POTENTIAL OF USING
MICROORGANISMS
FOR SUSTAINABLE
BIOFUEL PRODUCTION
Roman Netzer
Research Scientist
SINTEF – MK -
SFFE lunch lecture –
03.11.11
Technology for a better society
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STRUCTURING

Introduction
- SINTEF – Materials and Chemistry – Biotechnology
- What is biofuel and why using it?
- Statistical data and predictions
- Overview over different types biofuels
- History of biofuels

Biotechnology and 2G biofuels

Lignocellulosic biomass for 2G bioethanol production
- Role of microorganisms for ethanol production
- Important biochemical pathways for biotechnological biofuel
production
- Modification of yeast strains for biofuel production and biorefinery
- Consolidated bioprocess for bioethanol production

The LignoRef project
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SINTEF – MK – DEPARTMENT OF
BIOTECHNOLOGY
FROM GENE TO PRODUCT
Strain
development
Cultivations in
well-plates
Robotized colonypicking and liquid
handling
Fermentors for
process
optimizations
High-throughput
screening and
analyses
Product isolation
and purification
Pilot scale
equipment
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WHAT IS BIOFUEL?
 Fuel whose energy is derived from recent biological carbon
fixation in contrast to fossil fuels such as coal, petroleum, or
natural gas that come from long since dead plants and
microorganisms.
 Biofuels address the main issues
1.
Carbon neutral
2. Renewable & Integral part of emerging economy
3. Sustainable
4. Local production (also small scale)
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WHY USING BIOFUELS?
 Reduction of GHG emissions
CO2 released by burning biofuel already existed as part of the modern carbon
cycle.
Potential to become carbon neutral in the future.
 Price
Prices for fossil fuels will increase due to limited availability and increasing
demand and production costs.
Prices for biofuels are suggested to decrease due to progress in production
technology.
 Independence
Compared to fossil fuels, biofuels are easy to produce  No
monopolistic/oligopolistic market.
 Availability
It's renewable. Biofuels can be produced from raw agricultural/waste materials.
Worldwide energy demand will increase in future.
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SCENARIO GLOBAL POPULATION DEVELOP
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SCENARIO FOR GLOBAL ENERGY CONSUMP
Energy Information Administration
http://www.eia.gov/forecasts/archive/ieo10/world.html
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DEVELOPMENT OF WORLDWIDE
BIOFUEL PRODUCTION
Energy Information Administration
http://www.eia.gov/forecasts/archive/ieo10/world.html
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Ethanol production will again double over
the next decade
World Ethanol Production
Mil Gal
45.000
40.000
All Others
35.000
India
30.000
China
25.000
EU-27
20.000
Brazil
15.000
U.S.
10.000
5.000
0
2000
2005
2009
2019
Source: OECD/FAO
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BIODIESEL PRODUCTION ALSO WILL
GROW IN FUTURE
World Biodiesel Production
12.000
All Others
India
Mil Gal
10.000
Brazil
8.000
Argentina
U.S.
6.000
EU(27)
4.000
2.000
0
Avg 2007-2009
2019
Source: OECD/FAO
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NEGATIVE ASPECTS OF (CURRENT) BIOFUE
 Current biofuels compete with food for the same
feedstocks.
 Food prices may further rise if areas once used to grow
food are converted to grow energy crops.
 Rain forests and other tropical areas could be cleared to
cultivate crops for biofuel production.
 Indirect land use change
 Increasing food prices could affect populations in
developing countries.
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PIONEERS IN BIOFUEL TECHNOLOGY
Henry Ford in front of his T-Model
Ford "Tin Lizzie" (1908) (designed for
ethanol)
"There is fuel in every bit of vegetable
matter that can be fermented."
Rudolph Diesel's compression-ignition
engine
(1893) (designed for peanut oil)
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HISTORY OF BIOFUELS
Bioethanol:
1824: Samuel Morey developed an engine that runs on ethanol and turpentine.
1860: Nicholas Otto uses ethanol as fuel in one of his engines.
1862: Special tax is placed on ethanol whiskey (Moonshine) in the U.S. to help pay
for the Civil War. Ethanol falls out of favor as a fuel.
1896: Henry Ford builds his first automobile, and the engine is designed to run on
pure ethanol.
1919-1933: Prohibition era in the U.S. makes it impractical to use ethanol cars.
1920s: Gasoline becomes the most popular fuel in the U. S. as well as in many other
parts of the world. In Brazil, sugarcane based ethanol is produced as major car fuel
1945: Gasoline becomes cheap and easily accessible. This reduces the interest in
ethanol cars.
From 1970s: Oil crisis in 1973 and 1979
Environmental concerns (Global warming/air pollution/GHG emission)
 Idea of using biofuels was revitalized in the U.S. and Europe
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IMPORTANT BIOFUELS
Bioalcohols

-
Ethanol
Most common and best established biofuel
Produced by fermentation processes from sugars by microorganisms and
enzymes
Can be used in petrol engines (existing car engines up to 15% additive to
gasoline)
Lower energy density than gasoline (1/3 lower), corrosive effect
Butanol
Compared to ethanol: higher energy density, less corrosive, less water soluble
(better engine fuel, requires less change regarding infrastructure and engine
design)
- Until 1950s produced by fermentation process (ABE fermentation) from starch
using
Clostridium acetobutylicum; substituted by petrochemical routes
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- Suggested as substitute for jet fuels

-
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IMPORTANT BIOFUELS
 Biodiesel (FAME)
•
Most common type of biofuel used in European countries
•
Produced by transesterification from animal fats, vegetable oils, soy, rapeseed,
jatropha, mahua, mustard, flax, sunflower, palm oil, hemp, field pennycress,
pongamia pinnata and algae.
 Green (renewable) diesel
Produced from renewable feedstocks by
• Fischer Tropsch Synthesis from SynGas
• Conversion of pyrolysis oil (BtL)
• Catalytic conversion of sugar or lipids (algae)
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1st GENERATION BIOFUEL

Produced from sugar, starch, vegetable oil, or animal fats

Feedstocks can in most cases be used as food or feed  Food vs. Fuel dilemma

Technologies: fermentation, transesterification

Most established biofuel production process

Lower impact on reduction of green house gas emission
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ADVANCED BIOFUELS (2nd AND 3rd GENERAT
 Biofuels produced from sustainable feedstocks (e. g. lignocellulosic biomass, algae).
 Significant impact on GHG emissions: reduction of CO2 emissions up to 90%
 Examples: Ethanol
Fischer-Tropsch fuels ((2n+1) H2 + n CO → CnH(2n+2) + n H2O)
Biohydrogen, Biogas
 Most processes are currently still under development
 3rd generation biofuels:
- made out of microorganisms, primary
microalgae
- indistinguishable from petroleum
counterparts
Renewable Fuels Association
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BIOTECHNOLOGY
Any technological application that uses biological systems,
living organisms, or derivatives thereof, to make or modify
products or processes for specific use.
The United Nations Convention on Biological Diversity
• Classic biotechnological applications
Fermentation processes used in food production:
bread, brewerage, milk products, conservation of food, …..
• Modern biotechnological applications
– Therapeutics: antibiotics, insulin, vaccines, antibodies,
enzymes, …
– Carotenoids, vitamins, organic acids, polymers, ….
– Processes for mining, wastewater treatment, oil spill recovery
and other bioremedation, …
– Amino acids: L-glutamate (1.200.000 t/a), L-lysine (600.000
t/a),…
– Biofuels
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2nd GENERATION BIOETHANOL
One material could cure our addiction to oil!?
Lignocellulose
•the most abundant naturally occurring organic material
•Accounts to ca. 50% of the biomass in the biosphere
•No direct concurrency with food production (but risk of ILUC)
•Annual production world wide: 10 to 50 × 109 t
 Enormous and sustainable source of energy
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COMPOSITION OF LIGNOCELLULOSIC
BIOMASS
•Cellulose: polysaccharide consisting of a linear
chain of several hundred to over ten thousand
β(1→4) linked D-glucose units.
•Hemicellulose: heteropolymer comprised of
xylose linked backbone branched with other
sugars like arabinose, mannose, galactose and
glucose.
•Lignin: cross-linked racemic macromolecule,
relatively hydrophobic and aromatic in nature.
The carbohydrate polymers (cellulose and
hemicelluloses) are tightly bound to the lignin.
Nature 454, 841-845
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LIGNOCELLULOSIC FEEDSTOCKS
Agricultural residues Wood residues
Corn stover
Wood chips
Bagasse
Sawdust
Rice straw
Paper mill
waste
Municipal waste Dedicated energy cro
Municipal paper
waste
Biodegradable
fraction of MSW
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Elephant grass
Willow
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PRETREATMENT OF LIGNOCELLULOSIC
BIOMASS
Lignocellulosic biomass: rigid, resistent to biotic and abiotic stresses
Pretreatment:
a)Liberation of cellulose
b)Efficient hydrolysis of cellulose and
hemicellulose to its constituent sugars
 Access for enzymes and
microorganisms
Methods:
1.Physicochemical: pulping and steam explosion
2.Chemical: acid hydrolysis: acids are corrosive and hazardous. Efficient but
expensive; Organosolv (90% cellulose conversion for forest biomass)
3.Biological: enzymatic hydrolysis: mild process without the formation of inhibitory
byproducts; safe and environmentally friendly method
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RUNFLOW FOR LIGNOCELLULOSIC
BIOMASS PROCESSING
Biomass
Biochemical conversion
Steam
explosio
n
NH3
NaS
Cellulose
Hemicellulose
Acid
catalysis
Thermochemical conversion
Organ
o
solv
Lignin
Enzyme
catalysis
Torrefactio
n
Oils
Comb
ustion
Syngas
Pyrolysi
s
Charcoal
Fischer
Tropsc
h
Upgrade
Diesel
C6 sugars
C5 sugars
Decarbox
y
lation
Fermentatio
n
Fuels
Food
Feed
Fine chemicals
Fine chemicals
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Alcohols
Catalytic
hydrogenatio
n
Fuels
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BIOLOGICAL PRETREATMENT OF
LIGNOCELLULOSIC BIOMASS
Conversion of cellulose into glucose
Chemical hydrolysis: diluted acid + heat/pressure => toxic by-products
In nature cellulose can be broken down by specialized enzymes: Cellulases
Concerted action of different cellulases:
(a) Endoglucanases (EG) cleave
cellulose chains at random
positions
(b) Exoglucanases
(cellobiohydrolases, CBH) cleaves
two (cellobiose) to four units from
the ends of the exposed chains.
Lynd et al., 2002
(c) β-Glucosidases (BG) hydrolyse
cellobiose to glucose
Ethanol fermentation by yeast
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CELLULOLYTIC ENZYMES FOR
LIGNOCELLULOSE PRETREATMENT
Cellulases/hemicellulases are of high industrial interest
•Mainly produced by fungi, bacteria and protozoa
•Often produced by symbiotic bacteria in the rumen of herbivores and
guts of termites
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MICROORGANISMS PRODUCING
CELLULOLYTIC ENZYMES
Current Opinion in Microbiology 2003, 6:219–228
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COMPOSITION OF LIGNOCELLULOSIC
HYDROLYSATES
sugars...
Hydrolysed with diluted acid:
HEXOSES
C H2O H
O OH
OH
glucose
HO
OH
Lignin 21-32%
CH 2 O H
O OH
Extractives 1-5%
OH
OH
mannose
HO
Cellulose 33-51%
HO
C H2 O H
O
galactose
OH
OH
OH
HO
O OH
C H3
OH
Hemicellulose 1934%
rhamnose
OH
PENTOSES
FERMENTATION
INHIBITORS
O OH
OH
HO
xylose
OH
O
OH
arabinose
OH
HO H 2 C
OH
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MICROORGANISMS CAPABLE OF
ETHANOL FERMENTATION
Microorganisms
pH range
Fusarium oxysporum
5-6
Neurospora crassa
5–6
Monilia sp.
5
Mucor sp.
5.4
Saccharomyces cerevisiae
3-7
Klyuvermyces marxians
3-7
Pachysolen tannophilus
2.5 - 7
Candida shehatae
3-7
Pichia stiptis
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3-7
Bacillus polymyxa
5.5 - 8
Aerobacter hydrophila
55 8
Glu
Xyl
Ara
Man
Cel
Temp. range (°C)
+
+
+
+
+
28 - 32
+
+
-
-
+
28 – 37
+
+
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30
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-
+
-
30 - 35
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+
+
+
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30 - 35
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28 - 32
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+
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35 - 37
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35 - 37
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S. CEREVISIAE IS A SUPERIOR
MICROORGANISM FOR INDUSTRIAL
PRODUCTION OF 2G BIOFUELS
 Among the best known microorganisms and genetic tools and methods are
established
 GRAS (Generally Recognized as Safe) status
 Well established in production processes with a high robustness
 Some strains are highly resistant to toxic inhibitors and fermentation
products
 Fermentation processes have a minimized contamination risk
 Fermentation process is easier to control compared to other
microorganisms
Use of yeast for butanol production:
ethanol producers
in facilities
Brazil, Europe
or Northuse
America
willand
most
likely have
use a
 Main
All ethanol
production
worldwide
yeasts
yeasts
yeasts rather than bacteria should they switch from ethanol to butanol
high acceptance among ethanol producers
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S. CEREVISIAE IS A SUPERIOR
MICROORGANISM FOR INDUSTRIAL
PRODUCTION OF 2G BIOFUELS
HEXOSES
CH 2 O H
O OH
efficiently
ferments hexose
sugars
glucose
OH
HO
OH
CH 2 O H
O OH
OH
mannose
OH
HO
C H2 O H
O
HO
galactose
OH
S. cerevisiae
OH
OH
HO
O OH
C H3
OH
rhamnose
OH
PENTOSES
O OH
but no
pentose
sugars
OH
HO
xylose
OH
O
OH
arabinose
OH
HO H 2 C
OH
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XYLOSE METABOLISM IN YEAST, FUNGI
AND BACTERIA
Type I (Yeast and fungi) Type II (Bacteria, some fungi)
D-Xylose
D-Xylose
Xylose reductase
(XR)
NADPH
NADH
NADP+
NAD+
Xylose isomerase
(XI)
Xylitol
Xylitol dehydrogenase
(XDH)
NAD+
NADH
D-Xylulose
D-Xylulose
ATP
ADP
Xylulokinase
(XK)
ATP
ADP
D-Xylulose-5-P
D-Xylulose-5-P
PPW/Glycolysis
PPW/Glycolysis
ETHANOL
ETHANOL
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XYLOSE METABOLISM IN S.
CEREVISIAE
S. cerevisiae wild-type
D-Xylose
NADPH
ScXR
NADP+
 Wild type S. cerevisiae strains unable to utilize xylose for
Xylitol
NAD+
ScXDH
growth
NADH
D-Xylulose
ATP
ScXK
ADP
 XR, XDH and XK encoding genes present in the genome
(very low expression levels)
D-Xylulose-5-P
 Very slow growth on xylose has been observed even
PPW/Glycolysis
when the endogenous genes are overexpressed
ETHANOL
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XYLOSE METABOLISM IN P. STIPITIS AND
SOME
OTHER YEASTS
P. stipitis wild-type
D-Xylose
NADH
NADPH
PsXR
NAD+
NADP+
Xylitol
NAD+
PsXDH
 Able to ferment xylose to ethanol under anaerobic
conditions
NADH
D-Xylulose
 General slower sugar consumption rate compared to S.
ATP
PsXK
ADP
D-Xylulose-5-P
PPW/Glycolysis
cerevisiae
 P. stipitis possess both NADPH- and NADH-specific XR
 No redox imbalance during xylose fermentation.
ETHANOL
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GENETIC ENGINEERING OF S. CEREVISIAE
(TYPE I PATHWAY)
S. cerevisiae wild
type
Xylose
fermenting S.
cerevisiae mutant
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GENETIC ENGINEERING OF S. CEREVISIAE
Xylose reductase (XR) and Xylitol dehydrogenase (XDH)
D-Xylose
NADPH
NADH
NADPH
PsXR
XR
+ +
NADP
NAD
NADP+
Xylitol
XDH
PsXDH
P. stipitis
Recombinant S. cerevisiae strains overexpressing PsXR
NAD
NAD++
and PsXDH
NADH
Higher levels of XDH and XR without causing redox
D-Xylulose
D-Xylulose
ATP
ATP
XK
ScXK
from
imbalance
ADP
ADP
D-Xylulose-5-P
Xylulokinase (XK)
Low levels of XK activity limits in part xylose
PPW/Glycolysis
fermentation ability of S. cerevisiae
Recombinant strain able to grow on xylose as sole carbon
ETHANOL
source!
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LIGNOCELLULOSIC ETHANOL
PRODUCTION MIGHT BE ONE OF THE
CORE TECHNOLOGIES TO SOLVE FUTURE
ENERGY PROBLEMS
Shredding
Biomass
Hydrolysis
Lignocellulose
Addition of
Enzymes or Hexoses + Pentoses
chemicals
Fermentation
Bioalcohols
Chemicals
Destillation
and Extraction
Addition of
genetic
engineered
yeast
Therapeutics
Amino acids
Food/feed
additives
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LIGNOCELLULOSIC BIOMASS FOR
BIOREFINERY
Modified yeast High value
strains
by-products
Hydrolysis
Lignocellulose
Carbohydrates
(Hexoses/Pentos
es)
Butanol
Propandiol
Lactic acid
Enzymes
Recombinant proteins
Fine chemicals
Ethanol
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MODIFICATION OF YEAST STRAINS
Modified yeast
strains
Methods:
Genetic engineering
Ethanol
Classical mutagenesis
Lactic acid
Adaptive Evolution
Propandiol
Butanol
Targets:
Enzymes
Pathways for utilization of pentose sugars
Recombinant proteins
Improved sugar uptake systems
Fine chemicals
Improved resistance against fermentation
inhibitors
Pathways for different bio-products
Modification of industrial yeast
strains
Challenges:
Polyploid
No auxotrophic selection markers
Difficult to transform
Unstable
Advantages:
Stress tolerance
Inhibitor tolerance
High ethanol yields
High fermentation rates
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EVOLUTION OF LIGNOCELLULOSIC
BIOMASS PROCESSING
Production
step
Cellulase
production
Processing Strategy (each box represents a
bioreactor)
SHF
SS
F
SSC
F
CBP
Cellulose
hydrolysis
Hexose
fermentation
Pentose
fermentation
Ethanol
CBP: Consolidated
SHF: Separate hydrolysis & fermentation
SSF: Simultaneous saccharification & fermentation
bioprocessing
SSCF: Simultaneous saccharification & cofermentation
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CONSOLIDATED BIOPROCESS
TECHNOLOGY
Genetic modified
yeast strain
Ethanol
Hydrolysis
Lignocellulose
Hexoses/Pentos
es
Cellulolytic enzymes
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ONGOING PROJECT:
LIGNOREF - LIGNOCELLULOSICS AS A
BASIS FOR SECOND GENERATION
Strain development:
BIOFUELS
AND THE FUTURE
Genetic engineering of different S.cerevisiae strains
BIOREFINERY
Development of new cloning and expression systems for S. cerevisiae
Heterologous gene expression
Fermentation
Other project partners:
PFI (Project owner): Pretreatment of lignocellulosic raw materials
UMB: New enzymatic processes – enzymatic conversion of lignocellulosic ma
UoB: Thermochemical processing – conversion of lignin to value-added produ
Statoil: Evaluation of bio-oil quality
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THANK YOU FOR YOUR AT
Technology for a better society
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