Case Study-Production of Biofuels from Biomass

Learning objectives

• Understand the importance of sustainable and renewable

biofuels
• Describe the properties, advantages/disadvantages of 1st and 2nd
generation biofuels.
• Describe the process for producing bioethanol from
sugar/starch/cellulose platforms
• Describe the process and challenges of generating
bioethanol/biofuels from lignocellulose materials
• Describe the advantages of Isobutanol as more flexible and
versatile renewable alternative to current biofuels
• Understand the metabolic pathways (Ehrlich pathway) that
generates Isobutanol in micro-organisms
• Describe microbial engineering approaches that can be used to
increase isobutanol production in micro-organisms
 Production of Biofuels from Biomass

= Metabolic engineering/
Synthetic biology

Cereal crops
Sugar crops

Lignocellulose

Peralta-Yahya 2012, Nature 488, 320–32

Challenges
 Biorefinery- Production of Biofuels from Biomass
Biomass- Structurally different to Biofuels
- Based on C H O rich components
Biorefinery- Production of Biofuels from Biomass
1st generation biofuels are inefficient fuels in comparison to traditional fuels
 Bioethanol

The “oldest” biofuel

 Biorefinery- Production of Bioethanol

Sugar platform • Extraction

Sugarcane, sugar
beet, sweet
sorghum

Starch platform • Saccharification

Corn, potato, rice,
wheat, sweet potato

Cellulose platform • Pretreatment

Wood, grass,
agricultural residues
 Bioethanol
Schematic diagram of Bioethanol production process (CropEnergies AG)

http://www.cropenergies.com/en/Bioethanol/Produktionsverfahren/
 Bioethanol
CropEnergies (AG) plant in Zeitz produces bioethanol from wheat, maize, barley or
triticale as well as from sugar syrups (feedstocks).

At this plant the production of bioethanol from starch-containing cereals takes

place in five steps:

1. Milling -the mechanical crushing of the cereal grains to release the starch
components
2. Saccharification- Heating and addition of water and enzymes for conversion
into fermentable sugar
3. Fermentation of the mash using yeast, whereby the sugar is converted into
bioethanol and CO2
4. Distillation and rectification, i.e. concentration and cleaning the ethanol
produced by distillation
5. Drying (dehydration) of the bioethanol

Bioethanol can also be produced directly from sugar syrups. This dispenses
with steps 1 and 2, which serve to prepare the grain for fermentation.
 Saccharification of Starch
Starch Liquefaction Saccharification
Mixed with Starch process Glucoamylase
water dissolves in α-amylase added added
water to
60°C form a mash 70-80°C 60-65°C
5-10 min 2 hours 30 min

Degrade starch into

oligosaccharides and Convert
dextrin oligosaccharides
and dextrin into
glucose
 Glycolysis-Fermentation
• Aerobic conditions pyruvate is further oxidised enters Krebs cycle/oxidative
phosphorylation
• Anaerobic conditions pyruvate is converted to a reduced end product (lactate in
muscle) = homolactic fermentation.
• In yeast = alcoholic fermentation yield ethanol and CO2
 Glycolysis-Fermentation

Using the equation for the anaerobic production of ethanol in yeast. Calculate the
theoretical yield of ethanol (in litres) from 1 metric tonne (1000 kg) of Sugar.

NB you will need to look up MW of glucose, ethanol and density of ethanol! Get googling!
 Fermentation
• Initially, yeast cell concentration is low and yeast growth is dominant
• Glucose is mainly utilized to support the growth of yeast cells, so little
ethanol and CO2 are produced and the glucose conversion rate is
relatively low.
• The length of initial stage depends on yeast inoculation ratio and the
fermentation temperature.
• At normal inoculation ratio (5-10%), 30°C, it takes approximately 6-8 h.
• Next stage = Cell growth (increase in biomass) over 108 cells/mL.
• The fermentation becomes very active, resulting in rapid ethanol, CO2 and
energy production, which is indicated by vigorous bubbling and heat
production.
• At this time, cooling is required to maintain the fermentation temperature
at 30 °C.
• Active fermentation lasts about 12h, then the fermentation activity slows
down because less glucose is available.
• During the slow fermentation period, the yeast cells do not grow
anymore, the biochemical reactions are limited by the substrate (glucose)
concentration.
 Industry Developments
Many companies such as Dupont are developing solutions to
increase yield and productivity for producers throughout the
biofuels value chain e.g Xcelis

• Process optimization
• Industrial enzymes
• Strain engineering

https://www.dupontnutritionandbiosciences.com/bioenergy.html

http://xcelis.com/solutions/saccharification-fermentation/
Case Study-Production of Biofuels from Biomass
Learning objectives

• Understand the importance of sustainable and renewable

biofuels
• Describe the properties, advantages/disadvantages of 1st and 2nd
generation biofuels.
• Describe the process for producing bioethanol from
sugar/starch/cellulose platforms
• Describe the process and challenges of generating
bioethanol/biofuels from lignocellulose materials
• Describe the advantages of Isobutanol as more flexible and
versatile renewable alternative to current biofuels
• Understand the metabolic pathways (Ehrlich pathway) that
generates Isobutanol in micro-organisms
• Describe microbial engineering approaches that can be used to
increase isobutanol production in micro-organisms
 Bioethanol
• In 2014, the global production of bioethanol reached
24.5 billion gal, up from 23.4 billion gal in 2013

• More than half (about 60%) of global bioethanol production is

based on sugar cane conversion and the rest (40%) comes from
other crops .

• United States and Brazil are the global producers as they

produce more than 70% of the global bioethanol production

• Even the main source for bioethanol production is considered

to be the corn from US and sugar cane from Brazil, any country
with agro-industrial economy can be involved in bioethanol
fermentation. This is feasible cause of the current progress in
bioconversion of non-food crops in large scale production
 Bioethanol
Disadvantages and concerns:

Researchers studying the impact of biofuels over their entire life cycle — from
crop to car — predicted surprisingly large greenhouse-gas emissions.

A report by researchers at Princeton University published in Science in 2008

estimated that greenhouse gases caused by increased cultivation of corn for
ethanol would exceed the greenhouse-gas emissions of petrol.

Biofuels of all flavours took another hit when spiking food prices sparked food
riots in Mexico in December 2007, which subsequently spread to impoverished
communities throughout the developing world.

Annual output of bioethanol and biodiesel had exploded from 16 million litres
worldwide in 2000 to more than 100 million litres in 2010, outpacing growth in
supply of corn, sugarcane and vegetable oil.
Bioethanol
 Biofuel production from lignocellulose
Although lignocellulosic biomass is a promising
feedstock for biorefineries, its structure and complexity
make up an economic and technical constraint to
lignocellulosic-based biofuel production.

The three constituents of biomass

(cellulose, hemicellulose and lignin) enhance its
compactness and strength.

There are strong linkages between molecules resulting

in a complex structure of lignocellulosic material. As a
consequence, it is necessary to use specific enzymes as
a pre-treatment for fermentation
 Biofuel production from lignocellulose

See homework Q

• Helps in separation of main biomass components (cellulose, hemicellulose

and lignin)
• Increase available surface area
• Reduce particle size
• Ideally pretreatment:
– Solubilizes hemicellulose
– Increases enzymatic hydrolysibility of cellulose
 Biofuel production from lignocellulose

See homework Q

Cellulose is a long-chain homogeneous polysaccharide of D-

glucose units, linked by β-1,4 glycosidic bonds.

It is composed of over 10,000 glucose units.

 Biofuel production from lignocellulose
Physical

Pretreatment
Chemical
Methods
Physical (mechanical)
Chemical (acid and base) Biological
Biological (fungi or bacteria)
• Lignin-solubilizing microorganisms
• No chemicals
• Slow
• Consumption of cellulose and hemicellulose

Combination (synergistic effects)

• Chemical followed by biological
• Physical followed by biological
 Bioethanol
Vivergo Fuels plant is the UK’s largest and Europe’s second largest producer of
bioethanol

The bioethanol plant can product up to 420 million litres of bioethanol and the
bioethanol is made from 1.1 million tonnes of feed wheat, sourced from nearly
900 farms mostly across the East and North Yorkshire and Northern
Lincolnshire regions.

https://vivergofuels.com/
 Alternative Advanced biofuels

• Advanced biofuels produced by microorganisms have

similar properties to petroleum-based fuels

• Can ‘drop in’ to the existing transportation

infrastructure.

• Producing these biofuels in yields high enough to be

useful requires the engineering of the microorganism’s
metabolism

• Challenges need to be met to move advanced biofuels

towards commercialization, and to compete with more
conventional fuels.
Case Study-Production of Biofuels from Biomass
Learning objectives
• Understand the importance of sustainable and renewable
biofuels
• Describe the properties, advantages/disadvantages of 1st and 2nd
generation biofuels.
• Describe the process for producing bioethanol from
sugar/starch/cellulose platforms
• Describe the process and challenges of generating
bioethanol/biofuels from lignocellulose materials
• Describe the advantages of Isobutanol as more flexible and
versatile renewable alternative to current biofuels
• Understand the metabolic pathways (Ehrlich pathway) that
generates Isobutanol in micro-organisms
• Describe microbial engineering approaches that can be used to
increase isobutanol production in micro-organisms
 Alternative Advanced biofuels

• The first-generation biofuels,

ethanol and biodiesel
• High level of natural production
by microbes. Fermentation of
cornflour (US) or cane sugar (in
Brazil)
• Ethanol has only 70% of the
Peralta-Yahya 2012, Nature 488, 320–32
energy content of petrol, it has a
high tendency to absorb water
from the air, which leads to
Biodiesel is the main biofuel in corrosion in engines and pipes;
Europe, (transesterification of
and its distillation from the
vegetable oil) or animal fats with
fermentation broth is energy
methanol has its own limitations
intensive
 Microbial engineering for the production
of advanced biofuels –Isobutanol
• Progress in metabolic engineering, and synthetic and systems
biology, have allowed the engineering of microbes to produce
advanced biofuels with similar properties to petroleum-based
fuels

• The main challenge in using native hosts to convert feedstocks

into advanced biofuels is to overcome the endogenous
regulation (inside the cell!) of biofuel-producing pathways to
achieve high yields.

• Reconstruction of advanced biofuel pathways in hosts, such as

Escherichia coli and Saccharomyces cerevisiae, can work, but
this approach presents its own challenges in balancing the
enzyme activities and expression to maximize metabolic flux.
 Isobutanol
Isobutanol =- next-generation product, builds on the
foundation and provides additional solutions to various
challenges not met by first-generation products.

Advantages:

Blend properties in gasoline/petrol

Volatility
Phase separation
Energy content
Blend wall
Ideal platform molecule to produce renewable iso-paraffinic
kerosene (IPK), a blendstock for jet fuel
Blend Properties in Gasoline/Petrol
• Isobutanol has several blend property advantages (can be easily
added to conventional petrol/gasoline)
• Does not increase vapour pressure when added to gasoline/petrol,
avoiding the need for special gasoline/petrol base blends

Phase separation
• Isobutanol is less polar than ethanol, and tends to act like a
hydrocarbon with very limited amounts moving from the gasoline
phase to the water phase-no dilution of octane value, and
operational issues related to water content are reduced
• Can be more easily transported through pipelines, unlike existing
biofuels

Energy Content
• Isobutanol has approximately 82 percent of the energy value of
gasoline-higher energy content = greater fuel economy.
 Commercial production of Isobutanol

Gevo has developed a renewable method to produce a 98+

percent–purity products using sugars from any available source.

Convert existing U.S. cornstarch ethanol plants into isobutanol

plants.

Butamax -BP and DuPont, who are the partners behind Butamax™
Advanced Biofuels, have been developing biobutanol technology
for several years-constructing a biobutanol technology
demonstration plant in Hull, UK.

http://www.butamax.com/bio
fuel-technology.aspx http://www.butamax.com
www.gevo.co
 Isobutanol

www.gevo.co
 Isobutanol pathway

Complete isobutanol pathway

should consist of three parts of
different pathways

1)Glycolysis to provide pyruvate

2)Valine biosynthesis to
metabolise pyruvate to KIV

3)Ehrlich pathway which is

required for degradation of KIV to
isobutanol

The KDC-dependent synthetic pathway for isobutanol production.

AlsS acetolactate synthase, IlvC acetohydroxy acid isomeroreductase, IlvD dihydroxy-acid
dehydratase, Kdc 2-ketoacid decarboxylase, Adh alcohol dehydrogenase
Isobutanol pathway

As KIV-decarboxylase (KDC) activity links

valine metabolism and Ehrlich pathway, a
high activity of this enzyme reaction is
essential for high levels of isobutanol
production

Can take advantage of the amino

acid biosynthesis capability in
Escherichia coli to produce various
2-keto acids
Engineering the isobutanol biosynthetic pathways

New Research!
Peralta-Yahya et al., Microbial engineering for the
production of advanced biofuels Nature. 2012 Aug
16;488(7411)

Atsumi S, Hanai T, Liao JC (2008) Non-fermentative

pathways for synthesis of branched-chain higher
alcohols as biofuels. Nature 451(7174):86–89

Atsumi et al., Engineering the isobutanol

biosynthetic pathway in Escherichia coli by
comparison of three aldehyde reductase/alcohol
dehydrogenase genes. Appl Microbiol Biotechnol
(2010) 85:651–657.

Avalos et al., Compartmentalization of metabolic

pathways in yeast
mitochondria improves the production of branched
chain alcohols. Nature Biotech (2013) 31: 335-341
Engineering the isobutanol biosynthetic pathway
in Escherichia coli
Research to increase Isobutanol
production

1) Introducing a promiscuous 2-
keto-acid decarboxylase and an
alcohol dehydrogenase into E.
coli produced isobutanol in high
yields through the 2-keto-acid
pathway.

1) Overexpression of 2-
ketoisovalerate biosynthetic
genes.
Engineering the isobutanol biosynthetic pathway
in Escherichia coli
4) Deletion of multiple pathways
competing for pyruvate
consumption and the replacement
of the endogenous acetolactate
synthase enzyme with one from B.
subtilis — which has a higher
specificity for pyruvate — resulted
in increased isobutanol.

5) Overexpress alcohol
dehydrogenase enzymes including
Adh2 from S. cerevisiae and AdhA
from Lactococcus lactis for
isobutanol production in E. coli
 Engineering the isobutanol biosynthetic pathway
in Escherichia coli

• The final challenge, however, would be to overcome the toxicity

of isobutanol on microbial organisms.

• Most promising way is to find effective methods to extract

isobutanol already during the fermentations.