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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.