Dec 12, 2012 (4 years and 4 months ago)


“SSF Experimental Protocols

Lignocellulosic Biomass Hydrolysis and Fermentation”

Laboratory Analytical Procedure (LAP)

Issue Date: 10/30/2001

N. Dowe and J. McMillian

National Renewable Energy Laboratory (US Department of Energy)

The method discussed in this article is simultaneous saccharification and fermentation,
SSF. Cellulase is utilized to breakdown to break down the cellulose and yeast to ferment the
sugars. These are lab methods used by researchers to test a variety of
substrates. While an interesting reference, this article provides step
step lab
procedures focused on determining the optimal substrate. This would be useful for a research
and development group but has little to do with developing th
e large scale production line.

“Lignocellulose Biotechnology: Future Prospects”

Editors: R.C. Kuhad and Ajay Singh

Anshan Publishing


“Bioethanol from Crop Residues, Production Forecasting and Economics: An Indian
Perspective”. By Rajeev Kumar Kapo
or, Anuj Kumar Chandel, Sarika Kumar, Rishi Gupta and
Ramesh Chander Kuhad. Pages 247


Approximately 313 x 10

tons of crop residue is left after the crops have been harvested.
This could be converted to 89.42 x 10

liters of bioethanol. In India, l
ignocellulosic biomass is
abundantly available. The technology has been broken down into three steps,
depolymerization of the polysaccharide into fermentable sugars, fermentation of the sugars
into ethanol and ethanol recovery.





Steam Explosion


Ammonia Fibre Explosion


Biological Pretreatments


Acid Pretretment

Releases xylose as main sugar in hydrolysates. The
hydrolysates also contain furan derivative inhibitors. Methods must be
explored to remove these inhibitors su
ch as:




Charcoal Adsorption


Ion Exchange


Enzymatic Detoxification with Laccase


Biological Detoxification

Enzymatic Hydrolysis

After pretreatment, lignocellulosics can be saccharified

to get fermentable sugars. Typically a mixture of enzymes including
cellulases, xylanases and mannanses is used. Methods to be explored include
simultaneous saccharification and fermentation (SSF) and simultaneous saccharification
and co
fermentation (SSC

Fermentation of Lignoce
llulosic Hydrolysate

The sugar mixture obtained from
enzymatic hydrolysis can be used for ethanol fermentation. Ideally an organism that is
capable of utilizing the variety of sugars formed during the hydrolysis (both pentoses
and hexoses can form) should be used.
Saccharomyces cerevisiae

is only capable of
converting hexoses to ethanol. Recombinant plasmids have been have been developed
for co
fermentation of glucose and xylose. One of the major problems yet to be solved is
ntaining stable performance of genetically engineered yeast for commercial scale
, development of more efficient pretreatment technologies as well as
developing an economically feasible ethanol production system.

Review: Overall this article is
helpful. It is a little vague when it comes to the technical
description of how the process is carried out, but could be useful for some background
information as well as determining which areas we need to do additional research in.

“Genetic Improvement o
f Bioenergy Crops”

Editor: Wilfred Vermerris


Composition and Biosynthesis of Lignocellulosic Biomass


Pages 89


By Wilfred Vermerris

The process efficiency of lignocellulosic biomass on the large production scale
depends on the chemical compo
sition of the biomass. If there is interest in
this more in

for our project a more thorough discussion can be included.

“Selection of Promising Biomass Feedstock Lines Using High
Spectrometric and Enzymatic Assays”

Pages 143


By Mark F. Davis, Ed Wolfrum and Tina Jeoh

This was also discussed in the first article reviewed. This subject is not critical to
the process design, but would be a good subject to include as a process optimization if
we have time.

“Current Techonol
ogies for Fuel Ethanol Production from Lignocellulosic Plant

Pages 161


By Yulin Lu and Nathan S. Mosier

A study predicted that use of bioethanol could reduce net carbon dioxide
emissions from vehicles by 90% and sulfur dioxide emissions
by 60
80%. Argonne
National Laboratory claims that an 86% reduction of greenhouse gas emissions would
result per gallon of lignocellulosic ethanol when displacing an energy
equivalent amount
of gasoline.

An eight page discussion of pretreatment strategie
s is included. Jeff, this would
be a good resource for you to review also if you need it.

Ethanol Fermentation: Strain Development for Sugar Co

Microorganisms used for fermentation must be robust and have a high productivity.
Also, the micr
oorganism selected must be able to convert a mixture of different sugars to
ethanol. Other favorable traits include high tolerance to ethanol or other inhibitors and
resistance to contamination by undesired microorganisms.


Saccharomyces cerevisiae

S. ce

has been considered the
workhorse for ethanol fermentation for years.


This microorganism has high ethanol tolerance and operates at low
pH fermentation

conditions which can aide in minimizing the
potential for contamination. Wild type strains can ferment glucose,
mannose and fructose as well as disaccharides like sucrose and
maltose, but they cannot ferment pentoses which make up 40% of
total biomass car
bohydrates. Recombinant DNA technologies have
been applied to increase this function.


Major enzymes required for xylose fermentation must incorporate
xylose flux into the pentose phosphate pathway, these enzymes are
xylose reductase and xylitlo dehydrogen
ase which can be found in
yeasts like
P. stipitis

Candida shehatae


Additionally, xylose must be transported across the cell membrane. It
is believed that this is carried out by glucose transporters. These
transporters have a higher affinity for gluco
se which delays the
transport of xylose until the majority of the glucose has been
consumed which decreases the effectiveness of the co
process. This delay results in an increase fermentation time which in
turn can increase capital costs (larg
er tank volumes).


A detailed description of the plasmid development and genetic
engineering is included in the article.


Zymomnas mobilius

The Entner
Doudoroff pathway is utilized to
anaerobically produce ethanol from sugars. Since only one mole ATP is
enerated per mole sugar consumed, glucose metabolism must be high to
compensate for low energy yield which results in high ethanol productivity.
Compared to traditional yeast, a 5
10% increase in yield and a 5 fold higher
volumetric productivity is observe
Z. mobilius

must be metabolically
engineered to ferment xylose in addition to hexoses. Some success has been
achieved. Major hurdles include industrial adapataion of the recombinant
type, increased likelihood of contamination due to operation at neutral

and less robust cells.


Escherichia coli

Wild type E. Coli
can ferment a mixture of sugars to acids
with ethanol as minor by
product. Only half the theoretical yield of ethanol
can be achieved. By over
expressing pyruvate decarboxylase and alcohol
ehydrogenase the ethanol production and tolerance can be increased.

Ethanol Recovery: Distillation and Dehydration

Ethanol vaporizes at 78°C and water vaporizes at 100°C. If distilled an azeotropic
mixture of ethanol and water will form the distillate (95.
6% ethanol to 4.4% water). Since the
water mixture is not ideal a multi
stage process must be implemented.

Typically the fermentation mixture will be fed to a “beer” column. Carbon dioxide leaves
out the top with water coming out the bottom and eth
anol is removed from a side
draw. This
ethanol is fed to a rectifying column, the product of which is the aseotropic mixture. This
mixture cannot be further purified using distillation. The effluent is therefore fed to a
dehydration unit. Typically a molec
ular sieve adsorption unit can be used. The zeolites in the
molecular sieve unit selectively adsorb water from vapor mixtures, further purifying the

The energy input of the distillation/dehydration unit must be minimized to make the
process econo
mically feasible.
Pressure swing adsorption can be implemented to remove the
water from the azeotropic mixture. This enables to the system to operate at a constant
temperature and the heat of adsorption can be essentially stored for used later at the
eration stage.