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DSM enzymes and process conditions for cellulosic ethanol
Herman PelDSM Biotechnology CenterDelft, The Netherlands
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Chemical synthesis & biotechnology
Energy, chemistry & polymer technology
Biotechnology
Hoffman La Roche’s Vitamins (1930s)
DSM (1902)
Gist-Brocades (1869)
Vitamins Omega’s
CarotenoidsPremixes for food & feed
EnzymesMinerals
Cultures & YeastsNutraceuticals
PharmaceuticalsCellulosic bioethanolBiomedical materials
Bio-plasticsHigh Performance PlasticsPolyamides and precursors
Resins for coatings and composites
Functional MaterialsSolar – advanced surfaces
Life Sciences
Materials Sciences
Building on an impressive history
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1000 30002000
Oil
cons
umpt
ion
Living off the landA brief
momentin history
Living off the land
At DSM sustainability is not just a buzz word
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In the end we need to biorefine
Source: http://www.biocore-europe.org/page.php?optim=biocore-in-brief
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Overview large scale bio-ethanol plant
700-1000 short tons / day 20-30 M gallons / yearGlucose, xylose
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It is real! POET-DSM JV Project Liberty
• First commercial scale cellulosic ethanol plant of POET-DSM JV• Starting capacity 75 million liter Ethanol per year from corn
residues• Shared infrastructure with existing grain ethanol plant in
Emmetsburg Iowa• Local corn residues• Power for both plants
• Construction completedSeptember 2014
• Startup in progress
http://poetdsm.com/liberty
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Feedstock / net wrap removal
http://poetdsm.com/liberty
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Fermentation / Solid-liquid separation
http://poetdsm.com/liberty
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The substrate is complex
38% Glucan
21% Xylan
18% Lignin
23% Other
Variable…Viscous…
Sand & Stones…
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Xylose
Glucose
Acid pretreatment
Multi-enzyme hydrolysis A
cce
ssib
ilit
y
In 3 steps from Corn Residues to Ethanol
YeastFermentation
Ethanol
Glucose
Xylose
Glucan hydrolysis
Xylan hydrolysis
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Main cellulases
LPMO
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Removal of acetyl esters from xylo-oligosaccharides
Acetyl xylan esteraseAXH-m
Arabinofuranosidase active on mono substituted arabinoxylan
AXH-d3
Arabinofuranosidaseactive on the 3 position of double substituted arabinoxylan
Ferulic acid esterase
Removal of ferulic acid
Glucuronidase
Removal of glucuronic acid from xylo-oligosaccharides
Galactosidase
Removal of galactose from side chains
HemicellulasesEndo-xylanase
β-xylosidase
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Requirements for enzyme hydrolysisEnzyme production process:• Low cost• Flexible raw materials
Hydrolysis process:• High sugar yield• Fast liquefaction• Low contamination
High yield – low cost requires integral approach
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DSM thermostable enzyme cocktail for biofuel productionEnzyme cocktail build around enzymes derived from thermophilic filamentous fungus Rasamsonia emersonii
Enzyme development target: • Increase enzyme performance
Enzyme cocktail improvement strategies:• Classical strain improvement• Enzyme screening• Protein engineering
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Natural selection of improved cocktailsClassical Strain Improvement (CSI)
Culture supernatant:Protein composition
analysis
Add to suspension of pretreatedwheat straw
Incubation:pH 4.5
time = 20 hrsTemp 65oC
SugarAnalysis
Cellmutagenesis Growth
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CSI and Process Optimization
Parent0
0.2
0.4
0.6
0.8
1.0
Strain 1 Strain 2
Rela
tive
cel
lula
seac
tivi
ty
4x
2x
8-fold increase inrelative cellulaseactivity
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CSI effects enzyme cocktail compositionStrong changes in amount and ratio of main cellulases
BG0
4
8
12
16
CBH
Rela
tive
pro
tein
con
tent
EG
ParentStrain 1
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Enzyme ScreeningRecombinant produced single enzymes
• Spiking to base mix
• Compose artificial mixes
• Identify weak links
• Identify enzymes with
highest impact on
cellulose degradation
BG
CBHEG/LPMO
Others
26%42%
13%
Classes of enzymes tested
18%
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Enzyme Mix OptimizationExperimental set-up
• Enzyme mix varied in 4 cellulases• Statistical mixture design:
– 55 incubations– same total protein dose– variation in ranges:
• BG 0.04-0.12• CBH 0.10-0.60• EG 0.18-0.68
• Conditions wheat straw assay:– pH 4.5– Temperature 65°C– Time 20h 0
10
20
30
10
Glucose released
(mM)
20 30 40 50
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Optimized Enzyme Cocktail
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Enzyme cocktail is active above 60°C
*
High performance in broad temperature range
Active at acidic pH
Improved control of contamination in hydrolysis
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The effect of scale on oxygen supply
• LPMOs require oxygen for their oxidative action on crystalline cellulose
• Production scale: hardly any transfer of oxygen from head space due to unfavorable surface area to volume ratio!
• IP filed on oxygen/air supply and control on production scale
• Lab scale: good transfer of oxygen from head space due to favorable surface area to volume ratio!
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The effect of scale on oxygen supply
Oxygen supply beneficial
Lab Pilot Production
Minimal oxygen level for LPMO
Reactor volume
Oxy
gen
tran
sfer
fro
m h
ead
spac
e
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Example: the effect of oxygen at pilot scale
0
10
20
30
40
50
60
70
80
90
0 20 40 60 80 100 120 140
Glucose con
version (%
)
Hydrolysis time (h)
Pilot scale20 % NREL pCS
With air
Without air
Controlled aeration speeds up glucan conversion
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Fast feedstock liquefaction
0 24 48 72 96 120
Hydrolysis time [h]
viscosity
glucose
Glu
cose
conv
ersi
on (%
)
Rel
ativ
e vi
scos
ity
Fast viscosity reduction 20% acid-pretreated wheat straw, 62*C
100 %
20 %
40 %
60 %
80 %
0 %
100 %
20 %
40 %
60 %
80 %
0 %
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DSM enzymes for biofuel production
DSM thermostable enzymes
• Fast liquefaction allows high dry matter content up to 25%
• Thermostability allows for less energy use for cooling
• Thermostability gives improved control of contaminations
CAPEX savings due to lower required capacity of process equipment
OPEX savings due to reduced energy costs
Reduced antibiotic costsIncreased revenues because less sugars are lost
Cost savings!
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On-Site Manufacturing of DSM enzymes
• The OSM unit is co-located with a lignocellulosic ethanol plant
• OSM makes optimal use of the existing infrastructure like utility systems, waste water treatment and rail/road connections
• OSM will use carbon sources directly available on site
• The OSM product is a whole broth enzyme and has not gone through the usual recovery step (filtration, concentration, formulation & packaging)
The main attributes of the OSM unit
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Global commercialization
Examples of public partnerships on various continents
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Acknowledgements
Partnership betwen
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Thank you !!