribble)
Lignocellulosic biomass is an important food source
for marine organisms
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Termite guts are microbial bioreactors
Image: John Breznak, Michigan State University
Image: Genome Management Information System, Oak Ridge National
Laboratory
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Limnoria guts are sterile
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DNA pyrosequencing of
hepatopancreas cDNA libraries
• Total of 106 million bp of sequence
• 418,749 DNA sequences
• Average length of 247 base pairs
• 12,306 contiguous sequences
• 4,336 had close sequence similarity to known
genes
Exploring lignocellulose degradation in
Limnoria
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Glycosyl hydrolases dominate EST representation
Singletons
18%
Proteases
2%
Others
31%
Fatty acid
binding
protein
1% Lipase
0.2%
Glycosyl Hydrolases
27%
Haemocyanin
17%
Ferritin
1%
Oxygenase
2%
GH7
53.2%
GH9
37.0%
GH5
3.9%
GH38
0.1%
GH2
0.1%
GH16
0.4%
GH13
0.5%
GH35
2.7%
GH31
0.2%
GH20
0.1%
GH30
1.5%
GH18
0.3%
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GH7 family - cellobiohydrolases
• GH7 family enzymes found only
in fungi and protists
• Limnoria GH7 has 41% identity
to the closest relative
• Three distinct genes that
account for 14.4 % of all transcripts
0.1
USP - Md
P. grassii
USP - Cp 2
USP - Hs 2
USP - Rs 6
L. quadripunctata GH7C
L. edodes
C. rolfsii
Pleurotus sp.
V. volvacea I - I
P. arcularius
S. commune
H. jecorina
A. nidulans A. terreus
A. clavatus
A. fumigatus N. fischeri
T. aurantiacus A. niger
C. thermophilum
G. zeae
L. quadripunctata GH7A
L. quadripunctata GH7B
USP - Rs 5 USP - Rs 2 USP - Rs 3
USP - Rs 4 USP - Rs 1
USP - Hs 1
USP - Hs 3
PROTISTS
FUNGI
V. volvacea I - II
USP - Cp 1 USP - Cp 3
100
87
97
64 43
0.1
USP - Md
P. grassii
USP - Cp 2
USP - Hs 2
USP - Rs 6
L. quadripunctata GH7C
L. edodes
C. rolfsii
Pleurotus sp.
V. volvacea I - I
P. arcularius
S. commune
H. jecorina
A. nidulans A. terreus
A. clavatus
A. fumigatus N. fischeri
T. aurantiacus A. niger
C. thermophilum
G. zeae
L. quadripunctata GH7A
L. quadripunctata GH7B
USP - Rs 5 USP - Rs 2 USP - Rs 3
USP - Rs 4 USP - Rs 1
USP - Hs 1
USP - Hs 3
PROTISTS
FUNGI
V. volvacea I - II
USP - Cp 1 USP - Cp 3
100
87
97
64 43
0.1
USP - Md
P. grassii
USP - Cp 2
USP - Hs 2
USP - Rs 6
L. quadripunctata GH7C
L. edodes
C. rolfsii
Pleurotus sp.
V. volvacea I - I
P. arcularius
S. commune
H. jecorina
A. nidulans A. terreus
A. clavatus
A. fumigatus N. fischeri
T. aurantiacus A. niger
C. thermophilum
G. zeae
L. quadripunctata GH7A
L. quadripunctata GH7B
USP - Rs 5 USP - Rs 2 USP - Rs 3
USP - Rs 4 USP - Rs 1
USP - Hs 1
USP - Hs 3
PROTISTS
FUNGI
V. volvacea I - II
USP - Cp 1 USP - Cp 3
100
87
97
64 43
0.1
USP - Md
P. grassii
USP - Cp 2
USP - Hs 2
USP - Rs 6
L. quadripunctata GH7C
L. edodes
C. rolfsii
Pleurotus sp.
V. volvacea I - I
P. arcularius
S. commune
H. jecorina
A. nidulans A. terreus
A. clavatus
A. fumigatus N. fischeri
T. aurantiacus A. niger
C. thermophilum
G. zeae
L. quadripunctata GH7A
L. quadripunctata GH7B
USP - Rs 5 USP - Rs 2 USP - Rs 3
USP - Rs 4 USP - Rs 1
USP - Hs 1
USP - Hs 3
PROTISTS
FUNGI
V. volvacea I - II
USP - Cp 1 USP - Cp 3
100
87
97
64 43
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Acknowledgements
University of York
Simon McQueen Mason
Caragh Whitehead
Poppy Marriott
Rachael Simister
Susannah Bird
Marcelo Kern
Katrin Besser
Andrew Hunt
Tom Attard
SCRI-Dundee
Claire Halpin
Robby Waugh
Reza Shafiei
VIB-Gent
Wout Boerjan
Ruben Vanholme
INRA-Versailles
Richard Sibout
Herman Hofte