1

Biscol KO meetingFrom Sophied to Biscol

Christian-Marie Bols

Plan

1.Wetlands incubator sprl– Products– R&D

2.Sophied project– Patents– Lab pilot

3.Biscol project– WP3– Exploitation strategy

Plan

1.Wetlands incubator sprl– Products– R&D

2.Sophied project– Patents– Lab pilot

3.Biscol project– WP3– Exploitation strategy

Wetlands : a Walloon company inside Europe

Wetlands: key figures• Created in 2001• Own 6 500 m2 of building in science park • Turnover 1Mi€• Sales in 10 countries• R&D budjet > 0.5Mi€• 2 patents

Wetlands: Activities• Lab equipment (UmicLab range)

•Novel probes for environmental measurement (UmicProbe range)

EN 13432ISO 14855ISO 16929OECD 208.

Same technology as used for pH and O2 biosensor

Excitation, Emission Spectra

0

20

40

60

80

100

120

350 400 450 500 550 600 650 700 750

Wavelength (nm)

Inte

nsity

Oxygen sensitive coating

Substrate

Light Source +Filter

Detector +Filter

Excitation Emission

Same technology as used for pH and O2 biosensor

Wetlands: A strong R&D department

• R&D fields: • microbiology, toxicity, biodegradability;• fermentation, bioremediation, composting;• lab equipment, bioreactors, automation

• focused on laccase (enzyme) use for biosynthesis of novel products

FP6-IP SME: Sophied

• UCL coordinator, Wetlands SME Leader• 26 partners

– 16 SMEs (low and high tech)– 8 Universities– 2 research centres

• 10 countries• Budget 10 000 000 €

• 4 years

FP6-STREP: Quorum

• Wetlands SME Leader• 8 partners

– 1 SME – 7 Universities

• 7 countries• Budget 2 500 000 €• 3 years

Wetlands: About new projects linked with the topic• ERA-NET Matera LANCE: Nanoparticle-based technology for the

removal of micropolluants from waste water

• FP7 BugworkerRW Walaid, Hypharco...

Wetlands: Networks through local and international collaboration• Local (Regional) level: Agrobiopole wallon: « Biorefinery

Euroview »; Wagralim • Belgian level: Essenscia, Bio.be (board)• European level: Europabio (lead market); ETP Suschem

(IB board); ETP Biofuel; ETP Euratex; Cost Biobio (board)

Plan

1.Wetlands incubator sprl– Products– R&D

2.Sophied project– Patents– Lab pilot

3.Biscol project– WP3– Exploitation strategy

Sophied proposes solutions at 3 levels :

o New safe processes for the production of dyes.

o New dyes which are less toxic and synthesised biotechnologically for high added value markets.

o New bioremediation technology to detoxify coloured wastewaters.

1. Industrial needs

R&D situation

Bottlenecks

Requests from partners:

Chemical classes

Colours

Types of fibres

Characteristics…

Potentialities of bioprocesses

Perspectives of market development

Class Market size(million euros)

Acid 770

Basic 260

Directs 320

Disperse 1080

Reactive 970

Others : Sulfur, vat, metallic…

and Natural690

2. determination of Target dyes and target WW

3. Collection

Solomon Island, French Guyana, Cuba

Under Rio convention

FUNGAL BIODIVERSITY

Internal “virtual” culture collection

600 Microorganisms

4. Identification and preservation

Time (Days)0 5 10 15 20 25

Lacc

ase

activ

ity (U

/l)

0

20000

40000

60000

80000

100000

120000

140000

pH

0

1

2

3

4

5

6

Tota

l car

bohy

drat

e (m

g/m

l)

5

10

15

20

25

30

Tota

l ext

race

llula

r pro

tein

(mg/

ml)

0.0

0.2

0.4

0.6

0.8

1.0

a

b

c

5. More enzymes…Microbial physiology and fermentation technology

• 15 strains studied• Conditions for microbial production of selected enzymes, elicitation • Production of novel low cost renewable fermentation media• Downstream processes

Coriolus polyzona MUCL 38443 in 6 liter fermentor

Laccase activity(●); pH(○); total carbohydrate (▲); total extracellular protein(□).a, b & c: Addition of molasses as supplement.

6. Laccases benchmarking

Pycnoporus sanguineus PS7

Perenniporia ochroleuca PO33

Bacillus subtilis cotA

Phoma PS344

Pleurotus ostreatus POXC+ POXA3

Bioscreen

Coriolus polyzona CP36

Catalogue of enzymes

7. Synthesis of ecocolourants

Enzymatic processes

NO6

Selection of precursors

Chemical synthesis of precursors

+

+

Reactions of precursors

+ Mechanistic study

Lab scale optimisation

0%

20%

40%

60%

80%

100%

0 2 4 6 8 10 12 14 16time (hours)

yield (%)

Structure characterisation

O

O

SO3NaNH2

NO

O

SO3NaN

NH

Scaling up

Whole cells processes

8. Screening of precursors

Efficient methodology for the selective synthesis of sulfonated precursors

New original precursors synthesised

45 precursors were tested

500 coloured molecules obtained

• 280 microbial strains screened

• Tests on solid and liquid media

• 37 Strains selected

Example of advanced work with Cerrena unicolor

9. Whole cells production of biodyes

10. Synthesis of new dyesBiomimetic synthetic approach

3HOALAO1

Pycnoporus sp produce phenoxazinone pigments: how to make more and better ?

LAO1

11. Evaluation of the applicability and quality

• 45 dyes tested

• Grey scale (SDC)

• Washing fastness 40 C ;30 min ISO105C06:S

• Water fastness 37 C; 34hr ISO 105;EO1

• Chlorinated Waterfastness ISO 105 E03

• Light fastness ISO105 B02

• Perspiration fastness…

exposure to toxic sample

12. Toxicitya) Development of a fast, reliable and cheap toxicity screening procedure

Caco-2 cells

• Development of new optic sensor technologies for toxicity assessment

• Development and validation of a Mini Ames test to save costs and time

• Development and validation of a fish eggs test to replace animal testing

Zebra fish embryo test

AmesMini Ames Test

b) Development of novel toxicity methods (no animal)

New optic sensor technology

13. Upscaling

Biodyes

• 4 dyes scaled up

• Started at µg

• Scaling up 1-3 kg

• Microorganisms and enzymes production started at 200µL

• Scaling up 50mL, 1L, 20L, 50L and 150L

Enzyme production

14. Life cycle assessment

Impact analysis: Ecoindicator-99 single score contribution of each input (kPt)

Analyzing 1 kg material 'pigment curie-22'; Method: Eco-indicator 99 (E) V2.03 / Europe EI 99 E/E / weighting

pigment curie-22 NaOH (100%) Water demineralized ETH S Sodium sulphate B250Benzene I Electricity LV use in E + imports U

Carcinogens Respiratory organics

Respiratory inorganics

Climate change

Radiation Ozone layer Ecotoxicity Acidification/ Eutrophicati

Land use Minerals Fossil fuels

kPt

1,7

1,6

1,5

1,4

1,3

1,2

1,1

1

0,9

0,8

0,7

0,6

0,5

0,4

0,3

0,2

0,1

0

Dye synthesis

Collection and Preservation

Advanced characterization and improvement

Year 1

Year 4

Year 2-3

Year 2-3

300 strains

Biodiversity preservation into Biological Ressources Center

Bacteria Aquatic White rot fungi Yeasts

5 colours 3 industrial

sectors

300 strains

37 more promizing strains

500 dyes

20 promizing precursors

5 enzymes 10 precursors

12 dyes

Industrial bioprocesses

3 tinctorial

classes

4 chemical

classes

45 precursors

4 dyes scaled up

10 enzymes

Different microorganisms

Targets

Screening

Ranking and selection

Proof of concept and benchmarking

Issues to be faced to ensure a future sustainable economical development

1) how to protect the health of the citizens ?

3) how to struggle against global climate changes?

4) how to develop alternatives towards fossil feedstock shortage?

5) how to be competitive at a global scale ?

2) how to protect the environment ?

Less toxic and non mutagenic dyes

Wastewater treatmentNo dangerous substancesMild conditions

Reduced Energy processes

Renewable raw material InnovationsNew niche market products with added value

Breakthroughs

Demonstration potential of optic sensor device

Proof of concept of EccocolourantsTM

Future developments of EccocolourantsTM will be more achievable

Proved that LCA can be carried out as a useful tool to evaluate

sustainable bioprocesses

Proved possibility to use wastes as raw material

Proved that wild type strains can be used to produce enzymes instead of

GMO’s

Testing of coloured samples (toxicity), important in REACH context

Breakthroughs

Solved a lot of industrial issues in the same project with a large vision

Philosophy (the process, the way to control it) shown to be efficient (and

apreciated by L’Oréal)

A new way to manage interactions academics/companies (IP/SME): looking

together in the same direction

High level of integration of people from several origins with diverse

backgrounds

Efficient IP sharing procedure

Added value for the industries

First EU project

Company moved from small regional company to international

networking company

Hiring of high level people

Increased RTD level

First patents

Possibility for growing up the company

New markets

Set of clients

Thank you Sophie and all of you…

Plan

1.Wetlands incubator sprl– Products– R&D

2.Sophied project– Patents– Lab pilot

3.Biscol project– WP3– Exploitation strategy

WP3 Pilot production line

• Sophied pilotsReady to useSimpleRobust

Low ccDryingBy-products

• 2 Steps bioreactor

Better yieldOther solvantsHigher ccRisk assessm.

ComplexEarly stage

Equipment summary :

- Working volume : 500 ml – 2000 ml;

- Continuous spectral absorbency record (until 1.5);

- 149 ml of reactor for immobilized laccase or living biomass;

- Pump : 0-1600 ml/min;

- Conductivity, Temperature, pH, Oxygen logging;

- Temperature control by double jacket circuit (beaker and reactor);

- Software for an easy calibration and log of the equipment.

SOPHIED Lab :

Column 149 ml with thermostated

jacquet

Pump control panel

Conductivity and pH panel transmitter

Oxygen and conductivity sensor at the output of

the cartbridge

Temperature and pH sensor in the beaker

Thermostated bath with output recirculation

VIS Spectrophotometer

Peristaltic pump with flow up to 1600 ml/min

Pressure switch preventing over-

pressure damages

Dual position of the fitting

Tempered 2000 ml beaker

Circulation path

Fluidized bed

Packed bed

Common path

Liquid tested path

Warming liquid path

Temperature control

Tempering liquid

Software interface

Spectrophotometer calibration and settings

Trailer lab; ; Xavier; ; Lare one 1 gr/l speed 2; ; 11/03/2008;11/03/2008;11/03/200815:33;15:35;15:37Fluidized bed;Fluidized bed;Fluidized bedpH;3.99;4.38O2 (mg/l);2.49;2.50

Temperature (°C);20.93;21.06Conductivity (mS/cm);0.00;552.47Spectrum; ; nm; ; 345.74;NaN;NaN345.95;NaN;NaN

Record settings and user informations :

Easy data export …

First trials

0,6

0,8

1

1,2

1,4

1,6

1,8

2

Spectrum evolution

abs.

19

20

21

22

23

Temperature (°C)

3

4

5

6

7

pH

560

580

600

620

640

660

Conductivity (mS/cm)

Acid injection events

Exploitation strategy

• Dye structures (patents)

– SME• Niche 

market

• Low volume

• High price

– Big player• Impact

• Large volume

• Production line– Biocatalyst

• Consumable

• IP secret‐patent

– Bioreactor• Durable

• Patent‐IT