Astrosurf · Screening Production Presentation of the new created company Speakers Claire Coutel,...

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Presentation of the new created company

Speakers

Claire Coutel, Kathrin Feil, Chloé Maetz, Anne Schlegel, Ines Schulze

December 2006Amphi Ebel

Ecole Supérieure de Biotechnologie Strasbourg

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Cellulozyme is Supplier of cellulose degradation systems

For bioethanol producers

For other industrial cellulases’ users

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ChloChloéé MaetzMaetz

Claire CoutelClaire Coutel

Ines SchulzeInes Schulze

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To create To create a new highly active a new highly active

cellulose degradation systemscellulose degradation systemsto reduce of bioethanol to reduce of bioethanol

production costsproduction costs

Anne SchlegelAnne Schlegel

Kathrin FeilKathrin Feil

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Preprocessing

Pretreatment

Enzymatic or chemical cellulose hydrolysis

Fermentation

Distillation

Feedstock (wheat straw, corn stover, softwood)

Ethanol

Enzymatic cellulose degradation

system from cellulozymecellulozyme

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Humanressources

Legal steps

Developmentof the project

Investments

Localization

Development of our product

Production study

20072004 2005 2006

LaboratoriesSEMIA

Buisness incubator Schirmeck

Patent

Dr Maetz, Dr Fiel R&D proof of conceptIng. Schulze: production

ConnectusFunds

Creation of

Mrs Schlegel

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Oséo, personnal funds, grants

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R&D new products

Scale-up

Schirmeck

Production and commercialisation ofCropsBreaker

Privateinvestors

Dr Maetz, Dr Feil : production, R&DIng. Schulze: productionMrs Schlegel : Business manager and marketingMrs Coutel : CFO (volunteer)

Technician(s), trainee

��20102007 2008 2009

Humanressources

Legal steps


Investments

Localization

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Dr Maetz, Dr Feil : production, R&DIng. Schulze: productionMrs Schlegel : Company managementMrs Coutel : employed in 2011Technician(s), trainee

Production and commercialisation of CropsBreaker

R&D new products

��20142011 2012 2013

Humanressources

Legal steps


Investments

Localization Schirmeck

Privateinvestors « Point mort »

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Oil becomes more and more expensive

Increase of ethanol demand

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A lot of countries promote the use of bioethanol:� In USA, N°1 growth market

expected growth : 20% in the next few years.

� In Europe, directive 2003/98/EC :

5,75% in 2010

20% in 2020 (currently 2%)

Tax exemption for biofuel

� China focuses on biomass-based energy for the future

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EU quotas for bioethanol utilization

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� The demand for bioethanol will enormly increase in thenext few years

� More and more ethanol production facilities will appear

1st generation bioethanol : corn, wheat, sugar beet and sugar cane

In Europe and North America, the maximum capacity of cropsproduction is nearly reached…

--> 2nd generation biofuels have to be developed from non-food cellulosic biomasses

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� In USA:

Enough cellulosic feedstock to replace approximately 40% ofcurrent gasoline consumption.

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Total: 180 million tons/year

�115 billion liters bioethanol

Agricultural waste isabout a third cheaper than crops.

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� In Europe:

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There are large amounts of unused agricultural wastes

Straw and corn stover are the main cellulosic biomasses

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Cellulosique biomasses chosen:

Straw and corn stover

Targeted Market:

Europe and north America

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With the increasing demand for ethanol, more and more ethanolproduction facilities could stand out

The 2nd generation bioethanolmarket is emerging

For the moment, most companies produce starch-based or sugar-based ethanol

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� European Companies

About 10 big ethanol producers:

• from sugar beet: Südzucker (Germany), Tereos (France)

• from cereals: Agroetanol (Sweden), Vireol (UK)

• from wood: Sekab (Sweden)

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� Three examples for pilot plants:

BC International Corp.(USA): wood wastes, rice straw

Abengoa Bioenergy (Spain): straw

IOGEN Corporation (Canada): Wheat straw and corn stover & production of own enzymes

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� The market for industrial enzymes

7% growth by 2010

Cellulase market: $500 million and 20% of market share

Other industries concerned: textiles, detergent, feed

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�0�Main players:

� Place for us: largemarket and fewcompanies

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� Novozymes

World leader in industrial enzymes

Global sales: 840 million � in 2005

Net profit of 115 million �

Cellulase for bioethanol production: the strongest growtharea in 2005

Celluclast®:Enzymes from the fungus Trichoderma reesei

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� Genencor:

Big biotech company

Worked together with the NREL and achieved a cost of cellulases of 0,15� per gallon of ethanol

� 30 fold improvement in enzyme cost

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Genencor Novozyme Cellulozyme

Comparison of hydrolysis efficiency of cellulose degradation systems

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Products already available: mixture of several cellulases

CropsBreaker:�purified Multi-enzymatic complex�20 times more efficient�no need for pretreatment

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Products already available: mixture of several cellulases

Cost of feedstock's bioconversion into fermentable sugar at equal enzymatic activity

Competitors CellulozymePri

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vest

ed fo

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Return on investments of pretreatment

Pretreatment chargesCost of return on investments of pretreatment Cost of functioning charges of pretreatmentCost of enzymatic hydrolysis

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500�Base: 1kg of CropsBreaker

Cost of feedstock's bioconversion into fermentable sugar at equal enzymatic activity

Competitors CellulozymePri

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Return on investments of pretreatment

Pretreatment chargesCost of return on investments of pretreatment Cost of functioning charges of pretreatmentCost of enzymatic hydrolysis

Cost of return on investments of pretreatment Cost of functioning charges of pretreatmentCost of enzymatic hydrolysis

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CropsBreaker:�purified Multi-enzymatic complex�20 times more efficient�no need for pretreatment

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EthanolPlant material Sugars

Cellulases

Corn stover & straw:

Cellulose

Hemicellulose

Lignin

Glucose

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�� Crystal cellulose

Endo-Glucanase

�Endo

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Glucoseß-Glucosidase

BglA

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ExoGlucanaseCelK

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Multienzyme complex :

Different types of cellulases are secreted and extracellularly bound to a scaffolding molecule, the scaffoldin

� Cellulases are bound randomly to the scaffoldin, depending on concentrations

� CBD domain to bind substrate

�Synergy effect �Higher activity than single enzymes

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CBD3 Coh Coh Coh Coh Coh Coh CohCoh Coh

Creation of a scaffoldin module with 9 identical cohesin domains& a CBD with high affinity to cristalline cellulose

Endo*

Endo*

CelKCelKBglA

BglA

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Chosing cellulases with high synergistic activity to degrade the desired biomass

Endo*

Araf51

XynZ

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HemicellulasesSynergy!

Endo*

Doc

1. Directed evolution to improve activity & stability of Endoglucanase

(CelR) from C. thermocellum byFamily shuffling

� HTS to identify the most activeendoglucanase

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�� 1st SCREENING for improved Endoglucanaseswith colorigenic pNP-Glucosides

� 2 highly active clones

2nd SCREENING: define the stoechiometric composition of enzyme types in the final cellulosome

� Improved Cellulosome!

111223

ScafArafXynZBglACelKEndo*

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CLONING & SMALL SCALE PRODUCTION of a chimeric macromolecular complex degrading lignocellulosic biomass:

CropsBreaker

Endo*Araf51

XynZ

Endo*Endo*

CelKCelKBglA

BglA

CBD3

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Coh Coh Coh Coh Coh Coh CohCoh Coh

20 times higher activitythan currently available enzyme mixtures

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�Imitation of natural assembly: Extracellular expression

�Separate protein expression of each component in Bacillus subtilis

Concentrated protein solution ( 100 g/l )

� Extracellular expression of each component in

100 L batch fermentor (10 g/l)

� Protein concentration by ultrafiltration

waste: liquid & proteins smaller than concentrated protein

Waste:

cells

� Cell separation by microfiltration

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Our equipment: only three 100 L Reactors available (in 2007)

� Need to produce proteins in stock

Protein 1

Concentrated Protein 1

Protein 2


Protein 3


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311221

Endo*

XynZArafBglACelKScaf

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waste:

liquid, all unbound cellulases & other secreted proteins

SCAFF

CelK

BglA

Araf

XynZ

Endo*

binding buffer

Protein yield : 4-5 kg / week

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Purification?

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Endo*Araf51

XynZ

Endo*Endo*

CelKCelKBglA BglA

CBD3



CropsBreacker

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2010-2011Endo*

Araf51

XynZ

Endo*Endo*

CelKCelKBglA BglA

CBD3



CropsBreacker

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CropsBreakerBottle of 1LConcentration 100g/l

www.cellulozyme.com

How buy CropsBreaker ?

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�� $ �� Another industrial area for cellulases

Feed industry

Detergent industry

Textile industry

Endo*Endo*

Sale endo* individually

Endo*

Endo*

Endo*

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� For the laboratory : Connectus founds

� In SEMIA Buisness-incubator : OSEO, grants, personnal founds

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Investissments

1 097 000 �

Building renovation

Production equipment

Quality-Control, R&D equipment

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1 097 000 �

Capital+

Grants485 000 �

Price« Concours d'aide à la Création d'entreprise

innovante »

GrantsPersonnal founds

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Loans+

Private investors612 000 �

Capital+

Grants485 000 �

Investissments

1 097 000 �

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� ��.�" ��Profits of Cellulozyme

-300000

-250000

-200000

-150000

-100000

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100000

150000

200000

2007 2008 2009 2010 2011 2012 2013

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New products �Family schuffling sur d’autres enzymes?

Nouvelles biomasse?

Ou la je met la slide alternative strategy?

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Low coast of investissement but

profitability quickly

CropsBreacker is 20 times more efficientthan cellulase of our

competitors

Purified enzymes

No need expensive pretreatment of the

biomass

Highly competent and motivated team

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Cellulozyme21 route de Steinbach67 130 Russ – France

Tél : +33 (0)3 88 97 01 73Internet : www.astrosurf.com/astrodonon/

e-mail : [email protected]

CelluloZyme team at la remise des prix du « Concours d'aide à la

Création d'entreprise innovante »November 6th 2006 - Paris

CelluloZyme21 route de Steinbach67 130 Russ – France

Tél : +33 (0)3 88 97 01 73Internet :www.cellulosyme.fre-mail : [email protected]

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INCOME STATEMENT

2007

PRODUCTS 0Sell of CropsBreacker 0

CHARGES PurchaseMaintenance of equipementConsumablesMedium for productionEquipment

Operating chargesRentWater 500ElectricityInsurancePhone-InternetAdvertising

Imposition and taxProfessional tax 0Habitation taxTurnover tax 0

Employed chargesSalariesSocial costs

Depreciation and amortisationTangible fixed asset 216Intangible fixed asset

BuildingMaterial

PatentsOEB patent applicationAnnual installment OEBUSA patenAnnual installment USA

Financial costLoan interestPrivat investor interest

TOTAL PRODUCTS 0

TOTAL CHARGES

PROFIT or LOSS

13 500

10 0002 5001 000

12 8006 000

1 5002 0001 8001 000

5 000

5 000

69 23560 2059 030

144 616

25 000119 400

7 0003 800

3 200

24 00024 000

276 151

-276 151

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BALANCE SHEET 2007

ASSETS LIABILITIES

Gross Depreciation After-TaxIntangible fixed assets CapitalStart-up costs 216 216 0 Common equity

Patents Profits

GrantsBourse création entreprises innovantes

Tangible fixed assets Region AlsaceADEME

Building renovationProduction equipmentR&D equipment

DebtsBank loanPrivate investors

Financial assets 0

Available funds Overdraft

TOTAL ASSETS TOTAL LIABILITIES

75 000

35 000 7 000 28 000 -276 151

450 00020 00015 000

250 000 25 000 225 000572 000 114 400 457 60025 000 5 000 20 000

437 500100 000

90 749

821 349 821 349

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10 g/l production in 3 x 100 L fermentors availbale

Time of 1 fermentation cycle: 2 days

(including cell separation & protein concentration, autoclavation) :

for 100 L : 100 L x 10 g/l = 800 g [protein]

� 3 x 800 g = 2400 g [protein] in 2 days

� 1200 g [protein] in 1 day

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Time Table:

...XynZEndo*11-12

ArafBglAEndo*9-10

ScafCelKCelK7-8

Endo*ScafXynZ5-6

Endo*ArafBglA3-4

Endo*ScafCelK1-2

Reactor 3Reactor 2Reactor 1Day

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Astrosurf · Screening Production Presentation of the new created company Speakers Claire Coutel,...

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