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Smart biomass for futurematerials
Rishi Bhalerao
SLU/Umeå Plant Science Center
We have great challenges ahead of us!
• Population growth
• Climate change
• Conversion from non-renewable
fossil fuels/materials to renewable bio-based ones
The Conclusion is Clear!
During the next century we have to produce more foodand biomaterials on the same or less land as today!
We need better trees!
In what way better?
Grow more on less land
Have multiple uses other than traditional one(viz, paper, pulp and timber!)
We need smart biomass!
Whats our approach
modify expression of as many(unknown) genes as possible
PLANT
GROWTH
WALL
CHEMISTRY
SACCHARIFICATIONANALYSIS OF THE LINES
IDENTIFICATION OF SUPERIOR
LINES
PRODUCTION OF TRANSGENIC Populus LINES
Field trials Breeding programs
Identify interesting genes GENE DISCOVERY
SUCROSE
1. Biomass production
Photo Hannele Tuominen
Ove Nilsson Rishi Bhalerao
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DNA microarrays PopGenIE
Bark Phloem Kambium Xylem
Hertzberg et al. PNAS 2001Schrader et al. Plant Cell 2004 Sjödin et al. New Phytologist 2009
The
app
roac
h
Gene Discovery
=> 2000 genes expressed specifically in the xylem forming tissues
Functional characterisation of ~1000 genes by genetic engineering of the model tree species Populus tremula x tremuloides-done by SweTree Technologies
Production of transgenic Populus lines
Photos Hannele Tuominen
The
app
roac
h
1. Populus tremula x tremuloides T89 hybrid aspen2. Populus tremula x tremuloides local clones3. Populus tremula4. Populus trichocarpa
1. Greenhouse grown trees2. Natural aspens
SwAsp 112 and UmAsp 350 collectionsCloned, in orchards, in Umeå (and in Ekebo/Sweasp)
3. Hybrid aspen grown in field trials18 genes modified, in two locations in southern Sweden, 2010, 2011, 2013
The feedstocks
Hei
ght
of
the
tree
s (%
co
ntr
ol)
0
20
40
60
80
100
120
140
Biomass production
Res
ult
s Ex
p1
Wood-a source for biorefinery
• Change cellulose cristallinity– effects
on bioprocessing
• Modified cellulose for more efficient
production of nanocellulose
• Better separation of xylan from lignin
and cellulose
• Lignin levels/polymerisation – effects
on bioprocessing
• Modification of wood density
S3
S1
PM
S2
CML
Before lignification
rosette
After lignification
MF
xyloglucan
cellulosa
pectin
xylan
lignin
mannan
Ewa Mellerowicz
Source Chris Somerville
Cellulose
➢The amount➢Cellulose crystallinity➢Degree of polymerisation
Totte Niittylä
Identify and genetically improve the best trees for
nanocellulose production
Tree Woodfibre
Cellulosemicrofibril
CellulosenanofibresW: <100nm
L: > m
Cellulosenanocrystals
W: <5nmL: <300nm
Nanocellulosic materials:+ Stronger than steel and stiffer than Kevlar+ Potentially a cheaper alternative to glass and carbon fiber+ Safe and green alternative for petrol based plastics
One of the biggest challenges in nanocellulose production is related to the efficient separation of cellulose fibrils from the raw material.
Tunnare cellulosa fibriller i invertase modifierade asp
Rende et al. 2017
XylanGlucuronoxylan
Source: Scheller and Ulvskov 2010. Annu. Rev.Plant Biol
➢The amount➢Chain length➢ acetylation➢ Linkages to lignin
Ewa Mellerowicz
Improving xylan for biorefinery
Ewa Mellerowicz
Xylan – second most abundant biopolymer
Poplar wood
xylan
other
lignin
cellulose
Miscanthus (grass)
xylan
glucan
lignin
cellulose
UPSC
Custom-tailoring xylan using fungal enzymes from wood degrading fungi
Held et al 2006 Wood degradation by soft rot fungi
UPSC
Custom-tailoring xylan using fungal enzymes from wood degrading fungi
Held et al 2006 Wood degradation by soft rot fungi
UPSC
Trimming xylan with different enzymes
O
OH
OH
O
O
OH
OH
CH3-O
OH
O
OOH
OH
O
O O
OH
O
O
OH
OH
O
O O
OH
O
O
OH
OH
O
H3COOH
OH
CO
H3COOH
OH
COOH
O
O
OAcOAc
OAc
OLIGNIN
OAcOAc
Glucuronoyl esterase, CE15
Acetyl xylanesteraseCE1, CE5
a-glucuronidaseGH67, GH115Xylanase
GH10,GH11
UPSC
Example 1: Xylan deacetylation by CE1 improvessaccharification and ethanol yield
0
20
40
60
80
100
Hot water Acid Alkali
Su
ga
r p
rod
uc
tio
n r
ate
(n
mo
lm
g-1
h-1
) WT A B C D
+1
4%
+1
3%
+1
9%
+1
0%
+1
8%
+7
%
+11
%
+1
9%
P ≤ 0.0006 P ≤ 0.2
P ≤ 0.001
+1
8%
** ** ** **
**
****
** ** **
Pawar et al., Plant Biotechn J 2016
UPSC
Hot water AlkaliAcid
Sugar production rate
0
0,4
0,8
1,2
1,6
0 5 10 15 20
Eth
anol (g
L-1
)
days
PD vs WT ≤ 0.0001
Ethanol yield
+70%
Altered cell wall architecture !
+10%
+16%
+8%
Trimming xylan with different enzymes
O
OH
OH
O
O
OH
OH
CH3-O
OH
O
OOH
OH
O
O O
OH
O
O
OH
OH
O
O O
OH
O
O
OH
OH
O
H3COOH
OH
CO
H3COOH
OH
COOH
O
O
OAcOAc
OAc
OLIGNIN
OAcOAc
Glucuronoyl esterase, CE15
Acetyl xylanesteraseCE1, CE5
a-glucuronidaseGH67, GH115Xylanase
GH10,GH11
UPSC
Example 2: CE15 decreases Glc conversionwithout pretreatment... but increases it after acid pretreatment
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
Lin
e 4
Lin
e 1
0
L
ine 2
1
Lin
e 2
2
Lin
e 2
3
WT
Lin
e 4
Lin
e 1
0
Lin
e 2
1
Lin
e 2
2
Lin
e 2
3
WT
Glc
/ C
ellu
lose
[g g
-1]
PcGCE overexpressors CE15 overexpressors
without pretreatment with acid pretreatment
Glucose yields in g per g of cellulose in hydrolysates
-30%
+12%
Gandla et al., 2015, Phytochemistry
+12%
UPSC
Reduced lignin
cross-linking!
Lab conditions are fine, but whathappens under natural conditions?
Starting year Number of genes
2010 16
2011 13
2011 7
2012 7
2013 22
2014 10
2016 9
2016 13
Total 965-years-old hybrid aspen field (B-2011) in Våxtorp collected
in July 2016.
Courtesy of Ewa Mellerowicz
Fältförsök med genetisk modifierade hybridaspar
Lignin
Source: Vanholme R et al. Plant Physiol. 2010;153:895-905
Hannele Tuominen
➢Total content➢Degree of polymerisation➢ composition
The approach
modify expression of as many(unknown) genes as possible
PLANT
GROWTH
WALL
CHEMISTRY
SACCHARIFICATIONANALYSIS OF THE LINES
IDENTIFICATION OF SUPERIOR
LINES
PRODUCTION OF TRANSGENIC Populus LINES
Field trials Breeding programs
Identify interesting genes GENE DISCOVERY
FT-I
R s
core
0
2
4
6
8
10
12
14
16
18
20
wood chemistry – FT-IR
Res
ult
s Ex
p1
Saccharification potential
0
20
40
60
80
100
120
140
160
180
Suga
r yi
eld
(%
of
wild
typ
e)
In collaboration with Leonardo Gomez and Simon McQueen-Mason, York University, UK
Hei
ght
of
the
tree
s (%
co
ntr
ol)
0
20
40
60
80
100
120
140
Biomass production
Res
ult
s Ex
p1
Oil production by metabolic engineering in woody biomass
Which plants are largest producer of oils in Sweden?
Can trees be used as production systems for oil?
Right answer: Pine and spruce (tall oil)
350 00 ton of fatty acids/year
Compare with rape =100 000 ton oil
T89: 0 w phloem, cambium, xylem
T89: 10 w phloem, cambium, xylem
Does SD signal influence lipid accumulation?
What is the molecular mechanism underlying lipid accumulation?
Blocking SD perception blocks lipid accumulation
0
5
10
15
20
25
30
35
40
45
50
0w T89 6w T89 10w T89 0w FT 6w FT 10w FT
nm
ol/
mg
FWtotal lipid
Downregulation of single gene can increase oil content 20%
Blocking hormonal responses can increase oil content 30%
Conclusions
• We can increase biomass
• We can manipulate biomass for:
• Ethanol
• Cellulose chemistry
• Oil
Umeå Plant Science Centre
>200 researchers of 45 nationalities
One of the strongest research centers in Europe for
experimental plant research and plant biotechnology.
Increasing oil content in salix to 10% would yield 1.5 ton of oil/ha and year in energy forests.
Fatty acids
Extractives from wood
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