Achievements and Perspectives To Overcome The Poor Solvent Resistance in Acetone and Butanol-Producing Microorganisms

Appl Microbiol Biotechnol (2010) 85:16971712
DOI 10.1007/s00253-009-2390-0
MINI-REVIEW
Achievements and perspectives to overcome the poor solvent

resistance in acetone and butanol-producing microorganisms
Thaddeus Ezeji & Caroline Milne & Nathan D. Price &
Hans P. Blaschek
Received: 20 August 2009 / Revised: 27 November 2009 / Accepted: 28 November 2009 / Published online: 22 December 2009
# Springer-Verlag 2009
Abstract Anaerobic bacteria such as the solventogenic

clostridia can ferment a wide range of carbon sources
(e.g., glucose, galactose, cellobiose, mannose, xylose,
and arabinose) to produce carboxylic acids (acetic and
butyric) and solvents such as acetone, butanol, and
ethanol (ABE). The fermentation process typically
proceeds in two phases (acidogenic and solventogenic)
in a batch mode. Poor solvent resistance by the
solventogenic clostridia and other fermenting microorganisms is a major limiting factor in the profitability
of ABE production by fermentation. The toxic effect of
solvents, especially butanol, limits the concentration of
these solvents in the fermentation broth, limiting solvent
yields and adding to the cost of solvent recovery from
dilute solutions. The accepted dogma is that toxicity in
the ABE fermentation is due to chaotropic effects of
butanol on the cell membranes of the fermenting microorganisms, which poses a challenge for the biotechnoH. P. Blaschek (*)
Center for Advanced BioEnergy Research,
University of Illinois Urbana-Champaign,
1207 W. Gregory Drive,
Urbana, IL 61801, USA
e-mail: blaschek@uiuc.edu
T. Ezeji
Department of Animal Sciences and Ohio State Agricultural
Research and Development Center (OARDC),
The Ohio State University,
305 Gerlaugh Hall, 1680 Madison Avenue,
Wooster, OH 44691, USA
C. Milne : N. D. Price
Department of Chemical and Biomolecular Engineering,
University of Illinois,
119 Roger Adams Laboratory, Box C-3, 600 S. Mathews Avenue,
Urbana, IL 61801, USA
logical whole-cell bio-production of butanol. This minireview is focused on (1) the effects of solvents on
inhibition of cell metabolism (nutrient transport, ion
transport, and energy metabolism); (2) cell membrane
fluidity, death, and solvent tolerance associated with the
ability of cells to tolerate high concentrations of solvents
without significant loss of cell function; and (3)
strategies for overcoming poor solvent resistance in
acetone and butanol-producing microorganisms.
Keywords Clostridium . Solvents . Tolerance .
Butanol toxicity . Acetone
Introduction
Concern for the long-term availability of the non-renewable
feedstocks currently used to produce fuels and chemicals
have been raised as their demand and prices increase and
deposits diminish. Developing cost-effective and energyefficient methods to produce energy-rich biofuels and
sustainable chemicals such as butanol requires research
that draws upon both science and technology knowledge
bases. Butanol is an important chemical and has many
promising characteristics as a renewable liquid fuel (Ezeji
and Blaschek 2007; Durre 2008). In addition, butanol is a
potential fuel and fuel extender for airplanes, as was
demonstrated during the World War 2 when Japan
converted its sugar refineries into plants to produce butanol
as aviation fuel (Schwarz et al. 2007).
General physiological responses to solvent (ABE,
toluene, etc.) stress are found to occur in different types
of microorganisms ranging from prokaryotes (e.g.,
bacteria) to eukaryotes (e.g., yeast). Solvent response
mechanisms involving solvent exclusion systems and
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energy-dependent efflux pumps, which export toxic

solvents from cells, have been characterized in many
microorganisms (Ramos et al. 2002; Ingram 1990). Efflux
pump responses to toluene stress in Escherichia coli,
Pseudomonas putida, and Pseudomonas aeruginosa
(Ramos et al. 2002), for instance, are the most precisely
understood efflux pump response mechanisms (Taylor et
al. 2008). Several investigators examining global gene
expression in solvent-tolerant yeast strains have shown
that multiple synergistic responses are responsible for
improved ethanol tolerance (Taylor et al. 2008), and over
250 genes are implicated in solvent tolerance, including a
number of genes encoding heat shock proteins and
ATPases in yeast (Hu et al. 2007; Aguilera et al. 2006).
Unlike the ethanol fermentation by microorganisms such
as Saccharomyces cerevisiae and Zymomonas mobilis with
unique advantages in terms of ethanol tolerance and wellunderstood fermentation pathways, a major limitation of
biomass conversion to butanol is butanol toxicity toward
the producing microbial cells (Ezeji et al. 2004a, b,
2007c). Butanol toxicity results in a lower butanol
concentration and negatively impacts fermentation time,
productivity, and yield when compared to the ethanol
fermentation. Development of butanol-tolerant solventogenic clostridia together with in situ butanol recovery
processes have been proposed by many investigators as
potential solutions for overcoming butanol toxicity
(Hermann et al. 1985; Nielson et al. 1988; Lin and
Blaschek 1993; Ezeji et al. 2004b, 2007c). Butanol-tolerant
mutants, however, do not necessarily produce more butanol
than the lesser tolerant strains (Baer et al. 1987; Ezeji et al.
2004b).
A number of factors may be involved in the response
of microbes to stress brought about by solvents, which
include disruption of (1) nutrient transport, (2) ion
transport (sodium-potassium pump), (3) phospholipid
composition of cell membranes, and (4) cell metabolism. Survival of microorganisms in the presence of
greater solvent stress will depend on the ability to
activate a broad range of adaptation mechanisms, which
function synergistically to nullify the effects of solvent
toxicity on cell membranes, metabolic enzymes, and
ultimately prevent loss of cell functions. A precise
understanding of these adaptive mechanisms will obviously help researchers unravel ways to overcome poor
solvent resistance in fermentations of biotechnological
importance, especially in the acetonebutanolethanol
(ABE) fermentation. In the present review, we focus on
progress made towards overcoming poor solvent resistance in the ABE fermentation. The solventogenic
clostridia will be heavily drawn upon for example and
discussion, as they are commonly studied natural ABEproducing microorganisms.
Transport of substrates and organic acids

and their effect on solventogenic clostridia
cell metabolism
Gram-positive bacteria, such as solventogenic clostridia,
differ from Gram-negative bacteria with respect to physiology, cell structure, and pathology (Saier and Stiles 1975)
in that they do not have an outer cell membrane and,
consequently, lack a periplasmic space, unlike the Gramnegative bacteria that possess both features. Gram-positive
bacteria, however, have a more intricately developed
peptidoglycan layer than do Gram-negative species. There
has not been conclusive research as to whether the absence
of an outer cell membrane in Gram-positive bacteria and a
well-developed peptidoglycan layer are advantageous or
disadvantageous to these species with regard to responsiveness and adaptability to greater concentrations of solvents
in fermentation media. Transport of substrates and nutrients
from the surrounding environment into the cell cytoplasm is
a prerequisite for cell development and growth in both
Gram-positive and Gram-negative bacteria. Efficient carbon
utilization has been associated with an increase in sugar
uptake, sugar accumulation in the cell cytoplasm, and ABE
production (Annous and Blaschek 1991; Lee et al. 2001,
2005). Different transport mechanisms such as passive
transport (osmosis, diffusion, and facilitated diffusion) and
active transport (high-energy-dependent adenosine triphosphate (ATP); phosphoenolpyruvate (PEP); or ion gradientH+, Na+, K+, Cl, etc.) exist in microorganisms for uptake
of molecules across cell membranes and accumulation of
molecules in the cytoplasm. Gram-positive bacteria, especially obligate anaerobes such as solventogenic clostridia,
rely heavily, but not exclusively, on the PEP-dependent
phosphotransferase system (PTS) as a means for transporting sugars and sugar derivatives into the cell cytoplasm
for subsequent metabolism (Mitchell 1998).
The PTS consists of two common cytoplasmic proteins,
enzyme I and HPr (a heat stable histidine-phosphorylatable
protein), as well as an array of sugar-specific enzyme II
complexes (EIIs). Enzyme I and HPr are encoded by the genes
ptsI and ptsH, respectively (Mitchell and Tangney 2005).
These enzymes are hydrophilic and are usually referred to as
the general PTS proteins because they are common, with few
exceptions, in all cell phosphotransferases (Mitchell and
Tangney 2005). The phosphoryl group from PEP is
sequentially transferred to enzyme I, heat stable histidinephosphorylatable HPr, EIIs and, finally, to the incoming
substrate as it is translocated across the membrane. It should
be noted that non-PTS system uses free energy released upon
hydrolysis of ATP to activate ATP-dependent solute uptake
system, which belongs to ATP-binding cassette family, and
uses binding proteins to capture solutes for onward translocation into the bacteria cell.
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and Kashket 1986). Butanol addition into the fermentation

medium inhibits glucose uptake and growth of Clostridium
acetobutylicum cells (Bowles and Ellefson 1985; Moreira et
al. 1981; Ounine et al. 1985). The possible butanol-sensitive
step of glucose catabolism in C. acetobutylicum was not
defined until Hutkins and Kashket in 1986 investigated
whether the first step of glucose uptake is inhibited by
butanol addition. It is interesting to note that fructose PEPPTS activity is inducible, and more than tenfold increases in
activity for both Clostridium beijerinckii BA101 and C.
beijerinckii 8052 PTS were observed when fructose instead
Sugar transport and accumulation in the cytoplasm of

solventogenic clostridia is the first step towards ABE
production in that sugars must enter the glycolytic cycle for
conversion to pyruvate before subsequent reduction of
pyruvate to ABE (Fig. 1ac). Interference of the glycolytic
process will invariably result in interference of cell growth
and ABE production. Decreased rates of glycolysis caused
by end products (ethanol and butanol) of fermentation on
sugar catabolism have since been implicated in inhibition of
the cell growth of ethanol and butanol-producing microorganisms (Herrero et al. 1985; Lovitt et al. 1984; Hutkins
STARCH
1
NADH
GLUCOSE
2
2 ATP, 2NADH
PYRUVATE
3
13
NADH
LACTIC ACID
FERREDOXIN
NADH, NADPH
12
CO2
NAD, NADP
FERREDOXIN H2
ACETYL-CoA
ACETYL10
PHOSPHATE
4
ACETYL - CoA
NAD+
STARCH
1
GLUCOSE
NAD+
13
ATP
ACETIC
ACID
11
4
NAD+
18
ACETYL - CoA
ACETOACETYL - CoA
5
ACETALDEHYDE
19
ETHANOL
NADH
ACETOACETATE
16
17
ACETONE
CO2
-HYDROBUTYRYL - CoA
6
CROTONYL-CoA
CROTONYL - CoA
BUTYRYL-CoA
NADH
NAD+
BUTYRYL - CoA
14
BUTYRYL-PHOSPHATE
9
NADH
NADH
-HYDROBUTYRYL - CoA
6
NADH
LACTIC ACID
FERREDOXIN
NADH, NADPH
12
NAD, NADP
FERREDOXIN H2
ACETYL - CoA
ACETOACETYL-CoA
5
NAD+
2 ATP, 2NADH
PYRUVATE
CO2
3
NADH
BUTYRALDEHYDE
15
NADH
ATP
BUTANOL
BUTYRIC ACID
STARCH
1
GLUCOSE
2 ATP, 2NADH
13
LACTIC ACID
FERREDOXIN
NADH, NADPH
12
NAD, NADP
FERREDOXIN H2
PYRUVATE
3
CO2
19
ETHANOL
ACETAL
DEHYDE
18
ACETYL-CoA
4
NADH
17
ACETO
ACETATE
ACETONE
NADH
16
ACETIC ACID
16
ACETYL-CoA
ACETOACETYL-CoA
5
NADH
3-HYDROBUTYRYL-CoA
6
CO2
CROTONYL-CoA
7
NADH
BUTYRYL - CoA
NADH
14
BUTYRALDEHYDE
NADH
15
16
BUTYRIC ACID
BUTANOL
Fig. 1 ac Simplified metabolism of biomass by solventogenic

clostridia. 1, Starch hydrolysis (-amylase, -amylase, pullulanase,
glucoamylase, and -glucosidase); 2, glucose uptake by the
phosphotransferase system (PTS) and conversion to pyruvate by
the EmdenMeyerhofParnas pathway; 3, pyruvate-ferrodoxin oxidoreductase; 4, thiolase or acetyl-CoA acetyltransferase; 5, 3hydroxybutyryl-CoA dehydrogenase; 6, crotonase; 7, butyryl-CoA
dehydrogenase; 8, phosphate butyltransferase (phosphotrans-butyrylase);
9, butyrate kinase; 10, phosphate acetyltransferase (phosphostransacetylase); 11, acetate kinase; 12, NADH and NADPH-ferredoxin
oxidoreductase; 13, lactate dehydrogenase; 14, butyraldehyde
dehydrogenase and alcohol/aldehyde dehydrogenase; 15, butanol
dehydrogenase; 16, acetoacetyl-CoA:acetate/butyrate:CoA transferase;
17, acetoacetate decarboxylase; 18, acetaldehyde dehydrogenase; 19,
ethanol dehydrogenase
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of glucose was used as the growth substrate (Lee et al.

2001). C. acetobutylicum, when energized with fructose, was
found to transport and phosphorylate the glucose analog 2deoxyglucose by PEP-dependent PTS, and butanol up to 2%
did not inhibit phosphotransferase activity (Hutkins and
Kashket 1986). There is, however, a chaotropic effect
(disrupts the cell membrane) of butanol on the cell
membrane of C. acetobutylicum causing cellular PEP and
the 2-deoxyglucose-6-phosphate to be released across the
membrane. Enzyme I (cytoplasmic protein) has been
reported to be firmly associated with the cell membrane
(Mitchell et al. 1991; Mitchell and Booth 1984). Disruption
or fluidization of cell membranes by butanol, therefore, will
directly or indirectly affect sugar uptake by the PEPdependent phophotransferase system and will subsequently
result in decrease of glycolysis and pyruvate generation. This
assertion is compatible with the findings of Bowles and
Ellefson (1985) where exponential cultures of C. acetobutylicum cells had decreased cytoplasmic ATP and glucose
uptake because butanol disrupted the membrane integrity
and, in so doing, inhibited membrane-linked functions.
Ounine et al. (1985) investigated the inhibition of C.
acetobutylicum metabolism of glucose and xylose by end
products of fermentation and concluded that C. acetobutylicum, when grown in xylose, is inhibited to a greater extent
by ABE than is C. acetobutylicum grown on glucose.
Among the end products of fermentation, butanol is the
most potent inhibitor, and results obtained by Ounine et al.
(1985) indicates that when glucose and xylose are used as
substrates in the C. acetobutylicum fermentation, growth
inhibition on both substrates is correlated with the inhibitory
effects of butanol on sugar transport. This phenomenon is
most pronounced when growth occurs on xylose rather than
on glucose. C. beijerinckii cells had enhanced glucose
utilization when the butanol concentration in the medium
was maintained below the threshold of butanol toxicity
(Ezeji et al. 2003, 2004a). Re-absorption of organic acids
(acetic and butyric acid) by C. beijerinckii was increased,
and acids did not accumulate in the fermentation medium
even though a large amount of glucose was fermented during
batch and fed-batch fermentations (Ezeji et al. 2003, 2004a).
The cell membrane and the solventogenic switch Typically,
in a batch-fermentation mode, the ABE fermentation
proceeds in two phases: the first is called the acidogenic
phase and is growth associated, and the second is a
solventogenic phase characterized by the uptake of acids,
ABE production, and is relatively non-growth associated.
The shift in metabolic activity, which occurs when cells
switch from the acid-producing phase to the solventproducing phase, is accompanied by a corresponding shift
in cellular content of enzymes involved in acid- and
solvent-producing pathways (Yan et al. 1988). Organic acid
concentration increases as acids are produced during the

acidogenic phase of ABE fermentation and decreases as
acids are absorbed by the cells to produce ABE. Formation
of organic acids during ABE fermentation, however, cannot
be avoided because the solventogenic clostridia obtain part
of their metabolic energy (two ATP molecules from one
mole of pyruvate) during organic acid formation (Fig. 1a).
Excessive accumulation of undissociated acetic and butyric
acid caused by poor buffering capacity of the fermentation
medium or addition of excess acetic and butyric acid to
fermentation medium can be detrimental to solventogenic
clostridia nutrient uptake and growth.
In cell membranes, proteins may be embedded in the outer
layer, the inner layer, or they may span the two layers as
depicted in Fig. 2. The individual phospholipids and proteins
can freely move around within the membrane layer similar to
that which would occur in a liquid phase, resulting in rapid
diffusion of molecules such as acetic and butyric acid in and
out of microbial cells. The correlation between hydrophobicity of aliphatic alcohols and inhibition of cellular growth
and nutrient uptake suggests the membrane is the site of
inhibitory interactions (Linden and Moreira 1982). Moreira
et al. (1981) reported that addition of aliphatic alcohols to C.
acetobutylicum ATCC 824 cultures resulted in an instantaneous inhibition of membrane-bound ATPase activity.
Membrane-bound ATPase is essential for maintenance of
internal pH and an electrochemical gradient (Okamoto et al.
1977), and butanol inhibits the activity of this enzyme by
Fig. 2 Diagrammatic representation of the movement of undissociated acetic and butyric acid across phospholipids bilayer of a Grampositive (solventogenic clostridia) cell membrane. Acetic and butyric
acid can destroy cell function by acidifying clostridia cells cytoplasm
below the maximum pH of tolerance. Broken arrow and cross show
that dissociated acetic and butyric acid are non-membrane permeable
and cannot freely diffuse across cell membranes (model is a modified
form of Russell and Diez-Gonzalez (1997) model)
increasing the activation energy of the enzyme and disrupting phospholipids of the cell membrane (Bowles and
Ellefson 1985). The undissociated form of acetic and butyric
acid are membrane permeable and can pass across bacterial
cell membranes (Russell and Diez-Gonzalez 1998), unlike
the dissociated forms which are non-membrane permeable
(Fig. 2). The intracellular acid concentration associated with
the solventogenic Clostridium cells is usually different from
the measured concentration in the culture medium (Monot et
al. 1984). Because butyric and acetic acid are produced and
secreted by solventogenic Clostridium cells in the undissociated form (Terracciano and Kashket 1986), there will be a
concentration gradient across the cell membrane. Moreover,
with growing clostridia, the internal pH is greater than the
external pH due to a proton translocation process catalyzed
by a membrane-bound ATPase (Booth and Morris 1975;
Riebeling and Jugermann 1976; Herrero 1983; Monot et al.
1984). When 104 M N-N dicyclohexylcarbodiimide
(DCCD), a known specific inhibitor of the membranebound ATPase, was added to the culture medium, DCCD
completely inhibited the growth of C. acetobutylicum. When
a lesser concentration (105 M) of DCCD was added at the
beginning of the growth phase, there was cellular growth, but
metabolic activity of the cell was modified due to inhibition
of the membrane-bound ATPase and decreased intracellular
pH (Riebeling et al. 1975). Because butanol reduces the
intracellular concentrations of ATP in solventogenic clostridia
(Bowles and Ellefson 1985) and inhibits membrane-bound
ATPase, invariably intracellular pH maintenance and growth
will be negatively affected when there are greater concentrations of butanol present.
Biochemistry of solvent production by solventogenic

clostridia
Glucose is fermented via the EmdenMeyerhofParnas
pathway to pyruvate (Fig. 1ac). Solventogenic clostridia
produce two major types of products, solvents (ABE) and
gases (carbon dioxide and hydrogen), and one major type of
fermentation intermediate product, organic acids (acetic and
butyric acid). Solventogenic clostridia are obligate anaerobes
and generate only two net molecules of ATP during
glycolytic metabolism of glucose to pyruvate (Fig. 1ac).
The acetic and butyric acid pathway reactions have important
roles in solventogenic clostridia metabolism because synthesis of these acids is accompanied by generation of ATP,
which is important for cell growth and metabolism. Although
greater concentrations of total acetic acid (acetic acid from
P2 medium+acetic acid produced by C. beijerinckii BA101)
than butyric acid are always measured in the bioreactor
during butanol fermentation by C. beijerinckii BA101 (Ezeji
et al. 2003, 2005b, 2007a, b), more butyric acid than acetic
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acid is produced both during the acidogenic (exponential

growth phase) and solventogenic phase. Butyric acid is
quickly re-assimilated for butanol production during the
solventogenic phase. The formation of butyric acid during
the acidogenic phase is important for maintenance of the
redox equilibrium because nicotinamide adenine dinucleotides (NADHs) produced during glycolysis are only oxidized
in the butyric acid formation pathway, not in the acetic acid
formation pathway, resulting in the regeneration of NAD+
(important for continuation of glycolysis and generation of
pyruvate by C. beijerinckii (Fig. 1a)). A mechanism that
involves metabolism of pyruvate for acetic and butyric acid
production (Fig. 1a; two molecules of ATP produced) and
uptake of acetic and butyric acid for ABE production
(Fig. 1c) is energetically more attractive than a reaction
scheme (no ATP produced) that results in direct production
of ABE (Fig. 1b). Under certain conditions, lactic acid may
be produced (Fig. 1a), and some of the two precursors of
butyric acid, acetoacetyl-CoA, and butyryl-CoA are directly
converted by solventogenic clostridia to the neutral
productsacetone and butanol, respectively (Fig. 1b). The
CoA moiety from acetoacetatyl-CoA is transferred to acetate.
The resulting acetoacetate is decarboxylated to acetone by a
reaction catalyzed by acetoacetate decarboxylase (Fig. 1a, c).
However, direct production of ABE from pyruvate occurs
because acids and small amounts of ABE are produced
during acidogenic phase growth of C. beijerinckii in batch
fermentations (Ezeji et al. 2003; Shi and Blaschek 2008).
Addition of reducing compounds such as viologen dyes or
carbon monoxide purging of the fermentation medium may
cause metabolic shifts and result in a favorable carbon flow
to butanol production instead of butyrate production (Meyer
et al. 1986; Rao and Mutharasan 1987; Tashiro et al. 2007).
Although part of the NADH produced by the Emden
MeyerhofParnas pathway is acted upon by NADHferredoxin reductase to produce reduced ferredoxin and
hence molecular hydrogen (Peguin and Soucaille 1995), the
reaction may shift from pyruvate to lactic acid formation to
facilitate rapid NADH and NAD+ regeneration (Fig. 1a). A
reduced ferredoxin is the physiological electron donor of
hydrogenase in C. acetobutylicum, and bacterial-type ferredoxins are low molecular weight carrier proteins containing
two [4Fe4S] clusters involved in low-potential oxidationreduction reactions (Guerrini et al. 2008; Quinkal et al.
1994). Five open reading frames (ORFs) have been
annotated in the C. acetobutylicum genome for coding
putative ferredoxins (Nolling et al. 2001), and ORF
CAC0303 was shown to express the major ferredoxin in
the solventogenic C. acetobutylicum cells (Demuez et al.
2007). Pyruvate oxidation to acetyl-CoA requires ferredoxin
(Fd) reduction. Reduced Fd (FdH2) is oxidized by hydrogenase (NADPH) or (NADH), which regenerates Fd, NAD+,
or NADP+ and releases molecular hydrogen. The reaction is
1702
reversible, and depending on culture conditions, either FdH2

or NADH may be formed. The reverse reaction (NADH
formation from FdH2) is inhibited by NADH and proceeds
only after rapid removal of the NADH (Petitdemange et al.
1976; Rao and Mutharasan 1987). Despite the critical role of
ferrodoxin reductase in the maintenance of a low redox
potential during ABE fermentation in solventogenic clostridia (Ezeji et al. 2007c), there is no information in the literature
that describes how NADH-dependent or NADPH-dependent
ferrodoxin reductase is affected by presence of butanol and
butanol toxicity.
Autolysin production by the solventogenic clostridia and
the effect of butanol on clostridial cells during the solventogenic phase of butanol fermentation is worthy of mention.
Barber et al. (1979) reported the production of greater
concentrations of autolysin toward the end of the exponential
growth phase of C. acetobutylicum (onset of solventogenesis) and was accompanied by lysis of culture cells and
decrease in ABE-producing cells. This study was the first
where direct linkage of autolytic degradation in solventproducing cells of C. acetobutylicum and presence of butanol
during growth was established, and that inhibitory concentrations of butanol were involved in the induction of release
of cell-free autolysin during the solventogenic phase (Barber
et al. 1979; Jones and Woods 1986). Van Der Westhuizen et
al. (1982) reported the effects of acetone and butanol on the
growth and stability of swollen-phase bright-stationary-phase
cells (clostridial forms) of C. acetobutylicum P262 and an
autolytic-deficient mutant (lyt-1) strain where it was determined that butanol concentrations between 7 and 16 g/L,
which are within the range obtained in ABE fermentations,
increased butanol-induced degeneration of strain P262
clostridial forms but had no effect on the stability of
autolytic-deficient mutants that never underwent autolysis.
The conclusion was that there is a relationship between
butanol tolerance and autolytic activity. Although the
autolytic-deficient mutant (lyt-1) strain is butanol tolerant,
the mutant did not degenerate in the presence of butanol
(between 0 and 38 g/L); the ability of the mutant to produce
ABE when greater butanol concentrations existed was
compromised. Because greater concentrations of butanol
inhibited butanol production by an autolytic-deficient mutant
(lyt-1) but not growth of the mutant strain, examination of
global gene expression in lyt-1 alongside enzyme kinetics
studies may provide clues on the target of butanol inhibition,
whether it is at the transcriptional or enzyme level (kinetics).
Cell membrane fluidity, cell death, and solvent tolerance

in acetone and butanol-producing microorganisms
The major challenge with the ABE fermentation is that it
suffers from a number of limitations (e.g., low concentra-
tion, yield, and productivity) due to butanol stress and

toxicity to the microbial cells (Ezeji et al. 2004a, b).
Researchers have made several parallel observations between butanol stress and other forms of stress that can be
imposed on the microbial cell. Heat shock stress is probably
the most examined form of stress and results in the
repression of synthesis of many cellular proteins, while a
specific set of about 20 heat shock proteins (hsps) are
induced in response to temperature increases. These
proteins are involved in the cellular response and adaptation
to stress, as many of these proteins are also induced by
exposure of bacterial cells to other forms of stresses,
including alcohols (Michel and Starka 1986; Terracciano
et al. 1988). Solventogenic clostridia respond to the
perturbing effect of butanol as a result of expression of
several heat shock protein genes and modifying the fatty
acid (saturation) composition of membrane lipids. Expression of stress proteins was enhanced in C. acetobutylicum
by increasing concentration of butanol by as little as 11 mM
(about 6% of the total butanol produced by the cells) during
the acidogenic phase (Terracciano et al. 1988). The de novo
synthesis of similar proteins during the solventogenic phase
and in the absence of an added stress suggests a connection
between the stress response and solventogenesis (Terracciano
et al. 1988).
The toxicity of alcohols appears to increase with chain
length, with long-chain alcohols being more toxic at a
lesser concentration than short-chain alcohols (Lepage et al.
1987). Alcohol toxicity to microbial cells has been
suggested to occur as a result of damage to the cell
membrane (Osman and Ingram 1985) and direct inhibition
of metabolism (Herrero et al. 1985).
Vollherbst-Schneck et al. (1984), during their evaluation
of the effect of butanol on lipid composition and fluidity of
C. acetobutylicum ATCC824, found that when grown in the
presence of butanol, C. acetobutylicum synthesized greater
amounts of saturated acyl chains of fatty acids at the
expense of unsaturated chains. The presence of added
butanol affected the cellular acyl fatty acids chain composition in the same way as did growth at the peak (stationary
phase) of butanol production (Vollherbst-Schneck et al.
1984). This increase in saturation of cellular lipids may
have occurred in order to compensate for the increase in
fluidity of cell membranes that is induced by butanol (Baer
et al. 1987). Increase in cell fluidity facilitates leakage of
cellular contents and subsequently cell death.
The observed membrane change makes it the principal
target for cellular adaptation to potentially toxic amounts of
extracellular alcohol (Taylor et al. 2008). This assertion was
highlighted in 1976 by Ingram, who reported that the
membrane fatty acid composition of E. coli K-12 was
radically altered when the strain was grown in the presence
of butanol and other alcohols. The proportion of longer-
chain (18:1) fatty acids was found to increase at the

expense of relatively shorter-chain (<C18) fatty acids
(Ingram 1976). Development of a butanol-tolerant clostridial mutant that is able to produce greater amounts of
butanol in batch culture systems has yielded variable results
(Liyanage et al. 2000; Zhao et al. 2003). Liyanage et al.
(2000) found that antisense inhibition of gene expression of
glycerol dehydrogenase in C. beijerinckii wild type resulted
in a 25% decrease in glycerol dehydrogenase activity and
an increase in butanol tolerance. It was initially considered
that overexpression of the cyclopropane fatty acid synthase
gene (cfa) of C. acetobutylicum, ATCC 824, and the
concomitant change in lipid composition might increase
solvent tolerance and allow for greater solvent production
in C. acetobutylicum. Zhao et al. (2003) overexpressed the
cfa gene in the wild type, and the generated strain had
greater cyclopropane fatty acid content during the early logphase cycle as well as greater initial acid and butanol
resistance than the wild type. Contrary to all expectations,
solvent production in the cfa-overexpressing strain was
decreased considerably, while the amounts of acetate and
butyrate remained highsuggesting that overexpression of
the cfa gene results in changes in membrane properties that
decrease the complete induction of solventogenesis (Zhao
et al. 2003). Additional achievements in strain design will
be discussed in Genetic strain improvement section.
Amelioration of solvent toxicity in acetone

and butanol-producing microorganisms
Genetic strain improvement
Development of solvent-tolerant strains to ameliorate solvent
toxicity has typically followed one of two approaches: (1)
enhancement of butanol toxicity defenses in solventogenic
clostridia and (2) metabolic engineering of well-characterized
microorganisms (E. coli and S. cerevisiae) for ABE
production. The recent availability of genomic sequence
information for two of the solvent-producing clostridia,
namely, C. acetobutylicum ATCC 824 and C. beijerinckii
8052, enables global strategies for improving resistance of
these microbes to butanol and acetone. These strategies may
involve the use of gene expression microarrays as well as
various synthetic biology-based approaches (Shi and
Blaschek 2008). Additionally, investigations with the aim
of improving ABE tolerance and productivity are ongoing
for the identification of solvent-resistant microorganisms as
alternative solvent production hosts.
Enhancing ABE resistance in solventogenic clostridia The
solventogenic clostridia have historically been the most
studied acetone-butanol-producing organisms. Two genome-
1703
sequenced solventogenic microorganismsC. acetobutylicum 824 and C. beijerinckii NCIMB 8052have been the
focus of significant analysis and adaptive engineering in an
effort to increase resistance to solvents (particularly butanol)
in the fermentation broth. While significant advancements
have been made, low solvent concentration remains a hurdle
for commercialization of biologically produced butanol and
acetone. Typically, the concentration of total solvents in the
bioreactor during the ABE fermentation rarely exceeds 20 g/L,
with butanol concentrations rarely exceeding 13 g/L (Qureshi
and Blaschek 2001). Efforts in strain improvement have
increased butanol concentration to as much as 19 g/L, but
concentrations approaching 40 g/L could significantly
reduce the energy used for butanol recovery (Phillips and
Humphrey 1983).
C. acetobutylicum ATCC 824 One of the earliest developed
butanol-tolerant strains was a mutant of C. acetobutylicum
ATCC 824 called SA-1. This strain was developed using
serial transfer, a procedure where samples of C. acetobutylicum culture at OD585 0.8 (or the highest attainable) are
transferred into fresh media containing increasing concentrations of n-butanol. SA-1 had a 121% increase in
tolerance over wild type when grown on 6% extruded corn
broth (Lin and Blaschek 1983). Similar experiments later
led to strain SA-2, grown on brain heart infusion and P2
(minimal) medium. SA-2 had a 27% increase in butanol
tolerance over wild type and was hypothesized to have
adjusted its lipid membrane content in order to maintain a
stable environment for cellular functions (Baer et al. 1987).
In both SA-1 and SA-2, increased tolerance did not result in
a greater overall butanol yield, suggesting that tolerance is
not the only variable limiting butanol yield. A new method
for strain development in C. acetobutylicum was introduced
in 1994 by Mermelstein et al., employing a plasmid vector
system for introducing foreign DNA into ATCC 824 by
electrotransformation (Mermelstein et al. 1994). This
genetic transfer technique has been the most widely used
to investigate the effect of overexpressing or inactivating
various genes on ABE tolerance in solventogenic clostridia.
Solvent tolerance of engineered strains of C. acetobutylicum is typically evaluated by exposure to different
concentrations of butanol commonly referred to as a
butanol challenge.
Strain PJC4BKproduced by inactivating butyrate
kinasewas shown to produce 16.7 g/L of butanol, a final
concentration that surpassed the wild type toxicity limit of
13 g/L (Green et al. 1996). In 2000, Harris et al. compared
the solvent formation and tolerance abilities of PJC4BK to
a newly developed strain, pJC4BK(pTAAD), with overexpression of the alcohol dehydrogenase gene aad. Both
pJC4BK and pJC4BK(pTAAD) surpassed the wild type
toxicity threshold of 13 g/L without specific selection for
1704
butanol tolerance. This is noteworthy because it indicates

the production of butanol is not triggered by, or directly
correlated to, butanol concentration or tolerance limits.
Strain pJC4BK(pTAAD) did not produce increased levels
of butanol relative to pJC4BK, indicating that butanol
production is not limited by alcohol dehydrogenase activity.
However, pJC4BK(pTAAD) did produce increased
amounts of ethanol as well, corresponding to a total alcohol
tolerance of 21.2 g/L. Both ethanol and butanol are
hypothesized to disrupt the cell membrane; however, the
increased alcohol toxicity threshold with no specific
selection for alcohol tolerant strains suggests that this may
not be the primary effect (Green et al. 1996; Harris et al.
2000). Another study by Harris et al. (2001) again
investigated the effect of overexpression of alcohol dehydrogenase in SolRH(ptAAD) compared to the engineered
strain SolRH, both lacking the solvent formation repressor
SolR. SolRH achieved final solvent concentrations which
were essentially equal to the plasmid control, but SolRH
(pTAAD) produced 17.6 and 8.2 g/L butanol and acetone,
respectively. This study again demonstrated an increase in
solvent tolerance in connection with increased solvent
production without specific selection.
In 2003, a C. acetobutylicum strain 824(pGROE1) was
produced by overexpressing genes in the class I stress
response operon groESL (Tomas et al. 2003). This strain
demonstrated 85% less growth inhibition from butanol than
the control strain, resulting in 17.1 g/L butanol and 8.6 g/L
acetone. Furthermore, overexpressing groESL resulted in
longer active metabolism, increased expression of motility
and chemotaxis genes, and decreased expression of major
stress response genes. A follow-up study used DNA arraybased transcription profiles of 824(pGROE1) under 0.25%
v/v and 0.75% v/v butanol challenges as well as a 0.75%
challenge on the control strain 824(pSOS95del) in order to
differentiate genes that are likely associated with general
butanol stress response and those that are associated with
increased butanol tolerance (Tomas et al. 2004). Genetic
changes found to relate to general butanol stress response
included the overexpression of major stress protein genes,
solvent and butyrate formation genes, and the butyrylcoenzyme A biosynthesis operon genes, as well as
decreased expression of the fatty acid synthesis operon,
glycolytic genes and sporulation genes. Genes suspected to
be specifically involved in increased butanol tolerance
include rlpA, artP, and a hemin permease encoding gene.
A second transcriptional analysis study compared the
expression patterns of 824(pMSPOA)a strain with overexpression of the sporulation gene spo0Awith that of the
spo0A knock-out strain SKO1 and a plasmid control
(Alsaker et al. 2004). As with the previous study, most
differentially expressed genes were related to a general
stress response. The 824(pMSPOA) strain demonstrated
increased tolerance and prolonged metabolism when subjected to butanol stress, and genes associated with immediate butanol stress responsehence likely contributing to
increased tolerancewere identified. When 824(pMSPOA)
was compared to 824(pGROE1), 160 total differentially
expressed genes were identified under butanol challenge
conditions. The main difference seen between the two
strains was the time (during fermentation) at which genes
showed high expressionin one strain, increased expression
was seen early on, but in the other, the increased expression
had a more delayed response. Counterintuitively, both
studies found that butanol stress appeared to overexpress
solvent formation genes and underexpress fatty acid
synthesis genes.
Borden and Papoutsakis expanded the search for genes
to target C. acetobutylicum's ability to withstand greater
solvent concentrations using a genomic library. Plasmids
were inserted into wild type C. acetobutylicum cells via
electroporation, and the cells were challenged with various
amounts of butanol (Borden and Papoutsakis 2007).
Sixteen genes were identified as contributing to the cells
ability to withstand greater concentrations of butanol;
pCAC1869 in particular showed a 45% increase in
tolerance. Similarly, pCAC0003 was found to have a 24%
increase in butanol tolerance. CAC1869 is suspected to be a
transcriptional regulator (KEGG) and was found to have
maximal transcription preceding induction of the solventogenic genesaad, ctfA, and ctfB. This gene is actively
transcribed throughout the transitional phase.
C. beijerinckii NCIMB 8052 C. beijerinckii is phenotypically quite similar to C. acetobutylicum, but its genome
(which is 50% larger than C. acetobutylicum) was recently
sequenced, and genetic strain modification efforts are not
yet as advanced. However, in 1991, direct acting N-methylN-nitro-N-nitrosoguanidine was used as a mutagen to create
the C. beijerinckii mutant BA101capable of producing
greater amounts of solvent than any C. acetobutylicum
strain engineered thus far (Qureshi and Blaschek 2001;
Chen and Blaschek 1999). C. beijerinckii BA101 is both
stable and has hyper-amylolytic and hyper-butanologenic
characteristics (Annous and Blaschek 1991). When grown
on semi-defined P2 medium and in batch fermentation, C.
beijerinckii BA101 produces up to 19 g/L butanol and a
total solvent concentration of 29 g/L, over 100% improvement when compared to the wild type C. beijerinckii
NCIMB 8052. Even though specific selection for solvent
tolerance was not conducted, C. beijerinckii BA101
exhibits increased tolerance to butanol, with 100% cell
inhibition occurring at 23 g/L butanol rather than 11 g/L
characteristic of C. beijerinckii NCIMB 8052 (Qureshi and
Blaschek 2001). The exact mechanism for the observed
increase in tolerance is unknown.
Engineered microorganisms for ABE production and

resistance With the history of bio-butanol production by
the natural solvent-producing clostridia, there are some
clear inherent advantages in improving and using these
native clostridial systems for producing bio-butanol. However, there are several challenges to using these bacteria
relative to model fermentation organisms such as E. coli
and S. cerevisiae: (1) fewer tools are available for genetic
manipulation of Clostridium metabolism, (2) clostridia have
slower growth rates, and (3) clostridia are obligate anaerobes. For these reasons, an alternative model for economical production of solvents using well-characterized hosts
such as S. cerevisiae and E. coli for ABE production has
been investigated.
E. coli The first efforts towards using E. coli to produce
solvents was the expression of four C. acetobutylicum
ATCC 824 genes (adc, ctfA, ctfB, and thl) in E. coli for
enhanced acetone production (Bermejo et al. 1998). Strain
ATCC 11303 (pACT) produced 5.4 g/L acetone when
grown in glucose-fed shake flasksa concentration comparable to wild type C. acetobutylicum. Acetone's great
volatility limits toxic effects, and thus tolerance was not
found to be a concern (Bermejo et al. 1998). With the
addition of a secondary alcohol dehydrogenase and subsequent strain optimization, this acetone-producing strain was
adapted to produce 4.9 g/L of isopropanol (Hanai et al.
2007). E. coli has also been engineered to produce 1butanol using the pathway from C. acetobutylicum (containing genes thl, hbd, crt, bcd, etfAB, and adhE2; Atsumi
et al. 2008a). The engineered strain produced 13.9 mg/L
butanol, and by overexpressing E. coli's acetyl-CoA
acetyltransferase instead of the clostridia acetoacetyl-CoA
thiolase, butanol concentration was improved. By deleting
pathways competing for acetyl-CoA and NADH (ldhA,
adhE, and frdBC) as well as genes to increase pyruvate
dehydrogenase activity and decrease acetate production (fnr
and pta), butanol production reached 373 mg/L (strain
JCL88); this value was increased to 552 mg/L when grown
in rich media. Nielsen et al. (2009) later increased
production to 580 mg/L using individual expression of
pathway genes alongside co-expression of fdh1 from yeast
(for cofactor regeneration) and overexpression of the gapA
gene from E. coli to increase glycolytic flux and the acetylCoA pool. Without specific selection for butanol tolerance
or engineering for increased toxicity thresholds, E. coli
tolerated up to 1.5% butanola value competitive with
clostridia (Atsumi et al. 2008b).
After the initial success with solvent production by E.
coli using genes from C. acetobutylicum, Atsumi et al.
(2008a) developed a process for production of higher
alcohols (e.g., 1-butanol) that avoids CoA-mediated chemistry used by native solvent-producing organisms. By
1705
utilizing E. coli's amino acid biosynthesis pathway, isobutanol (Atsumi et al. 2008b; Atsumi and Liao 2008a), 1butanol (Atsumi and Liao 2008b), 2-methyl-1-butanol
(Atsumi et al. 2008a; Atsumi and Liao 2008a; Cann and
Liao 2008), 3-methyl-1-butanol (Atsumi et al. 2008b;
Atsumi and Liao 2008b; Connor and Liao 2008), and 2
phenylethanol (Atsumi et al. 2008a; Atsumi and Liao
2008a) are made from the valine, norvaline, isoleucine,
leucine, and phenylalanine biosynthesis pathways, respectively. By diverting 2-keto acid intermediates to alcohol
synthesis, this study demonstrated up to 22 g/L of
isobutanol (Atsumi et al. 2008b). To achieve this, only
genes for two additional enzymes (2-keto acid decarboxylase and alcohol dehydrogenase) were needed. To maximize
the production of each product in turn, genes to enhance the
intermediate 2-keto acid of interest were overexpressed, and
genes corresponding to competing reactions were deleted.
In some instances, native E. coli genes were replaced with
more active genes from other hosts. Native 1-butanolproducing organisms (clostridia) tolerate up to 2% w/v of
the product, and E. coli was found to initially be intolerant
to 1.5% w/v isobutanol (a less toxic solvent). After
performing serial transfers to enhance tolerance, the E. coli
strain survived in up to 2% w/v isobutanol.
Later, the initial isobutanol response network of E. coli
was characterized using gene expression, transcription
factor-gene interaction data, gene knockouts, and network
component analysis (Brynildsen and Liao 2009). An
additional comparison showed similar response networks
for both isobutanol and n-butanol. Notably, this study also
found that isobutanol stress leads to the disruption of
important membrane components (most importantly, quinones) that subsequently affected respiratory, phosphate,
and iron control. Network component analysis determined
67 transcription factors to be active as a result of isobutanol
stress, 16 (relating to stress mitigation, metabolism regulation, and nucleoproteins) of which were significantly
perturbed. Most significantly perturbed were transcription
factors effecting respiration (in particular, the transcription
factors ARcA, PdhR, and FNR), and further verification of
this supported the assumption that solvent toxicity leads to
cell membrane malfunction. Analysis of the response
networks showed that conditions affecting isobutanol
resistance apply to n-butanol as well, except that 1butanol had a greater repression effect on amino acid
synthesis in microorganisms than did isobutanol. A deletion
study removing arcA, fur, and phoB did not significantly
increase tolerance, supporting the hypothesis that tolerance
response is a complex result of multiple mutations.
Previous studies with C. acetobutylicum, which showed
that stress by increased 1-butanol concentrations elicits a
response similar to heat shock proteins, were supported by
the network analysis showing that E. coli displayed greatly
1706
enhanced activation of a heat shock sigma factor in

response to isobutanol.
S. cerevisiae S. cerevisiae naturally produces small amounts
of isobutanol and 3-methyl-1-butanol as byproducts of
fermentation, giving it a basal ability to resist solvent
toxicity. Recently, S. cerevisiae was engineered to produce
2.5 mg/L of n-butanol through the cloning of the 1-butanol
pathway and various isozymes chosen from C. beijerinckii,
E. coli, and Ralstonia eutropha into S. cerevisiae (Steen et
al. 2008)with C. beijerinckii genes showing the best
results. This amount of solvent production, however, is still
far less than concentrations achieved by the most effective
Clostridium strains for butanol production (approximately
20 g/L) and the most recently engineered E. coli strains.
Given that S. cerevisiae can tolerate up to 2% butanol
(Fischer et al. 2008; Knoshaug and Zhang 2009) and that
only 2.5 mg/L butanol was produced during fermentation,
butanol toxicity does not appear to be the limiting factor.
An earlier study investigating S. cerevisiae response to
butanol found that butanol inhibits translation at the
initiation step (Ashe et al. 2001).
Solvent-resistant microorganisms as potential
acetone-butanol production hosts
As an alternative approach, future efforts to genetically
engineer microorganisms for increased tolerance to butanol
may focus on investigations of host microorganisms that
have a natural resistance to solvent but that have not
demonstrated significant solvent formation. This approach
was reported recently by investigators who screened 24
hosts, spanning seven genera, with the aim of identifying
microorganisms that possess great butanol tolerance capability (Knoshaug and Zhang 2009). Of the yeast strains, S.
cerevisiae ATCC 26602 and ATCC 20252, as well as
Candida sonorensis, were able to grow on 2% of 1-butanol.
In all cases, growth was only 10% to 20% that of the
control (without butanol challenge) cultures. Of the nonyeast species, Lactobacillus delbrueckii and Lactobacillus
brevis grew on 2% butanol with relative growth rates of
55% and 58%, respectively, with L. brevis maintaining 30%
relative growth rate on 3% butanol. In another study,
Bacillus subtilis was found to tolerate up to 2% butanol
(Fischer et al. 2008). Knoshaug and Zhang (2009) found
tolerance to be dependent on temperature: at higher
temperatures, strains grew faster but had lower butanol
tolerance thresholds, while strains at lower temperature
had increased butanol tolerance thresholds but grew
slower.
A separate study investigated the butanol resistance of
three solvent-tolerant and one solvent-sensitive P. putida
strains (Ruhl et al. 2009). Each strain was adapted using
serial transfer, and a mutant strain capable of growth on 6%

butanol was generated. 13C tracer-based flux analysis was
performed to investigate the strain response to butanol
relative to the untreated strain. For untreated strains, glucose
uptake rate increased while growth decreasedwithout a
corresponding increase in byproductsand corresponded to
a redistribution of intracellular flux. Specifically, carbon was
redirected to the tricarboxylic acid (TCA) cycle, which
increased regeneration rates of redox cofactors. The authors
therefore hypothesized that butanol leads to an increased
need for cell maintenance energy. The solvent-tolerant strain
appeared to have an adapted cell membrane to lower cell
maintenance needsdemonstrating reduced glucose uptake,
TCA cycle usage, and redox cofactor regeneration rates.
Recently, Nielsen et al. (2009) explored the production
ability of two butanol-tolerant organisms, including two
strains of P. putida, using polycistronic expression of butanol
biosynthetic genes. The two P. putida strains produced 44
and 50 mg/L butanol, respectively, when grown on glucose
and 122 and 112 mg/L butanol, respectively, when grown on
glycerol. The study by Ruhl et al. showing tolerance
thresholds of up to 6% butanol, however, suggests potential
for increasing production in the solvent-producing strain.
Advanced fermentation techniques and downstream
processing
To realize the full potential of the altered candidate genes with
respect to butanol concentration, tolerance, and productivity,
developed strains may be used for simultaneous ABE
fermentation and recovery systems to overcome low solvent
(ABE) resistance of these strains. Various alternative in situ
butanol removal techniques including membrane-based systems, such as pervaporation (Groot et al. 1984; Qureshi et al.
1992, 1999; Qureshi and Blaschek 2000), perstraction
(Qureshi et al. 1992), reverse osmosis (Garcia et al. 1986),
adsorption (Ennis et al. 1987; Nielson et al. 1988), liquid
liquid extraction (Evans and Wang 1988), and gas stripping
(Ezeji et al. 2004b, 2005a) have been examined. A summary
of in situ butanol removal techniques and limitations is
provided in Table 1. Although there has been significant
progress in integration of fermentation with product recovery
techniques, there are few commercial applications of these
technologies. To advance butanol fermentation from laboratory scale production to industrial scale, the integrated
process must be microbial friendly, scalable, non-fouling,
and enhance butanol productivity.
Liquidliquid extraction
Liquidliquid extraction is a technique used to reduce
solvent (ABE) toxicity of solventogenic clostridia. In this
process, a water-insoluble organic extractant is mixed with
1707
Table 1 Summary of the integrated butanol fermentation and in situ butanol removal techniques
Process
Relieves
butanol toxicity
Increases
yield
Increases
productivity
Limitations
Adsorptiona,b
Yes
No
Yes
Gas strippingc,d
Liquidliquid extractione
Yes
Yes
Yes
No
Yes
Yes
Perstractionf
Yes
No
Yes
Pervaporationf,g,h,i
Yes
No
Yes
Reverse osmosisj
Yes
No
Yes
Loss of nutrients to adsorbent, clogging, loss of fermentation

(acetic and butyric acid) intermediate products
Low butanol stripping rate
Extractant toxicity to cells, formation of rag layer, emulsion,
loss of fermentation intermediate products to extractant
Loss of fermentation intermediate product to extractant,
expensive to operate, membrane fouling, and lack simplicity
Loss of fermentation intermediate products to extractant due
to diffusion across membrane, membrane fouling
Fouling, loss of nutrients, and lack simplicity
Ennis et al. (1987)
Nielson et al. (1988)
Ezeji et al. (2004b)
Ezeji et al. (2005a)
Evans and Wang (1988)
Qureshi et al. (1992)
Groot et al. (1984)
Qureshi and Blaschek (1999)
Qureshi and Blaschek (2000)
Garcia et al. (1986)
the fermentation broth. Butanol is more soluble in the

organic (extractant) phase than in the aqueous (fermentation
broth) phase; therefore, butanol selectively concentrates in
the organic phase (Ezeji et al. 2007c). ABE are concentrated in the extraction solvent and recovered by backextraction into another extraction solvent or by distillation
(Maddox 1989). The extractant of choice for in situ ABE
recovery among researchers has been oleyl alcohol because
it is relatively non-toxic as well as being a high quality
extractant (Evans and Wang 1988; Ezeji et al. 2006;
Karcher et al. 2005). Dibutyl phthalate, an important
extraction solvent, was found to be non-toxic to C.
beijerinckii BA101 during butanol fermentation and recovery by liquidliquid extraction (Karcher et al. 2005). In a
detailed investigation conducted by Roffler et al. (1987),
six solvents or solvent mixtures were tested in batch
extractive fermentations: kerosene, 30 wt.% tetradecanol
in kerosene, 50 wt.% dodecanol in kerosene, oleyl alcohol,
50 wt.% oleyl alcohol in a decane fraction, and 50 wt.%
oleyl alcohol in benzyl benzoate. Oleyl alcohol or a mixture
of oleyl alcohol and benzyl benzoate provided the most
desirable results and improved volumetric butanol productivity by as much as 60%. Common extractants, toxicity
data, and partition coefficients used in ethanol and butanol
fermentation are published elsewhere (Karcher et al. 2005).
There are some challenges associated with a liquid
liquid extraction system including extraction solvent toxic-
ity to butanol-producing cells, formation of an emulsion,

loss of extraction solvent, and the accumulation of
microbial cells at the extractant and fermentation broth
interphase (formation of rag layer; Ezeji et al. 2007c).
Perstraction
To avoid the toxicity problem brought about by the
extraction solvent, investigators have used perstractive
fermentation and recovery that employs a membrane
contactor (Roffler et al. 1987; Traxler et al. 1985; Qureshi
et al. 1992). Perstraction is an extractive fermentation
process designed to reduce extraction solvent toxicity and
butanol toxicity to the producing culture, while improving
butanol concentration, productivity, and selectivity. The
membrane contactor in perstractive process provides surface area where the two immiscible phases can exchange
the butanol. The total mass transport of butanol from the
fermentation broth to the organic side depends on the rate
of diffusion of butanol across the membrane (Ezeji et al.
2007c). Because there is no direct contact between the two
phases, extractant toxicity, phase dispersion, emulsion, and
rag layer formation are drastically reduced or eliminated
(Ezeji et al. 2006).
The major drawback of ABE fermentation and recovery
by perstraction is fouling of the membrane (Ezeji and Li
2009). Even without membrane fouling, the membrane
1708
does, however, present a physical barrier that can limit the

rate of butanol extraction. In addition, the concentration of
mineral salts in the aqueous phase, brought about by salt
accumulation due to long fermentation times (alkali from
media feed that reacts with acids or other components) can
lead to cessation of fermentation.
Gas stripping
Gas stripping is a simple technique that can be integrated
with ABE fermentation to simultaneously recover ABE
during fermentation. This technique is useful for keeping
butanol concentration in the bioreactor below the threshold
of butanol toxicity to the culture and allowing fermentation
to go on unimpeded until all the sugars present in the
bioreactor are utilized and converted to ABE (Ezeji et al.
2003, 2004a). Fermentation and recovery by gas stripping
involves bubbling oxygen free nitrogen or fermentation
gases (CO2 and H2) through the fermentation broth
followed by cooling the enriched gas (or gases) with ABE
in a condenser (Ezeji et al. 2003, 2004a, 2006). As the gas
is bubbled through the fermentor, it captures ABE, which is
condensed in the condenser followed by collection in a
receiver. After the solvents are condensed, the gas is
recycled back to the fermentor to capture more ABE as
depicted in Fig. 3. Application of gas stripping to batch and
fed-batch butanol fermentation systems have been reviewed
elsewhere (Ezeji et al. 2004b, 2006, 2007c).
Pervaporation
Pervaporation is a technique which allows selective
removal of volatiles from broths during fermentation using
a membrane. A pervaporation system is generally composed of feed pump, membrane module, condenser, and
Enriched
gas
Stripping gas
vacuum pump (Fig. 4). This technique has been used

extensively to keep butanol concentration in the bioreactor
below the threshold of butanol toxicity to solventogenic
clostridia during ABE fermentation (Qureshi et al. 1992;
Ezeji et al. 2004b, 2006, 2007c). The volatile or organic
component diffuses through the membrane as a vapor
followed by recovery by condensation. Membranes used in
pervaporation are either hydrophilic or hydrophobic. There
are three steps involved in mass transfer of permeates in
pervaporation: (1) adsorption of solvents into upstream
surface of membrane, (2) diffusion of dissolved solvents
through the membrane, and (3) absorption of dissolved
solvents into permeate vapor at the downstream surface of
membrane (Shao and Huang 2007). Generally, a vacuum or
an inert sweep gas such as N2 are applied on the permeate
side of the membrane to maintain a partial pressure
difference across the membrane which facilitates volatilization of permeates for subsequent condensation and
recovery (Fig. 4). The current benchmark pervaporation
membrane material is polydimethylsiloxane (PDMS). The
reported ethanolwater separation factor for PDMS membranes ranged from 4.4 to 10.8, while the reported butanol
water separation factor for PDMS ranged from 40 to 60,
which is six to ten times more than that of ethanolwater
(Vane 2005). Poly(1-trimethylsilyl-1-propyne) (PTMSP),
hydrophobic zeolite membranes, and composite membranes
have also been studied for ethanolwater or butanol water
separation in a pervaporation system (Ezeji and Li 2009).
The ethanolwater separation factors are largely ranked in
the following order: PDMS<PTMSP<composite membrane<zeolite membranes (Huang et al. 2008). The zeolite
membranes are more expensive than polymer membranes
on a unit area basis, but zeolite membranes have greater
separation factors and flux than polymer membranes (Ezeji
and Li 2009). Although zeolite membranes have not been
tested with butanol, zeolite membranes may be desirable for
simultaneous ABE fermentation and recovery. Application
of pervaporation to batch and fed-batch butanol fermentation systems have been reviewed elsewhere (Qureshi and
Ezeji 2008; Vane 2005; Ezeji et al. 2004b, 2006, 2007c).
Condenser
Bioreactor
Recovered
ABE
Fig. 3 Schematic diagram of simultaneous acetone, butanol, and

ethanol (ABE) fermentation and product recovery by gas stripping.
Arrows show direction of gas flow and recovered ABE
Fig. 4 Schematic diagram of simultaneous acetone, butanol, and

ethanol fermentation and product recovery by pervaporation using a
selective membrane
Concluding remarks
Solvent production by microorganismsin particular, the
production of butanolholds great interest as a means for
generating sustainable transportation fuel and commodity
chemicals. Current research efforts seek to better understand
the effects of solvent toxicity on the metabolism of microorganisms, as well as ameliorate tolerance limitations through
strain design and product recovery techniques. The solventogenic clostridia are commonly studied organisms that produce
solvents (ABE) from a variety of carbon sources using a twophase fermentation process (acidogenesis and solventogenesis). Butanol produced during solventogenesis in clostridia has
been shown to affect the PTS sugar transport system, ion
transport, and the integrity of the cell membrane. The metabolic
changes that occur in the presence of butanol have been
implicated in phenomena such as cellular response to stress
(e.g., heat shock) and fatty acid content of membrane lipids.
Strain design efforts have targeted over- and under-expression
of genes related to solvent stress in organisms of interest, as
well broader approaches including the use of mutagens to
create C. beijerinckii BA101. More recently, research efforts
in ABE production have turned towards both industrially
well-characterized organisms (e.g., E. coli and S. cerevisiae)
and organisms with known tolerance robustness to various
solvents (e.g., P. putida). These strategies often make use of
natural ABE production pathways from clostridia, though
they have not yet been able to match the production levels of
these native ABE-producing microorganisms. In situ recovery
techniques such as liquidliquid extraction, perstraction, gas
stripping, and pervaporation are being used alongside strain
design to maintain a low enough solvent concentration during
fermentation to sustain cell growth and product formation.
While significant progress has been made towards improving
tolerance and increasing ABE yield, it is clear that toxicity is
not the only factor limiting ABE production. Further
advancements must be achieved in order to make the butanol
production process economically competitive and deliver on
this highly promising avenue for biofuel production.
Acknowledgements This work was supported by funding from
Northeast Sungrant (Cornell University) Award/Contract number
GRT00012344, National Research Initiative of the USDA Cooperative
State Research, Education and Extension Service, grant number 200635504-17419, NSF CAREER award (NDP) to Nathan Price, and Seed
grant from Ohio Agricultural Research and Development Center
(OARDC), Wooster.
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