Review

Butanol, a superior biofuel

production from agricultural
residues (renewable biomass):
recent progress in technology
Nasib Qureshi, USDA, Peoria, IL, USA
Thaddeus C. Ezeji, Ohio State University, Wooster, OH, USA
Received March 3, 2008; revised version received May 16, 2008; accepted May 19, 2008
Published online June 12, 2008 in Wiley InterScience (www.interscience.wiley.com); DOI: 10.1002/bbb.85;
Biofuels, Bioprod. Bioref. 2:319330 (2008)
Abstract: This article reviews bioconversion of plant materials such as wheat straw (WS), corn stover (CS), barley
straw (BS), and switchgrass (SG) to butanol and process technology that converts these materials into this superior
biofuel. Successful fermentation of low-value WS makes butanol fermentation economically attractive. Simultaneous
hydrolysis, fermentation, and product recovery has been successfully performed in a single reactor using WS and
C. beijerinckii P260. Research on the production of butanol from other agricultural residues including CS, BS, and
SG has steadily progressed. Use of several product-recovery technologies such as liquid-liquid extraction, gas stripping, perstraction, and pervaporation has been successfully applied in laboratory-scale bioreactors. It is expected
that these recovery technologies will play a major role in commercialization of this fermentation. By employing in line/
in situ product-recovery systems during fermentation, butanol toxicity to the culture has been drastically reduced. In
addition to the use of low-cost plant materials for the production of this biofuel, process integration is expected to
play a major role in the economics of this product. 2008 Society of Chemical Industry and John Wiley & Sons, Ltd
Keywords: butanol; acetone-butanol (AB); agricultural residues; bioreactors; product recovery; process integration;
fermentation.

Introduction

ising oil prices and increased dependence on

foreign fuel have generated a strong interest in the
production of ethanol by fermentation from corn or

agricultural residues not only in the United States, but

worldwide. Ethanol is aimed at replacing motor fuel. Additionally, constant conflicts in the oil supply regions have
reminded the world that alternate energy sources need
to be sought for future energy independence. As a result,

Correspondence to: Nasib Qureshi, United States Department of Agriculture, National Center for Agricultural Utilization Research, Fermentation
Biotechnology Research Unit, 1815 N. University Street, Peoria, IL 61604, USA. E-mail: Nasib.Qureshi@ars.usda.gov

Mention of trade names or commercial products in this article is solely for the purpose of providing scientific information and does not imply

recommendation or endorsement by the United States Department of Agriculture.

2008 Society of Chemical Industry and John Wiley & Sons, Ltd

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N Qureshi, TC Ezeji

the USA has initiated various energy initiatives including

replacing 30% of transportation fuel by ethanol by 2030. In
2007, approximately 19.72 109 kg of ethanol was produced
from corn (in the USA)1 which is approximately 4.6% of
total gasoline consumption (435.5 109 kg) in the USA. It
is anticipated that up to 65.3 x 109 kg (15%) of ethanol can
be produced annually from corn without affecting food and
feed supply. Further increase in ethanol production would
require the use of cellulosic biomass, such as corn fiber (CF),
corn stover (CS), wheat straw (WS), barley straw (BS), or
energy crops like switchgrass (SG) and miscanthus.
Furthermore, attempts are underway to produce other
alternative fuels and chemical feedstocks from the renewable resources listed above. Butanol is one such fuel that
can be produced from agricultural crops such as corn,
molasses, and whey permeate (WP) using Clostridium acetobutylicum or C. beijerinckii. The advantage of using these
and some other butanol-producing bacteria is that they can
utilize both lignocellulosic hydrolysate sugars (hexoses and
pentoses) as opposed to traditional ethanol-producing yeast
strains that cannot use them. The reader may ask why focus
on butanol? Butanol has some interesting properties that
other fermentation-derived fuels do not have, including
ethanol. Butanols energy content is 30% more than ethanol
and is closer to gasoline, its low vapor pressure facilitates
its application in existing gasoline supply channels, it is not
sensitive to water, is less volatile, less hazardous to handle,
less flammable, and can be mixed with gasoline in any
proportion.2,3
Butanol (acetone butanol ethanol (ABE) or AB or solvents)
production by fermentation is one of the oldest fermentation processes employed for commercial production of a
chemical to benefit mankind. Production of butanol was
discovered by Pasteur in 1861.4 Its production by fermentation is second to ethanol in importance and history as
there were commercial plants that were operational during
World War I and World War II.5 During these periods, there
were large commercial plants in Great Britain/UK (Kings
Lynn and Bromborough), Canada, France, the USA (Terre
Haute, IN; Peoria, IL; and Philadelphia, PA), Japan, India,
China, Australia, South Africa, Taiwan, Egypt, Brazil, and
Soviet Union (Dokshukino) that produced acetone and
butanol on a large scale. It should be noted that after World

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War II, between the 1950s and 1960s, butanol fermentation

could not compete with petrochemically derived butanol
due to the development of petrochemical industries, and
hence many plants around the world were shut down. The
remaining biobased butanol plants ceased operations in
the early to late 1980s in South Africa (due to a shortage of
molasses, the feedstock for this fermentation, brought on
by drought), China, Egypt, and former Soviet Union. 5 One
might ask, if this was such a successful fermentation process
during the last century, why cannot it be revived again?
During those days, substrates (molasses and corn) were
economically available. Today, prices of these substrates are
high, making butanol production by fermentation uneconomical. Process economics could be improved if butanol
could be produced from agricultural residues and product
recovered using energy-efficient techniques in place of
distillation. Agricultural residues are both renewable and
economically available. The objective of this review is to
inform the reader of the research efforts that have been
made to produce butanol from agricultural residues. Additionally, a significant amount of progress has been made
on the development of new process technologies, including
studies with high-productivity reactors and energy-efficient
product-recovery systems. Initially, the recovery technologies were developed not to recover AB economically but to
relieve butanol inhibition in the reactor. This gave birth to
integrated technologies where production of butanol was
combined with product recovery.

Traditional substrates, cultures,

and technologies
Traditionally, butanol is produced in batch reactors and
the product is recovered using distillation. A typical batch
fermentation system is initiated with 60 g/L substrate (glucose
equivalent), usually molasses, cornstarch, glucose, or (WP).
The medium containing substrate and nutrients is autoclaved
at 121oC followed by cooling under a blanket of nitrogen or
carbon dioxide. Upon cooling, the reactor is inoculated with
the culture and fermentation is started. During the course of
fermentation, which takes 3672 h, total ABE up to 2025 g/L
are accumulated. Butanol is toxic to the culture and inhibition can be noticed at concentrations as low as 510 g/L.

2008 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. 2:319330 (2008); DOI: 10.1002/bbb

Review: Butanol production from agricultural residues

Batch reactors result in low AB or butanol productivity

partly due to butanol toxicity and partly due to downtime
which includes charging, sterilization, and cleaning the
reactor. As a result of toxicity to the culture, cell concentration > 34 g/L is not achieved. In a batch process, reactor
productivity > 0.50.6 g/L.h is rarely achieved. Another
problem with this fermentation is low ABE yield which is
of the order of 0.30. However, both yield and productivity
depend on the substrate and medium used. Th is fermentation differs from that of ethanol, in that during ethanol
fermentation a product concentration as high as 80 g/L can
easily be achieved. Additionally, substrate concentration
higher than 160 g/L (glucose or sucrose (glucose equivalent);
yield 0.490.50) can be used which reduces the amount of
water used.

N Qureshi, TC Ezeji

Agricultural residues as current substrates

The high cost of substrates including molasses, whey
permeate, corn, and starchy roots is a major factor affecting
the economic viability of butanol production by fermentation. In order to produce butanol cost-competitively, use of
more economical substrates must be identified and evaluated.
These substrates could include agricultural residues and
energy crops such as CS, CF, SG, WS, BS, rice straw (RS),
and miscanthus. It should be noted that butanol-producing
cultures are able to utilize a wide variety of carbohydrates,
such as cellobiose, sucrose, glucose, fructose, mannose,
lactose, dextrin, starch, xylose, and arabinose. The use of these
carbohydrates by butanol-producing cultures illustrates the
potential to ferment sugars derived from hydrolysates of agricultural residues to butanol. In a plant in Russia attempts were
made to use corncobs, hemp waste, and sunflower shells.5

Traditional substrates
Cane molasses was successfully used in the commercial
production of butanol in South Africa (National Chemical
Products (NCP) Germiston, South Africa) until the early
1980s.4 Other substrates, such as corn, millet, wheat, rice,
tapioca, WP, soy molasses, and potatoes have been used
successfully.6 Clostridia possess strong amylolytic activity,
and substrates containing starch have been used without a
need for hydrolysis using amylolytic enzymes.
Other substrates that have been used for this fermentation include cassava and Jerusalem artichokes.4,6 The use
of Jerusalem artichokes has been investigated as part of
the French research program on the production of fuel
extenders from biomass. Hydrolysates were prepared using
enzyme treatment with inulinase and, apart from ammonia,
no nutritional supplements were added to the fermentation
medium. After a period of 36 h, 2324 g/L AB was obtained.
Liquefied corn starch (LCS) is another potential industrial
substrate that can be used for AB production.7 It is a product
of corn wet-milling process and is composed of 3540% dry
solids as reducing sugars and dextrins. In one of the studies,
AB was produced from LCS, and the process resulted in the
production of AB comparable to that from glucose. This
substrate was also saccharified (saccharified liquefied corn
starch, SLCS) prior to use in a fed-batch reactor integrated
with product recovery.

Corn fiber
Corn fiber is a byproduct of corn-processing industries and
it sells at about $0.044/kg. Recently, it was demonstrated that
CF could be used to produce butanol.8 In this process, CF
was pretreated using dilute (0.5%, v/v) sulfuric acid for 1 h at
121oC. The mixture was then treated with enzymes (cellulase
and cellobiase) for hydrolysis. It should be noted that this
solution had to be treated to remove fermentation inhibitors
prior to fermentation.8 From an economical point of view,
removal of inhibitors from agricultural residue hydrolysates
is a disadvantage.
Wheat straw
Attempts have been made to produce butanol from WS
hydrolyzate (WSH). WS was pretreated using dilute (1%,
v/v) sulfuric acid and hydrolyzed using enzymes (cellulase,
xylanase, and -glucosidase). The hydrolyzate was then
fermented to butanol using C. beijerinckii P260. In these
studies, no inhibition due to salts or inhibitory products was
observed. In fact, fermentation was more rapid than control
batch fermentation where glucose was used as a substrate.9
These studies demonstrated that hydrolyzates of some of
the agricultural residues can be fermented to butanol with
little or no inhibition. In addition to the novel substrates
mentioned in this section, other substrates such as distillers

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dry grains with solubles (DDGS) have been used to produce

AB.10 At this stage, we are not clear as what makes WSH a
successful substrate for butanol fermentation. Currently,
investigations in this direction are underway at the United
States Department of Agriculture, National Center for Agricultural Utilization Research, Peoria, IL (USA).

Pretreatment inhibitors of agricultural

residues
Dilute acid (0.5%, v/v) pretreatment and enzymatic
hydrolysis of (CF) resulted in poor fermentation of this
substrate. In order to ferment CF hydrolyzate (CFH) to
AB, the CFH was treated with molecular sieve to remove
fermentation inhibitors prior to fermentation.8 Upon treatment, the CFH was fermented successfully, suggesting
that inhibitors prevented fermentation of CFH. In another
study on fermentation of corn cobs (CC) to AB, presence of
fermentation inhibitors was identified in the hydrolyzate.11
Th is fermentation was pretreated using steam explosion
(currently called steam expansion) and hydrolyzed using
enzymes. Furthermore, attempts were made to quantify
effect of inhibitors such as salts, furfural, hydroxymethyl
furfural (HMF), syringealdhyde, and acetic, ferulic,
-coumaric, and glucuronic acids on cell growth and
product formation.12 Furfural, HMF, and acetic acid (as
acetate) did not inhibit fermentation at the concentrations
that were studied. However, when 0.3 gL-1 each of ferulic
and -coumaric acids were added to the fermentation
medium cell growth and ABE production decreased significantly. Interestingly, fermentation with added furfural and
HMF was found stimulating to the culture. A comparison of
CC, CFH, and WSH demonstrated that generation of inhibitors is substrate specific. It was noticed that fermentation of
WSH was vigorous and no inhibition was observed.9

Microbial cultures and culture development

A number of butanol-producing strains have been
reported in literature. These cultures include Clostridium
acetobutylicum P262 (renamed as C. saccharobutylicum),
C. acetobutylicum ATCC 824, C. acetobutylicum NRRL
B643, C. acetobutylicum B18, C. beijerinckii P260,
C. beijerinckii BA101, C. beijerinckii LMD 27.6, C. butylicum,

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Review: Butanol production from agricultural residues

C. aurantibutyricum, and C. tetanomorphum. C. saccharobutylicum P262 and C. beijerinckii P260 are industrial
strains that were used for commercial solvent production in
South Africa [Germiston] until early 1980s. These butanolproducing strains are characterized based on the ratio and
type of solvent production.13
It should be noted that significant research efforts have
focused on developing or genetically improving butanolproducing cultures. These cultures can produce or tolerate
elevated levels of AB ranging from 20.030.0 g/L. In a recent
report, microbial cultures such as Escherichia coli have
been developed that produce butanol.1416 However, butanol
concentration was 13.9 mg/L.14 A different E. coli strain
has been developed that can produce 25 g/L isobutanol.17 It
should be noted that the newly developed butanol-producing
strain cannot tolerate butanol in excess of 15 g/L as butanol
is more toxic than isobutanol.14,17 It is likely to be a challenging task to further develop E. coli that can produce
butanol in excess of 15 g/L. Attempts are also made to
eliminate pathways that result in the production of acetone,
ethanol, and isopropanol (Personal communication).

Regulation of butanol production

in solventogenic Clostridia
No genus among the three domains (Bacteria, Archaea,
and Eucarya) is better equipped to produce butanol
than solventogenic Clostridium spp. The metabolism of
solventogenic clostridia such as C. acetobutylicum and
C. beijerinckii follows a series of biochemical reactions
where polysaccharides, and hexose and pentose sugars are
converted to Pyruvate, ATP, and NADH.18 C. acetobutylicum
and C. beijerinckii utilize pyruvate to undergo biphasic
fermentation where intermediate products, such as acetate
and butyrate, are produced (acidogenesis) during exponential growth phase and re-assimilated during late exponential
and stationary phases to produce AB (solventogenesis)
(Fig. 1). During acidogenic phase, solventogenic clostridia
cells grow exponentially due to production of high amounts
of ATP.4 Under appropriate environmental conditions,
acidogenic genes are induced and expressed. These genes
regulate formation of relevant enzymes that bring about the
catalysis of pyruvate to acetyl-CoA.19

2008 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. 2:319330 (2008); DOI: 10.1002/bbb

Review: Butanol production from agricultural residues

N Qureshi, TC Ezeji

Acetone
Acetate
Substrate

Pyruvate

Butanol
Butyrate

Fermentation

Acidogenesis

Ethanol
Solventogenesis

Figure 1. Phases of the ABE fermentation process showing interactions of fermentation intermediates directed toward ABE formation.
Arrow shows the direction of the metabolism in the solventogenic
clostridia fermentation pathways. Acetate can be converted
to butanol in the following sequence (acetate acetyl CoA
acetoacetyl CoA 3-hydroxybutyryl CoA crotonyl CoA butyryl
CoA butyraldehyde butanol).

There have been considerable efforts during the last two

decades to unravel the factors that are involved in triggering
the metabolic shift (transition from the acidogenic to solventogenic phase) and physiological changes in solventogenic
clostridia. Though the complex mechanisms involved in
this transition are not totally understood, it has long been
observed that the shift to solvent production is associated
with the induction of solventogenic enzymes and a decrease
in the activity of acidogenic enzymes.4 It should be noted
that acetyl coenzyme A is the branch point intermediate
leading to the synthesis of acids (acetic and butyric) and
solvents (AB) in the fermentation pathways.18 Hartmanis
and Gatenbeck, during their investigation of the levels
of seven intermediary enzymes involved in acetate and
butyrate formation (in batch butanol fermentations), showed
that acetyl-CoA can be converted in vivo to acetate and
butyrate.20 Re-assimilation of these organic acids leads to
the formation of AB. Threshold undissociated butyric acid
concentrations have been implicated in the switch to solvent
production, citing that 613 mM of undissociated butyric
acid as the requirement for initiation of butanol production when the external pH was below 5.0.21 The second part
of the reaction is the direct catalysis of pyruvate to acetylCoA and to AB by the solventogenic clostridia stationary
phase cells. Both mechanisms are depicted in Fig. 1. A close
examination of this figure reveals that under the appropriate

conditions, solventogenic clostridia can re-assimilate

both acetate and butyrate for acetone and butanol formation. Details of specific enzymes, intermediate products, and
scheme of reactions leading to acids and ABE production
during fermentations employing C. acetobutylicum and
C. beijerinckii have been published elsewhere.4,18,22
Obviously, enzyme synthesis and control of electron flow
in the glycolytic pathway are vital with respect to regulation of acetate, butyrate, and butanol formation.22 Since the
electron flow can be reversed, butanol yield should respond
to factors that influence the direction of electron flow.23 As
a result, the effect of numerous reducing compounds, such
as carbon monoxide gassing, addition of methyl viologen,
and neutral red into the fermentation medium during
the AB fermentation, has been investigated by different
research groups. In the presence of these electron carriers,
butanol formation was stimulated at the expense of acetone
synthesis.23 This holds that during solventogenesis, butanol
is produced from butyryl-CoA by two successive reductions involving butyraldehyde dehydrogenase and butanol
dehydrogenase.22 Vasconcelos et al. reported similar results
when a mixture of glucose and glycerol was used as carbon
source during continuous cultivation of C. acetobutylicum
at neutral pH.24 Under such conditions, glycerol, a more
reduced substrate than glucose, induced the formation of
butanol and decreased the production of acetate, butyrate,
and hydrogen. It is anticipated that this knowledge base will
provide a basis for the development of improved butanolproducing strain, fermentation medium, and process for
butanol production.

Current novel fermentation technologies

Since batch reactors result in low reactor productivities, use
of novel fermentation systems, such as free cell continuous
fermentations, cell recycle, and immobilized cell reactors,
have been explored. Continuous free cell systems offer
comparatively higher productivities because of the elimination of down time. In these reactors, high cell concentration
cannot be achieved as there is no means to retain cells in the
reactor and hence cell washout occurs at high dilution rates.
For this reason, methods to retain high cell concentration
inside the reactor, and still operate the reactor at high flow

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Review: Butanol production from agricultural residues

rates, have been developed.22,25 These methods are known

as cell immobilization and cell recycle systems. By using
cell immobilization methods, cell concentrations in excess
of 5070 g/L can be achieved in the reactor. Such reactors
can be operated at high flow rates with no cell washout. The
added advantage of these systems is that they offer high
reactor productivities, both due to the elimination of downtime and increased cell concentrations. Increased reactor
productivity results in the reduction of process vessel size
and capital cost thus improving process economics.
In immobilized cell reactors, productivities of the order
of 6.515.8 g/L.h have been achieved.2628 These productivity values are 4050 times higher than achieved in batch
reactors (0.070.35 g/L.h).29,30 Immobilized cell reactor
configurations can be packed or fluidized bed. Details of
these reactors have been given elsewhere. 27 Figure 2 shows
various types of reactor systems that can be used for butanol

Feed

Product

Product

Feed
(i) Batch
Reactor

(ii) Continuous
Reactor

(iii) Packed-bed
Reactor

Product
Feed

Cell recycle
Product

Membrane

Feed

Recycle

production. In another approach, a group from the Ohio

State University (Columbus, OH, USA) immobilized cells of
C. acetobutylicum on a fibrous support which was used in a
continuous reactor to produce ABE.31 This system resulted
in a maximum reactor productivity of 4.6 g/L.h.

Cell recycle membrane reactors

As described previously, microbial cells are immobilized
using one of three techniques: adsorption, entrapment, and
covalent bond formation.13 In all three cases, the culture
may experience substrate, nutrient, and product diff usion
limitations. In substrate diff usion limitations, the culture
may not get carbon source for its energy needs thus resulting
in death of the innermost cell layers due to starvation or
formation of spores. This will reduce the amount of active
cells that take part in the reaction. Starvation or diff usion
limitation may inactivate a significant amount of cells thus
resulting in reducing reactor productivity. At the same time,
product diff usion limitation may not allow diff usion of
toxic products from the cell surroundings thus affecting cell
viability. Both of these problems can result in significantly
reduced productivity.
As cell immobilization increases cell concentration inside
the bioreactor, cell recycle membranes are another means
of increasing cell concentration. Unlike immobilized cell
particles, the cells remain suspended in the liquid medium.
A membrane is used as a means of preventing the cells from
being removed with the out flow. Using membrane cell
recycle reactors, cell concentrations as high as 90 g/L can
be achieved.32,33 Using this approach, reactor productivities
up to 6.5 g/L.h have been achieved in the butanol fermentation.34,35 Some of the major limitations of cell recycle reactors include membrane fouling with fermentation broth, and
high membrane cost. A schematic diagram of membrane cell
recycle reactor has been shown in Fig. 2 (v).

Reactor

Product-recovery technologies
(iv) Fluidized-bed
Reactor

(v) Membrane Cell

Recycle Reactor

Figure 2. Schematic diagrams of various reactor types used in AB

fermentation. i) batch reactor; ii) continuous stirred tank reactor
(CSTR); iii) immobilized cell packed bed reactor; iv) fluidized bed
reactor; v) membrane cell recycle reactor.

324

There are two basic problems with butanol fermentation:

i) use of dilute sugar solution which results in dilute product
and large disposal loads; and ii) energy-intensive recovery of
butanol from dilute fermentation broth. A solution to these
problems can be addressed in two ways: i) use of genetic
engineering techniques to develop strains that could tolerate

2008 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. 2:319330 (2008); DOI: 10.1002/bbb

Review: Butanol production from agricultural residues

higher concentration of butanol and sugar and produce

higher concentrations of butanol; ii) use of engineering
techniques to ferment and remove product simultaneously
so that a toxic butanol concentration inside the reactor is
never reached. Employing the first approach, cultures have
been developed that can tolerate and produce up to 30 g/L
AB.6 However, the problem of dilute product stream still
persists. The culture broth still contains 97% water that has
to be removed before recovering the final product. In this
case dilute feedstock sugar solutions (80 g/L) are typically
used. The second solution involves application of engineering
techniques to relieve product inhibition and allows use
of concentrated sugar solutions. This approach has made
good progress at least in laboratory scale bioreactors. Using
simultaneous product-recovery techniques, application
of concentrated sugar feedstock solutions has been made
possible. Sugar solutions as concentrated as 500700 g/L
have been used as compared to 6080 g/L in conventional
batch systems.36,37 Product-removal techniques include gas
stripping, adsorption, liquid-liquid extraction, perstraction, pervaporation, and reverse osmosis. Details of some of
these techniques are published elsewhere.6,18,22 Simultaneous
removal of AB has been exercised in batch, fed-batch, and
continuous immobilized cell reactors. It should be noted
that removal of AB from batch and fed-batch systems can be
directly applied to existing fermentation industries which are
not likely to make changes to their existing infrastructure.

Batch process with concentrated sugar

solutions
Traditional batch fermentations with >6080 g/L sugar
in the medium result in a high residual substrate in the
effluent of the reactor, resulting in inefficient sugar utilization and increased biological oxygen demand (BOD) load
for wastewater treatment. However, recent developments in
simultaneous recovery of AB have made it possible to use
concentrated sugar solutions for this fermentation. During
the fermentation, the toxic products, such as butanol, are
removed simultaneously, thus relieving inhibition which
results in the utilization of more substrate. Employing a
butanol removal technique, sugar solution containing 227
g/L lactose (C. saccharobutylicum P262) has been successfully
fermented.38 In this process, product (AB) was simultaneously

N Qureshi, TC Ezeji

removed by a technique called perstraction. For AB removal,

a membrane was placed in between fermentation broth and
an organic extractant. AB that diff used selectively through
the membrane was extracted into oleyl alcohol (extractant).
Since solvent (AB) removal from fermentation broth was
continuous, it prevented accumulation of toxic butanol.
Hence, culture continued fermentation until all the sugar was
converted to AB. Use of concentrated lactose solution resulted
in the production of 99.3 g/L AB (as compared to 20 g/L or
less in a batch system). In such fermentations, high ABE yield
is possible as acids cannot leave the system unless converted
to AB. In another process, 200 g/L lactose was successfully fermented in a batch reactor of C. saccharobutylicum
P262 when integrated with product recovery.39 This system
resulted in the production of 70 g/L ABE with a productivity
of 0.32 g/L.h as compared to 0.07 g/L.h in the control batch
reactor. Studies reported in this section demonstrate that a
fermentation medium containing over three times the sugar
concentration as compared to a batch reactor can be successfully fermented when integrated with product removal techniques. It should be noted that no residual sugar was left in
these reactors.
In an attempt to produce AB from lignocellulosic
substrates such as WS, the substrate was first hydrolyzed
using dilute sulfuric acid and enzymatic treatments. The
hydrolyzate so obtained contained 68.3 g/L total sugars. To
this hydrolyzate, glucose (approximately 60 g/L) was added
which resulted in a total approximate sugar concentration of
128 g/L. The sugar solution was then used to produce AB in
a batch reactor from which product was recovered simultaneously. As a result of reduction in product inhibition due to
AB removal the culture was able to utilize all the sugars (WS
sugars + added glucose) completely.9 During this fermentation and product-recovery system 47.6 g/L total AB was
produced.

Fed-batch reactors
It is well known that application of the fed-batch technique
is usually restricted to processes where substrate is toxic to
the culture. In such a case, the reactor is initiated in a batch
mode with a low substrate concentration (usually 60100 g/L)
and low fermentation medium volume, usually less than half
the volume of the fermentor. Upon inoculation, the culture

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initiates utilizing substrate. As the substrate is used by the

culture, a fresh feed solution containing concentrated sugar
solution is added to the reactor at a rate that is equal to
utilization of substrate. Both the liquid volume and product
concentration increase with time. As the culture volume
reaches near 70% of the reactor volume, the fermentation is
stopped and product is harvested. This kind of system has
been applied to the production of chemicals that are not very
toxic to the culture, such as 2,3-butanediol.
Since butanol is toxic to C. acetobutylicum or C. beijerinckii cells, the fed-batch fermentation technique cannot
be applied as described above unless one of the previously
noted product-recovery techniques is applied for simultaneous recovery of the product. This technique has been
applied successfully in a number of studies to the AB
fermentation. In a fed-batch fermentation, up to 500 g
glucose in 1 L culture volume (500 g/L) was used as
compared to 60 g/L in a control batch process.40
In one study, a substrate of WP (lactose 350 g/L) was used
to produce butanol in a fed-batch reactor of C. acetobutylicum P262.29 In this process, four different techniques of
butanol separation were used to remove AB including liquidliquid extraction, perstraction, gas stripping, and pervaporation. Overall, application of these four techniques suggested
that fed-batch fermentation technique can be applied to the
AB fermentation provided AB is removed from the culture
broth simultaneously. In another study, AB was produced
in a fed-batch reactor of C. acetobutylicum (ATCC 824) and
recovered by pervaporation.36

Recovery using N2 or fermentation gases

(CO2 & H2)
Gas stripping is a simple technique that can be applied for
recovering AB from model solution or the fermentation
broth. Oxygen free nitrogen or fermentation gases (CO2
and H2: produced during solvent fermentation) are bubbled
through the fermentation broth followed by cooling the
gas (or gases) in a condenser. As the gas is bubbled through
the broth in the fermentor, it carries AB which is captured
in the condenser followed by collection in a receiver. Once
the solvents are condensed, the gas is recycled back to the
fermentor to capture more AB. This process continues
until the fermentation stops due to lack of sugar (as a result

326

Review: Butanol production from agricultural residues

of utilization of all the sugar). In some cases, a separate

stripper can be used to strip off solvents followed by recycling the stripper effluent that is low in AB. Gas stripping has
been successfully applied to remove solvents from batch,41
fed-batch,40 and fluidized bed continuous42 reactors. In addition to removal of solvents, a concentrated sugar solution
was fed to the reactors (fed-batch and continuous reactors)
to reduce the volume of process streams and economize the
butanol fermentation process. In addition to removal of AB,
other advantages such as increases in reactor productivities and product yield are associated with this technique.
Additional advantages of gas stripping include achieving a
high AB concentration in the condensed stream for further
recovery without membranes or chemicals for the recovery
process. During the gas stripping process all the three chemicals (A, B, and E) are removed from the fermentation broth.
Gas stripping can be carried out at fermentation or a higher
temperature. If the recovery is carried out at a higher temperature, a separate stripper is required. It should be noted that
under normal conditions, such as 35 oC temperature, the rate
of gas recycle is approximately four times that of fermentation broth. For example, to remove AB from 1L culture
volume continuously, 4L of gas is recycled per min. In the
gas-stripping experiments no indication of cell damage due
to recycling of large amounts of gas was observed.

Membrane-based recovery such

as pervaporation
Pervaporation is a simple technique that allows selective
removal of volatiles from model solutions or fermentation
broths using a membrane. The volatile organic component
(AB in this case) diff uses selectively through the membrane
as a vapor followed by recovery by condensation. During
this process, a phase change occurs from liquid to vapor.
Since it is a selective removal process, the desired component requires a heat of vaporization at the feed temperature.
A schematic diagram of the pervaporation process is shown
in Fig. 3(iii). Application of pervaporation to batch and
fed-batch butanol fermentation systems has been reviewed
elsewhere.13,18
In an attempt to improve membrane selectivity, liquidliquid extraction and pervaporation were combined to
recover butanol. The details of this technique have been

2008 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. 2:319330 (2008); DOI: 10.1002/bbb

Review: Butanol production from agricultural residues

Ferm broth low in AB

F

N Qureshi, TC Ezeji

Broth Recycle

Gas Recycle
C

ABVC

R
Fermentation broth
(i) Adsorption

SPM

Product
(ii) Gas Stripping

(iii) Pervaporation

Figure 3. Schematic diagrams of some of the product recovery

processes. i) adsorption [butanol containing stream is passed
through the column (flow from bottom to top) and effluent low in AB
(or free of AB) is obtained at the top]; ii) recovery by gas stripping
(F feed, R reactor, C condenser); iii) recovery by pervaporation
using a selective membrane (R reactor, SPM selective
pervaporation membrane, ABVC AB vapors to condenser).

published elsewhere.13 It was estimated that if this solidliquid pervaporation membrane with a selectivity (a measure
of selective removal of butanol (or acetone); it is defined
as [y/(1y)]/[x/(1x)], where x and y are weight fractions of
butanol (or acetone) in feed and product, respectively) of 180
were used for butanol separation, the energy requirement
would be only 10% of that required in a conventional distillation. Unfortunately, the membrane used was not stable as
the oleyl alcohol that formed a thin fi lm and was impregnated into the polypropylene fi lm pores diff used out of the
membrane during the recovery process. In order to improve
this liquid membrane, Thongsukmak and Sirkar developed
a new liquid membrane which had high selectivity (275) and
proved to be stable.43 Unfortunately, butanol fluxes were
low (maximum < 60 g/m2 .h). Other examples of pervaporation membranes are those of Qureshi et al.,44 Huang
and Meagher,45 and Liu et al.46 Qureshi et al. developed a
silicalite composite membrane with a selectivity of 209.44
Huang and Meagher made improvements on this silicalite
membrane and reported a selectivity of 90100 and total
flux of 600700 g/m2 .h (butanol flux 300 g/m2 .h).45 While
significant progress has been made to separate butanol from
fermentation broths by pervaporation, the cost of membrane
still remains a prohibitive factor which has been discussed
elsewhere.13

There are other product-recovery techniques that could be

applied for recovery of butanol, such as adsorption, liquidliquid extraction, perstraction (also known as ionic liquid
separation), super critical fluid extraction, aqueous twophase separation, and reverse osmosis. Description of these
techniques has been discussed elsewhere and hence is beyond
the scope of this review.13,25,47,48 Figure 3 shows schematic
diagrams of some of the product removal techniques.

Integrated processes
The main objective of this section is to report on process
integration as it combines processes, such as pretreatment,
hydrolysis, fermentation, and recovery to be performed in a
single unit. While use of economically available substrates
(such as agricultural residues) is the most important need at
this time, a combination of pretreatment, fermentation, and
product-separation techniques reduces process cost.49 As a
result, it has become the primary focus of the USDAs laboratory (United States Department of Agriculture, National
Center for Agricultural Utilization Research (NCAUR),
Peoria, IL, USA; Principal investigator N. Qureshi) to use
agricultural residues such as CF, corn fiber xylan, CS, WS,
BS, and energy crops including SG, for the production of
biofuels, such as butanol and ethanol. In addition to the
use of these substrates, we have combined hydrolysis of WS
to sugars, fermentation, and product recovery in a single
unit.50,51 C. beijerinckii P260 was used to produce butanol in
these studies. These studies represent a major breakthrough
in the conversion of cellulosic biomass to butanol in integrated systems.
With the advancement in technology, it has become
possible to produce solvents from agricultural residues such
as WS, CF, CS, BS, and SG. Production of solvents from
LCS, SLCS, and WP has also been successful. It should be
noted that some years ago, use of some of these substrates
appeared to be a difficult and challenging task. Application of these substrates, development of efficient hydrolytic
enzymes, and a combination of hydrolysis, fermentation, and
recovery technologies has made this fermentation appear to
be competitive with butanol obtained from petrochemicals.
Also, application of in situ product-recovery technologies
has allowed the use of concentrated sugar solutions. The
integrated processes listed in Table 1 resulted in production

2008 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. 2:319330 (2008); DOI: 10.1002/bbb

327

N Qureshi, TC Ezeji

Review: Butanol production from agricultural residues

Table 1. A brief summary of AB production in bioreactors coupled with various product-recovery systems.
Fermentation -product recovery
System, substrate

Substrate
conc. [g/L]

Control (Batch, glucose, no recovery)

Gas stripping (Fed-batch, glucose)

AB/ABE produced
[g/L broth]

Reference

48.9

20.1

500.0

232.8

40

Gas stripping (Batch, whey permeate)

200.0

70.0

39

Gas stripping (Batch, WSH)1

128.3

47.6

Perstraction (Batch, whey permeate)

227.0

99.3

38

Pervaporation (Fed-batch, glucose)3

342.0

119.0

36

Pervaporation (Batch, glucose)

121.2

51.5

30

Total sugars 128.3 g/L including hexoses and pentoses

Culture volume 1.38 L. Values in table are per L broth (total sugar 313.3 g, total ABE 137.0 g)

Culture volume 1-1.3 L. Values in table are per L broth (total sugar 444.6 g, total ABE 154.7 g)

of more AB per L culture volume than in the control batch

fermentation process (non-integrated).
During butanol production by fermentation, a number of
byproducts such as CO2 and H2 gases, cell mass, acetone,
and ethanol are produced. Upon separation of these gases,
H2 gas can be used as an excellent energy source. CO2 can be
fi xed to produce other chemicals. Additionally, byproducts
including vitamin B12 can be extracted from the fermentation broth.5 It has been reported that a total of 400600 g
of cobalamin was obtained per liter of fermentation broth
in a Russian plant.5 It is viewed that appropriate marketing
of these byproducts would generate significant amount of
revenues thus making this fermentation profitable.

Conclusions
The successful fermentation of some of the plant materials,
such as WS, has made butanol fermentation look economically attractive. Simultaneous hydrolysis, fermentation, and
product recovery has been successfully performed in laboratory scale single reactor when using WS and C. beijerinckii
P260. Production of butanol from other agricultural residues
including CS, BS, and SG has been making steady progress.
It is suggested that the problem of generation of pretreatment/hydrolysis inhibitors in the case of CS, BS, and SG be
addressed. In addition to dilute sulfuric acid pretreatment
method, there are other techniques (dilute alkali, ammonia
expansion, and hot-water treatment) that should be evaluated
for the generation of less inhibitory hydrolyzates. Use of
several product-recovery technologies such as liquid-liquid

328

extraction, gas stripping, perstraction, and pervaporation,

has been successful in laboratory-scale bioreactors and are
expected to play a major role in reviving butanol fermentation to a commercial scale. By employing in line/in situ
product-recovery systems during butanol fermentation,
substrate inhibition (due to high concentration of carbon
source) and butanol toxicity to the culture are drastically
reduced. Given that butanol is an excellent potential fuel and
the United States is rich in biomass, butanol production from
agricultural biomass appears to have a bright future.
Acknowledgement
N. Qureshi would like to thank Michael A. Cotta (United
States Department of Agriculture, National Center for Agricultural Utilization Research (USDA, NCAUR), Peoria, IL,
USA) for providing help and constant encouragements. NQ
would also like to thank John Michael Henderson for fi nding
ethanol production data for 2007 on internet (see ref. 1).
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