Vantaggi e problematiche dell’approccio

enzimatico per la produzione di bioetanolo da

biomassa lignocellulosica

22ndnd BioBio--EthanolEthanol ConferenceConference

University of Milano Bicocca

Department of BioTechnology and BioSciences

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced from

biomass (2nd gen)?

�What are the key

biological barriers

� What and How

at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

BioEthanol - 2nd Generation ??

The Future …

… is under

way !!!!

The Future …

… is under

way !!!!

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the keybiological barriers

� What and How at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

Requirements for industrial scale-up

Starting cheap – ending cheap?

The proper choice of the substrate is

crucial

Gain and/or Loss of functions

lead(s) the way to production

(gr/L), productivity (gr/l/hr) and

yield (gr/gr).Bio-process optimization: technical

support of natural or engineered

abilities

Starting cheap – ending cheap?

Purification of product adds

significantly to the production

costsContinuous R & D: adaptation

of (micro)organisms to technical

constraints (development of

superior hosts)

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the keybiological barriers

� What and How at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

19971997: : Yeast Strains for the Production of Lactic Acid, (Patents)Yeast Strains for the Production of Lactic Acid, (Patents)

20002000: : Ascorbic Acid Production from Yeasts, (Patents)Ascorbic Acid Production from Yeasts, (Patents)

20002000: : Production of Production of heterologous heterologous proteins from proteins from Z. Z. bailiibailii,,

(US Patent)(US Patent)

20022002: : Process for expression and secretion of proteins by the Process for expression and secretion of proteins by the

nonnon-- conventional yeast conventional yeast Zygosaccharomyces bailiiZygosaccharomyces bailii (Patents)(Patents)

20052005:: Ascorbic acid production from DAscorbic acid production from D--glucose (Patents)glucose (Patents)

20052005: : Improved strains for the production of organic acids Improved strains for the production of organic acids (Patents)(Patents)

2006: Improved strains for the production of organic acids (Patents))

2007: Increase in stress tolerance with ascorbic acid during

fermentation (PCT patent application)

2008: Methods for improving acid and low pH tolerance in yeast

(PCT patent application)

2008: 2008: Improved yeast strains for organic acid production Improved yeast strains for organic acid production

(EU Patent application) (EU Patent application) University of University of MilanoMilano--BicoccaBicocca

2009: 2009: A new one will be filed within 3 monthsA new one will be filed within 3 months

(EU Patent application) (EU Patent application) University of University of MilanoMilano--BicoccaBicocca

Patents and Patent Applications�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the keybiological barriers

� What and How at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

1. Pretreatment (Heat,

pressure, or acid treatments).

2. Solid-Liquid Separation.

3. Enzymes Production.

4. Cellulose Hydrolysis.

5. Fermentation of Cellulosic

and Hemicellulosic Sugars

(C5 and C6)

A. Milling.

B. Liquefying and Heating the Corn meal.

C. Starch Hydrolysis.

D. Fermentation of Starch Sugar (C6)

E/6. Distillation (96%)

F/7. Dehydration.

1. Pretreatment (Heat,

pressure, or acid treatments).

2. Solid-Liquid Separation.

3. Enzymes Production.

4. Cellulose Hydrolysis.

5. Fermentation of Cellulosic

and Hemicellulosic Sugars

(C5 and C6)

A. Milling.

B. Liquefying and Heating the Corn meal.

C. Starch Hydrolysis.

D. Fermentation of Starch Sugar (C6)

E/6. Distillation (96%)

F/7. Dehydration.

How is EtOH produced ?

1st Generation

2nd Generation

US Department of

Energy - Office

of Science

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced from

biomass (2nd gen)?

�What are the keybiological barriers

� What and How at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

How is EtOH (2nd Gen) produced ?

US Department

of

Energy - Office

of Science

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced from

biomass (2nd gen)?

�What are the keybiological barriers

� What and How at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

- Sequence DNA from

energy crops

- Identify genes and

pathways that improve

biomass productivity

- Develop crops

optimized for enzyme

degradation

- Sequence DNA from

energy crops

- Identify genes and

pathways that improve

biomass productivity

- Develop crops

optimized for enzyme

degradation

1. Biomass production and delivery: main challenges

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the key

biological barriers

� What and How at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

2. Pretreatment:main challenges

-Identify enzymes that

rescue the severity of

pre-treatments

-Minimize production

of inhibitory by-

products

-Identify enzymes that

rescue the severity of

pre-treatments

-Minimize production

of inhibitory by-

products

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the key

biological barriers

� What and How at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

3. Cellulose hydrolysis:main challenges

-Screen natural

environments for the

most efficient

enzymes produced by

fungi, bacteria and

animals

-Understand how

enzyme system(s)

interact with cellulose

-Increase the catalytic

rate and thermal

tolerance of enzymes

-Screen natural

environments for the

most efficient

enzymes produced by

fungi, bacteria and

animals

-Understand how

enzyme system(s)

interact with cellulose

-Increase the catalytic

rate and thermal

tolerance of enzymes

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the key

biological barriers

� What and How at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

4. Sugar fermentation: main challenges

-Integrate Biomass

hydrolysis and

fermentation

-Engineer metabolic

pathways to produce

diverse biofuels

-Increase product

tolerance and yield

-Develop strains

capable to efficiently

fermenting mixed

substrata

-Engineer strains to

increase tolerance to

by-products

-Integrate Biomass

hydrolysis and

fermentation

-Engineer metabolic

pathways to produce

diverse biofuels

-Increase product

tolerance and yield

-Develop strains

capable to efficiently

fermenting mixed

substrata

-Engineer strains to

increase tolerance to

by-products

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the key

biological barriers

� What and How

at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

WP3: Optimal enzyme

mixtures

- Hydrolysis efficiency

- Enzyme induction- Feasibility of production

Novel micro-organisms

for bioethanol production

WP7: Process calculations

- Economics & ecoefficiency- Optimal process regimes

WP1: Raw materials

- Raw material collection- Pretreatment

WP2: Efficient enzymes

- Screening from nature and culture collections

- Metagenomic andcDNA libraries

- Protein engineering

Novel enzymes for

biomass hydrolysis

WP5: Targeted strainengineering

- Pathway engineering

- Sugar uptake engineering

- Redox & energetics- Metabolic modelling

WP4: New strains through

mutagenesis and screening- Screening from nature and

culture collections

- Mutagenesis- Evolutionary engineering

Ecoefficient and economicprocess options for efficient 2nd generation

bioethanol production

• Efficient anaerobic co-fermentation of pentose and hexose sugars

• Process robustness and tolerance

towards high temperature, acid,ethanol, and hydrolysate inhibitors

• Thermo and mesophilic

enzymes with improved efficiency,

and reduced nonproductive binding• Optimal enzyme mixtures for

biomasses of European relevance

Novel enzymes Novel micro-organisms

WP6: Process assessment

- Biomass hydrolysis

and fermentation

- Process options

(SHF, SSF, modified)- Temperature regimes

- Pilot scale assessmentWP3: Optimal enzyme

mixtures

- Hydrolysis efficiency

- Enzyme induction- Feasibility of production

Novel micro-organisms

for bioethanol production

WP7: Process calculations

- Economics & ecoefficiency- Optimal process regimes

WP1: Raw materials

- Raw material collection- Pretreatment

WP2: Efficient enzymes

- Screening from nature and culture collections

- Metagenomic andcDNA libraries

- Protein engineering

Novel enzymes for

biomass hydrolysis

WP5: Targeted strainengineering

- Pathway engineering

- Sugar uptake engineering

- Redox & energetics- Metabolic modelling

WP4: New strains through

mutagenesis and screening- Screening from nature and

culture collections

- Mutagenesis- Evolutionary engineering

Ecoefficient and economicprocess options for efficient 2nd generation

bioethanol production

• Efficient anaerobic co-fermentation of pentose and hexose sugars

• Process robustness and tolerance

towards high temperature, acid,ethanol, and hydrolysate inhibitors

• Thermo and mesophilic

enzymes with improved efficiency,

and reduced nonproductive binding• Optimal enzyme mixtures for

biomasses of European relevance

Novel enzymes Novel micro-organisms

WP6: Process assessment

- Biomass hydrolysis

and fermentation

- Process options

(SHF, SSF, modified)- Temperature regimes

- Pilot scale assessment

Novel high performance Enzymes and Micro-Organism for

conversion of lignocellulosic biomass to bioethanol

EU Framework VII – NEMO

2009-20132009-2013

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the keybiological barriers

� What and How

at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0 12 24 36 48 60 72 84

time (hours)

OD

(6

60

nm

)

0,0

0,3

0,6

0,9

1,2

1,5

1,8

0 12 24 36 48 60 72 84

time (hours)

OD

(6

60

nm

)

0,0

0,4

0,8

1,2

1,6

2,0

2,4

0 12 24 36 48 60 72 84

time (hours)

OD

(6

60

nm

)

Lactic Acid 45 g/l pH 3

Acetic Acid 60mM pH 3pH 2.2

Lactic Acid 45 g/l pH 3

Acetic Acid 60mM pH 3

pH 2.2

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the keybiological barriers

� What and How

at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

EU Framework VII - NEMO

Innovative

DS Processing

7

1Novel Enzymes and

Microrganisms 2Microbial Genomics

and Bioinformatics

3Metabolic Engineering

and Modelling

6 Innovative

Fermentation Science

4Biocatalyst Function

and Optimization

5Biocatalyst Process

Design

Bioreactor

Source: SusChem

the 7 High-Tec Platforms requiredfor the future

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the keybiological barriers

� What and How

at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

BBC is a structure based on

the most advanced scientific

and technological platforms

required for upstream,

bioreactor production,

downstream and analysis of

the product (GMP-like env.)

BBC is part of CEBIB

(Centre of Excellence Bicocca

Industrial Biotechnology),

Regione Lombardia -

Università degli Studi di

Milano Bicocca.

Bicocca Bicocca BiotechnicumBiotechnicum

CenterCenter�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the keybiological barriers

� What and How

at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

BBC

5th Floor - Upstream and Production

4th Floor - Downstream and Analysis

What is a good Biofuel?

(a) … it is highly combustible, but not

explosive;

(b) … it is something with a high

energy to mass ratio;

(c) … it is stable for long-term storage;

(d) … it is transportable;

(e) … it is inexpensive;

… (f) and it is renewable!!

(a) … it is highly combustible, but not

explosive;

(b) … it is something with a high

energy to mass ratio;

(c) … it is stable for long-term storage;

(d) … it is transportable;

(e) … it is inexpensive;

… (f) and it is renewable!!

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the keybiological barriers

� What and How at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

Current and proposed Biofuels

Gallery of fuel

molecules in

current

(or proposed)

use in spark

ignition,

diesel, or

construction

vehicles

Gallery of fuel

molecules in

current

(or proposed)

use in spark

ignition,

diesel, or

construction

vehicles

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the keybiological barriers

� What and How at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

Not only Biofuels�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the keybiological barriers

� What and How at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

… depended on biological sources for energy and materials.

Plants were combusted for heat, used for building materials and

clothing.

Animals were used for building materials and animal power was

harnessed for transportation.

A little bit more than a century ago, society underwent a HUGE

transition: from horse to automobile, from whale oil to crude oil.

That time was similar to today in that many material and fuel

sources were being tested, production was somehow settled, but

this new industry was not yet integrated and consolidated.

... a similar situation is under way …

… depended on biological sources for energy and materials.

Plants were combusted for heat, used for building materials and

clothing.

Animals were used for building materials and animal power was

harnessed for transportation.

A little bit more than a century ago, society underwent a HUGE

transition: from horse to automobile, from whale oil to crude oil.

That time was similar to today in that many material and fuel

sources were being tested, production was somehow settled, but

this new industry was not yet integrated and consolidated.

... a similar situation is under way …

For millenniaFor millennia,, human societies …

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the keybiological barriers

� What and How at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

Conclusions

-Presidenza del

Consiglio dei MinistriComitato Nazionale per la Biosicurezza, le Biotecnologie e le Scienze della Vita

Gruppo di Lavoro per lo Sviluppo delle Biotecnologie Industriali

The Future … is under way …The Future … is under way …

- ItSusChem… sviluppare vere Bioraffinerie per favorire la produzione di fine-

chemicals, bioprodotti, biomateriali e biocombustibili dalle

proprietà superiori da impiegare nelle applicazioni esistenti, o con

caratteristiche innovative per nuove applicazioni industriali, con

particolare riferimento ai processi biotecnologici in uso

nell'industria chimica, nutraceutica, alimentare, farmaceutica,

cosmetica, tessile, etc…

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the keybiological barriers

� What and How at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.

ACKNOWLEDGEMENTS

It is a joint challenge ….

Paola Branduardi

Simone Passolunghi

…and ALL the past

and current (2009)

young Researchers:

Dato Laura

Fossati Tiziana

Codazzi Vera

Rossi Giorgia

Longo Valeria

Posteri Riccardo

Solinas Nicola

… thanks for your attention …

�The Furture is

… under way!!

�5+1 Requirements

for scale up

�How is EtOH

produced from

starch (1st gen)?

�How is EtOH

produced frombiomass (2nd gen)?

�What are the keybiological barriers

� What and How at Bicocca

�Not only EtOH !

�Not only Biofuels !

�Conclusions

�Acknow.