Technology for the Biobased EconomyTechnology for the Biobased Economy

Advanced Yeasts As A Robust Advanced Yeasts As A Robust Production Platform For Next Production Platform For Next Generation Fuels And ChemicalsGeneration Fuels And Chemicals

Marcel Marcel WubboltsWubboltsVP R&T DSM Innovation CenterVP R&T DSM Innovation Center

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DSM Company profile 2010

Global top 30 chemical industry• Net sales : € 7,243 billion • Net profit : € 337 million

22,738 employees• of which in Europe: approx 14,250• and in Netherlands: approx 7,300

>200 sites and offices on 5 continents

Ranked global no 1 in Dow Jones Sustainability Index

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DSM in motion: driving focused growth

People - Planet - Profit: creating value along three dimensions

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Climate & EnergyFossil Economy Issues

• Population growth• Resource scarcity• Climate change• Increased consumption

and waste • Energy security • Decline & costs of

fossil-fuel reserves • Deep well drilling risks

… Sustainability… Resource efficient economy

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Crude Oil Scenario’s

Source: http://www.trendlines.ca/scenarios.htmPeak Oil: 94 mbd in 2022The year we virtually run out of oil (excl BTL): 2257 (less than 5mbd)

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Oil will Run Out and Biomass is RenewableBiobased Economy - Back to the future?

1000 30002000

mbd

A briefmoment

in history

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Bio-Based Economy (BBE)

•• The bioThe bio--based economy is NOT just about based economy is NOT just about biofuelsbiofuels

•• There is more value in biomass than There is more value in biomass than burning valueburning value

•• BioBio--feedstocksfeedstocks are being used to create biomaterials are being used to create biomaterials and have massive potential: and have massive potential: -- Almost every fossilAlmost every fossil--derived carbon product derived carbon product couldcould be biobe bio--based or based or have a biobased alternative have a biobased alternative -- Many traditional chemistryMany traditional chemistry--based processes based processes couldcould be transformed to be transformed to biobio--based processesbased processes

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Bio-Based EconomyIntegration of Biotechnology and Chemistry

•• The The BioBio--Based Economy is NOT just about Based Economy is NOT just about biotechnologybiotechnology

•• Biotechnology Biotechnology –– using natureusing nature’’s tool kit s tool kit –– is a versatile is a versatile and powerful enabling technology and powerful enabling technology

•• BiobasedBiobased technologies are geared towards the technologies are geared towards the application of application of separationseparation technologies, technologies, chemistrychemistry and and biotechnologybiotechnology to make efficient use of biomassto make efficient use of biomass

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Job creation• Up to a million man-years of employment in Europe between 2010 and 2020• Predominantly in rural areas

Economic impact• Innovation and economic growth up to € 31bn in EU27 per year by 2020

Natural resources• 225m-270m tons of biomass residues available in EU 27 for bioproduct

conversion by 2020• Without change in today’s agricultural land use patterns or cultivating energy

crops

Environmental benefits• Biomass annually processed into 75bn-90bn liters of next-generation ethanol• 52-62% of EU27 forecast fossil gasoline consumption thus be replaced

Energy security• Greater transport fuel self-sufficiency for EU27

Bloomberg study: BBE offers lots of opportunities

Source: Bloomberg New Energy Finance

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LignocelluloseLignocellulose FeedstocksFeedstocks

Producing Food/Feed AND Producing Food/Feed AND FeedstocksFeedstocks for Chemistry / Energy for Chemistry / Energy on the same land by using on the same land by using byby--productsproducts of Agriculture and Forestryof Agriculture and Forestry

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Industrial Biotechnology

Feedstock ProcessBiobased Products & Solutions

Using NatureUsing Nature’’s Toolbox For Industrial Processess Toolbox For Industrial Processes

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Biomass from agricultural waste as a feedstock

• Agricultural waste material (e.g. corn stover, fiber, cob) or dedicated energy crops, but only when grown on land that is not used for food production

• Pre-treatment to open up the fibers, remove lignin and hydrolyze hemicellulose (chemical or enzyme catalyzed) to C5 sugars (xylose, arabinose)

• Enzyme treatment to hydrolyze cellulose to C6 sugars (glucose)

• Fermentation organisms (e.g. yeast) that can convert cellulosic C5 and C6 sugars into biofuels and other valuable products

SourceSource: CEN December 2008: CEN December 2008

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Biomass fermentation

• Cellulose degradationyields mainly glucose

• Hemicellulose consistsof heteropolymeric C5 and C6 sugars

• Lignin is a non-fermentablearomatic polymer

• Pretreatment, hydrolysis and fermentation

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DSM Cellulosic Enzyme System Performance on pretreated Wheat Straw

Unique: ThermoUnique: Thermo-- and Acid stable and Acid stable CellulaseCellulase CocktailCocktail

Less EnzymeLess EnzymeDramatically reduced viscosityDramatically reduced viscosity

Higher solids loading Higher solids loading No bacterial contaminationNo bacterial contaminationLowered antibiotics use?Lowered antibiotics use?

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Pilot Scale (2m3) Test of DSM Enzyme ABNT York Site

20% TS Abengoa Pretreated Corn Stover's Liquifaction & Saccharification Using DSM Cellulase Enzyme Product

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Time (hrs)

Visc

osity

(CP.

S)

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Viscosity (cp.s) Glucose (g/l) (sec Y axis)

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DSM Advanced Yeast Program

Unique: C5/C6 Fermenting YeastUnique: C5/C6 Fermenting Yeast

Industrial advanced yeast based on robust Industrial advanced yeast based on robust Saccharomyces cerevisiaeSaccharomyces cerevisiae engineered for conversion engineered for conversion

of biomass sugars of biomass sugars glucose, xylose, arabinose, glucose, xylose, arabinose, galactose and mannosegalactose and mannose at high yield to ethanolat high yield to ethanol

DSM Cellulosic Yeast product optimized with DSM DSM Cellulosic Yeast product optimized with DSM Cellulosic enzyme cocktail to offer a full package solution to Cellulosic enzyme cocktail to offer a full package solution to

industry industry vsvs peers.peers.

Business model: on site manufacturing, service, licensingBusiness model: on site manufacturing, service, licensing

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Why S. cerevisiae?

• Current organism of choice in the ethanol industry• High resistance to ethanol, lignocellulosic inhibitors, pH

and the like (robustness)• Genetics well described, easy genetic modification

• S. cerevisiae does not ferment pentoses• Industrial strains are considered to be more robust, but are

more difficult to transform

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Approach

• Construct cassettes allowing constitutive expression of a bacterial xylose isomerase and XKS1 in yeast (WO2009/109633; WO2009/109634)

• Constitutive expression of four non-oxidative PPP-genes (TAL1, TKL1, RPE1 and RKI1)

• Constitutive expression of the genes araA, araB and araD from L. plantarum(WO2008/041840)

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Enabling C5 metabolism in S. cerevisiae

XYL2

D-glucoseout

glucosein

G6P

F6P

F16DP

DHAP G3P

plasmamembraneHXT

EtOH

HXK

PGI

PFK

FBA

TPITDH PGK PGMENO PYK PDCADH

D-xyloseout

D-xyloseinXI

D-xyluloseXKS

X5PRPE

D-ribulose-5PRKI

D-ribose-5P

L-arabinoseout

L-arabinosein

L-ribulose

L-R5P

HXT GAL2

araA

araB

araD

TKLsedoheptulose-7P

TAL

erythrose-4P

TALarabitol

xylitolARAR AR

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Biological Activity Monitor (BAM)

DS58227 Preculture 2% GlucoseVerduyn 5% Glucose, 5% Xylose, 3.5% Arabinose, 1% Galactose

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0 20 40 60 80 100 120 140Time [hr]

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180 Glucose [g/l]

Xylose [g/l]

Arabinose [g/l]

Galactose [g/l]

Ethanol [g/l]

Glycerol [g/l]

OD600

CO2 [ml/hr] 2ndaxis

Strain ADS61147 Preculture 2% GlucoseVerduyn 5% Glucose, 5% Xylose, 3.5% Arabinose, 1% Galactose

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0 20 40 60 80 100 120 140Time [hr]

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Xylose [g/l]

Arabinose [g/l]

Galactose [g/l]

Ethanol [g/l]

Glycerol [g/l]

OD600

CO2 [ml/hr] 2ndaxis

Strain BDS61486 Preculture 2% GlucoseVerduyn 5% Glucose, 5% Xylose, 3.5% Arabinose, 1% Galactose

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Xylose [g/l]

Arabinose [g/l]

Galactose [g/l]

Ethanol [g/l]

Glycerol [g/l]

OD600

CO2 [ml/hr] 2ndaxis

Strain C

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Application tests in real hydrolysates

Corn Fiber, Corn Stover, Wheat Straw

• Hydrolysis• 10 % dry matter dilute acid pretreated biomass1)

• pH adjusted to 5.0• Addition of DSM Biomass Saccharification Thermostable Enzyme Cocktail• Liquefaction for 72 hours at 60 °C • Cooling to 33 °C

• Fermentation• Addition of salts and anti foam• pH adjusted to 5.5• Addition of DSM C5 Yeast• Fermentation at 33 °C until CO2 production was absent

1) In case of corn fiber, 13.8% dry matter was used

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Application tests in real hydrolysates

Corn Fiber

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-72 -48 -24 0 24 48 72

t (hours)

Con

cent

ratio

n (g

/l)

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0.1

0.2

0.3

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0.6

CO

2 (m

mol

/min

)

Galactose g/lMannose g/lGlucose g/lXylose g/lArabinose g/lEthanol g/lCO2 [mmol/min]

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Application tests in real hydrolysates

Corn Stover

0.0

5.0

10.0

15.0

20.0

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-72 -48 -24 0 24 48 72

t (hours)

Conc

entra

tion

(g/l)

0.00

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CO2

(mm

ol/m

in)

Galactose g/lMannose g/lGlucose g/lXylose g/lArabinose g/lEthanol g/lCO2 [mmol/min]

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Application tests in real hydrolysates

Wheat Straw

0.0

5.0

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30.0

-72 -48 -24 0 24 48 72

t (hours)

Conc

entra

tion

(g/l)

0

0.1

0.2

0.3

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0.5

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CO2

(mm

ol/m

in)

Galactose g/lMannose g/lGlucose g/lXylose g/lArabinose g/lEthanol g/lCO2 [mmol/min]

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Technical Accomplishments - Yeast

• Stable, marker-free chromosomal integration of genes encoding the xylose and arabinose pathways for xylose and arabinose utilization

• Adaptation improves the performance of the strains• Progress has been made in the fast conversion of the five

most abundant C5 and C6 sugars in real biomass hydrolysates, in the presence of undissociated acetic acid

• High ethanol yields, no or low by-product formation

• Opening up a platform for production of building blocks for materials from renewable resources

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Bio-based succinic acid will lead to a portfolio of products and applications

Solvents Coatings and pigments

Pharmaceuticals

PlasticizerFood

Freezing point depression agents

Renewable thermoplastics - PBS Pyrrolidones

Polyurethane1,4 BDO/THF

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pH 6-7 Prokaryotes+ Available Succinic strains+ Genetic tools available (e.g. E. coli)- Salt (e.g. Gypsum) formation- Infection risks (CAPEX)

pH ~3 Eukaryotes- Succinic strain to be developed+ Genetic tools available+ Free acid formation → Lower cost price+ No more Gypsum+ Robust organisms

At start of project

DSM technology - change of paradigm

Multiple host organisms, gene pathways andrecovery methods were explored and tested:

- Novel proprietary technology (more than 15 patents filed)

Prokaryotic

Eukaryotic

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White Biotechnology andWhite Biotechnology andIntegrated Integrated BiorefineriesBiorefineries

Business Model: Business Model: On Site Manufacturing of Enzymes and On Site Manufacturing of Enzymes and YeastYeast

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On Site Manufacturing ConceptLow Cost Production Concept

Traditional• Large scale production• Low cost geographies• Formulation (stabilization,

drying, dust prevention)• Logistics (handling, shipping)• Costly• Life Cycle Analysis?

On Site Manufacturing• Smaller scale installations• Production on location where

enzymes/yeast is applied, with option to supply neighboring plants

• No formulation required• Logistics simplified dramatically• More cost efficient• Better LCA (preliminary

analysis)

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Revolution from ‘Oil to Bio’Innovations Required

• Oil-refinery: – Crude oil (finite) as feedstock– Technology established– Very efficient use of (limited) feedstock

• Bio-refinery (1st generation):– Starch / Sugar (renewable) as feedstock– Technology established– Efficient use of (unlimited) feedstock– Food competition for some feedstocks

• Bio-refinery (2nd generation):– Biomass (renewable) as input– Sustainability– Logistics? Small scale or vicinity of harbor– Technology integration (energy, chemistry

and biotechnology) still in development– Valorization of co-products– Early stage, high risk. Partnerships

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The 2G Challenge

Feedstock Pre-treatment Hydrolysis Fermentation End Products

Ag. side streams:• corn fiber• wheat bran• other grain fibers

Ag. residues:• corn stover• wheat straw• cane bagasse• other residues

Energy crops:• switch-grass• energy cane• poplar

Waste materials:• municipal waste• forestry waste• paper fines

Aqueous:• steam explosion• hot water• combinations

Chemical:• acid• alkali• ammonia• oxidative

Solvent:• alcohols• esters• combinations

Biological:• enzymatic• microbial

Process• pre-sacc only• SSF• C6 only• C5 only• C5 & C6 combined• batch• continuous

Organism:• bacterial• fungal• yeast• GMO• non-GM• propagated• direct pitch

Process:• pre-sacc only• pre-sacc. + SSF• clean substrate• contaminated

substrate• physical conditions• ionic conditions

Enzymes:• natural cocktail• component

cellulases• enhanced cocktail• produced in situ• shipped in• plant expressed• combinations

Primary outputs• fuel ethanol• potable ethanol• technical ethanol• bio-based

chemicals• other biofuels

Co products:• valorize• energy generation• waste streams

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Delft Cellulose Biorefinery PilotA Unique Facility

• First truly multi-purpose biorefinery pilot (multiple feedstocks, fractionation, fermentation, down stream processing, digestion, recylcetests, integration of process technologies) suited for a variety of products

• Representative scale: >2.5 t/d biomass• Integration of existing facilities• All permits in place• Waste water treatment• Multiple pretreatments• Multi-client set-up• Independent governance

- Independent legal entity- Own management

Large investments• PPP academia, industry

government

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Acknowledgements

TU Delft IndustrialMicrobiology Group

Colleagues at DSM Biotechnology Center, Delft

Special thanks to: Paul Klaassen, Bianca Gielesen, Denise van Suylekom, Beate Wiedemann, Jan Roerig and Wim de Laat

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Change?