MINI-REVIEW

Achievements and perspectives to overcome the poor solventresistance 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 solventogenicclostridia can ferment a wide range of carbon sources(e.g., glucose, galactose, cellobiose, mannose, xylose,and arabinose) to produce carboxylic acids (acetic andbutyric) and solvents such as acetone, butanol, andethanol (ABE). The fermentation process typicallyproceeds in two phases (acidogenic and solventogenic)in a batch mode. Poor solvent resistance by thesolventogenic clostridia and other fermenting micro-organisms is a major limiting factor in the profitabilityof ABE production by fermentation. The toxic effect ofsolvents, especially butanol, limits the concentration ofthese solvents in the fermentation broth, limiting solventyields and adding to the cost of solvent recovery fromdilute solutions. The accepted dogma is that toxicity inthe ABE fermentation is due to chaotropic effects ofbutanol on the cell membranes of the fermenting micro-organisms, which poses a challenge for the biotechno-

logical whole-cell bio-production of butanol. This mini-review is focused on (1) the effects of solvents oninhibition of cell metabolism (nutrient transport, iontransport, and energy metabolism); (2) cell membranefluidity, death, and solvent tolerance associated with theability of cells to tolerate high concentrations of solventswithout significant loss of cell function; and (3)strategies for overcoming poor solvent resistance inacetone and butanol-producing microorganisms.

Keywords Clostridium . Solvents . Tolerance .

Butanol toxicity . Acetone

Introduction

Concern for the long-term availability of the non-renewablefeedstocks currently used to produce fuels and chemicalshave been raised as their demand and prices increase anddeposits diminish. Developing cost-effective and energy-efficient methods to produce energy-rich biofuels andsustainable chemicals such as butanol requires researchthat draws upon both science and technology knowledgebases. Butanol is an important chemical and has manypromising characteristics as a renewable liquid fuel (Ezejiand Blaschek 2007; Durre 2008). In addition, butanol is apotential fuel and fuel extender for airplanes, as wasdemonstrated during the World War 2 when Japanconverted its sugar refineries into plants to produce butanolas aviation fuel (Schwarz et al. 2007).

General physiological responses to solvent (ABE,toluene, etc.) stress are found to occur in different typesof microorganisms ranging from prokaryotes (e.g.,bacteria) to eukaryotes (e.g., yeast). Solvent responsemechanisms involving solvent exclusion systems and

H. P. Blaschek (*)Center for Advanced BioEnergy Research,University of Illinois Urbana-Champaign,1207 W. Gregory Drive,Urbana, IL 61801, USAe-mail: blaschek@uiuc.edu

T. EzejiDepartment of Animal Sciences and Ohio State AgriculturalResearch and Development Center (OARDC),The Ohio State University,305 Gerlaugh Hall, 1680 Madison Avenue,Wooster, OH 44691, USA

C. Milne :N. D. PriceDepartment of Chemical and Biomolecular Engineering,University of Illinois,119 Roger Adams Laboratory, Box C-3, 600 S. Mathews Avenue,Urbana, IL 61801, USA

Appl Microbiol Biotechnol (2010) 85:16971712DOI 10.1007/s00253-009-2390-0

energy-dependent efflux pumps, which export toxicsolvents from cells, have been characterized in manymicroorganisms (Ramos et al. 2002; Ingram 1990). Effluxpump responses to toluene stress in Escherichia coli,Pseudomonas putida, and Pseudomonas aeruginosa(Ramos et al. 2002), for instance, are the most preciselyunderstood efflux pump response mechanisms (Taylor etal. 2008). Several investigators examining global geneexpression in solvent-tolerant yeast strains have shownthat multiple synergistic responses are responsible forimproved ethanol tolerance (Taylor et al. 2008), and over250 genes are implicated in solvent tolerance, including anumber of genes encoding heat shock proteins andATPases in yeast (Hu et al. 2007; Aguilera et al. 2006).Unlike the ethanol fermentation by microorganisms suchas Saccharomyces cerevisiae and Zymomonas mobilis withunique advantages in terms of ethanol tolerance and well-understood fermentation pathways, a major limitation ofbiomass conversion to butanol is butanol toxicity towardthe producing microbial cells (Ezeji et al. 2004a, b,2007c). Butanol toxicity results in a lower butanolconcentration and negatively impacts fermentation time,productivity, and yield when compared to the ethanolfermentation. Development of butanol-tolerant solvento-genic clostridia together with in situ butanol recoveryprocesses have been proposed by many investigators aspotential solutions for overcoming butanol toxicity(Hermann et al. 1985; Nielson et al. 1988; Lin andBlaschek 1993; Ezeji et al. 2004b, 2007c). Butanol-tolerantmutants, however, do not necessarily produce more butanolthan the lesser tolerant strains (Baer et al. 1987; Ezeji et al.2004b).

A number of factors may be involved in the responseof microbes to stress brought about by solvents, whichinclude disruption of (1) nutrient transport, (2) iontransport (sodium-potassium pump), (3) phospholipidcomposition of cell membranes, and (4) cell metabo-lism. Survival of microorganisms in the presence ofgreater solvent stress will depend on the ability toactivate a broad range of adaptation mechanisms, whichfunction synergistically to nullify the effects of solventtoxicity on cell membranes, metabolic enzymes, andultimately prevent loss of cell functions. A preciseunderstanding of these adaptive mechanisms will obvi-ously help researchers unravel ways to overcome poorsolvent resistance in fermentations of biotechnologicalimportance, especially in the acetonebutanolethanol(ABE) fermentation. In the present review, we focus onprogress made towards overcoming poor solvent resis-tance in the ABE fermentation. The solventogenicclostridia will be heavily drawn upon for example anddiscussion, as they are commonly studied natural ABE-producing microorganisms.

Transport of substrates and organic acidsand their effect on solventogenic clostridiacell metabolism

Gram-positive bacteria, such as solventogenic clostridia,differ from Gram-negative bacteria with respect to physi-ology, 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 Gram-negative bacteria that possess both features. Gram-positivebacteria, however, have a more intricately developedpeptidoglycan layer than do Gram-negative species. Therehas not been conclusive research as to whether the absenceof an outer cell membrane in Gram-positive bacteria and awell-developed peptidoglycan layer are advantageous ordisadvantageous to these species with regard to responsive-ness and adaptability to greater concentrations of solventsin fermentation media. Transport of substrates and nutrientsfrom the surrounding environment into the cell cytoplasm isa prerequisite for cell development and growth in bothGram-positive and Gram-negative bacteria. Efficient carbonutilization has been associated with an increase in sugaruptake, sugar accumulation in the cell cytoplasm, and ABEproduction (Annous and Blaschek 1991; Lee et al. 2001,2005). Different transport mechanisms such as passivetransport (osmosis, diffusion, and facilitated diffusion) andactive transport (high-energy-dependent adenosine triphos-phate (ATP); phosphoenolpyruvate (PEP); or ion gradient-H+, Na+, K+, Cl, etc.) exist in microorganisms for uptakeof molecules across cell membranes and accumulation ofmolecules in the cytoplasm. Gram-positive bacteria, espe-cially obligate anaerobes such as solventogenic clostridia,rely heavily, but not exclusively, on the PEP-dependentphosphotransferase system (PTS) as a means for trans-porting sugars and sugar derivatives into the cell cytoplasmfor subsequent metabolism (Mitchell 1998).

The PTS consists of two common cytoplasmic proteins,enzyme I and HPr (a heat stable histidine-phosphorylatableprotein), as well as an array of sugar-specific enzyme IIcomplexes (EIIs). Enzyme I and HPr are encoded by the genesptsI and ptsH, respectively (Mitchell and Tangney 2005).These enzymes are hydrophilic and are usually referred to asthe general PTS proteins because they are common, with fewexceptions, in all cell phosphotransferases (Mitchell andTangney 2005). The phosphoryl group from PEP issequentially transferred to enzyme I, heat stable histidine-phosphorylatable HPr, EIIs and, finally, to the incomingsubstrate as it is translocated across the membrane. It shouldbe noted that non-PTS system uses free energy released uponhydrolysis of ATP to activate ATP-dependent solute uptakesystem, which belongs to ATP-binding cassette family, anduses binding proteins to capture solutes for onward translo-cation into the bacteria cell.

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Sugar transport and accumulation in the cytoplasm ofsolventogenic clostridia is the first step towards ABEproduction in that sugars must enter the glycolytic cycle forconversion to pyruvate before subsequent reduction ofpyruvate to ABE (Fig. 1ac). Interference of the glycolyticprocess will invariably result in interference of cell growthand ABE production. Decreased rates of glycolysis causedby end products (ethanol and butanol) of fermentation onsugar catabolism have since been implicated in inhibition ofthe cell growth of ethanol and butanol-producing micro-organisms (Herrero et al. 1985; Lovitt et al. 1984; Hutkins

and Kashket 1986). Butanol addition into the fermentationmedium inhibits glucose uptake and growth of Clostridiumacetobutylicum cells (Bowles and Ellefson 1985; Moreira etal. 1981; Ounine et al. 1985). The possible butanol-sensitivestep of glucose catabolism in C. acetobutylicum was notdefined until Hutkins and Kashket in 1986 investigatedwhether the first step of glucose uptake is inhibited bybutanol addition. It is interesting to note that fructose PEP-PTS activity is inducible, and more than tenfold increases inactivity for both Clostridium beijerinckii BA101 and C.beijerinckii 8052 PTS were observed when fructose instead

BUTYRIC ACID

STARCH

GLUCOSE

PYRUVATE

ACETYL-CoA

ACETOACETYL-CoA

-HYDROBUTYRYL - CoA

BUTYRYL-PHOSPHATE

CROTONYL-CoA

BUTYRYL-CoA

FERREDOXIN

2 ATP, 2NADHLACTIC ACID

CO2FERREDOXIN H2

NADH, NADPHNAD, NADP

ATP

ACETYL - CoA

ACETYL-PHOSPHATE

ACETICACID

1

2

3

4

5

6

7

8

9

10 11

12

13

ATP

A

NADHNAD+

NADH

NAD+

NADH

NAD+

BUTANOL

STARCH

GLUCOSE

PYRUVATE

ACETYL - CoA

ACETOACETYL - CoA

-HYDROBUTYRYL - CoA

BUTYRALDEHYDE

CROTONYL - CoA

BUTYRYL - CoA

FERREDOXIN

2 ATP, 2NADHLACTIC ACID

CO2

FERREDOXIN H2

NADH, NADPHNAD, NADP

ACETYL - CoA

ACETALDEHYDE ETHANOL

1

2

3

4

5

6

7

18 19

12

13

NADH

NADH

ACETOACETATE ACETONE16 17 CO2

15

14 NADH

NADHB

NADH

NAD+

NAD+NADH

STARCH

GLUCOSE

PYRUVATE

ACETYL-CoA

ACETOACETYL-CoA

3-HYDROBUTYRYL-CoA

CROTONYL-CoA

BUTYRYL - CoA

FERREDOXIN

2 ATP, 2NADH

LACTIC ACID

CO2FERREDOXIN H2

NADH, NADPHNAD, NADP

ACETYL-CoA

1

2

3

4

5

6

7

16

12

13

ACETIC ACID

16 BUTYRIC ACID

BUTANOL

BUTYRALDEHYDE15

14 NADH

NADH

ACETALDEHYDEETHANOL

19 18

NADHNADH

16ACETOACETATE17ACETONE

CO2

C

NADH

NADH

Fig. 1 ac Simplified metabolism of biomass by solventogenicclostridia. 1, Starch hydrolysis (-amylase, -amylase, pullulanase,glucoamylase, and -glucosidase); 2, glucose uptake by thephosphotransferase system (PTS) and conversion to pyruvate bythe EmdenMeyerhofParnas pathway; 3, pyruvate-ferrodoxin oxi-doreductase; 4, thiolase or acetyl-CoA acetyltransferase; 5, 3-hydroxybutyryl-CoA dehydrogenase; 6, crotonase; 7, butyryl-CoAdehydrogenase; 8, phosphate butyltransferase (phosphotrans-butyrylase);

9, butyrate kinase; 10, phosphate acetyltransferase (phosphostran-sacetylase); 11, acetate kinase; 12, NADH and NADPH-ferredoxinoxidoreductase; 13, lactate dehydrogenase; 14, butyraldehydedehydrogenase and alcohol/aldehyde dehydrogenase; 15, butanoldehydrogenase; 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, wasfound to transport and phosphorylate the glucose analog 2-deoxyglucose by PEP-dependent PTS, and butanol up to 2%did not inhibit phosphotransferase activity (Hutkins andKashket 1986). There is, however, a chaotropic effect(disrupts the cell membrane) of butanol on the cellmembrane of C. acetobutylicum causing cellular PEP andthe 2-deoxyglucose-6-phosphate to be released across themembrane. Enzyme I (cytoplasmic protein) has beenreported to be firmly associated with the cell membrane(Mitchell et al. 1991; Mitchell and Booth 1984). Disruptionor fluidization of cell membranes by butanol, therefore, willdirectly or indirectly affect sugar uptake by the PEP-dependent phophotransferase system and will subsequentlyresult in decrease of glycolysis and pyruvate generation. Thisassertion is compatible with the findings of Bowles andEllefson (1985) where exponential cultures of C. acetobuty-licum cells had decreased cytoplasmic ATP and glucoseuptake because butanol disrupted the membrane integrityand, in so doing, inhibited membrane-linked functions.Ounine et al. (1985) investigated the inhibition of C.acetobutylicum metabolism of glucose and xylose by endproducts of fermentation and concluded that C. acetobutyli-cum, when grown in xylose, is inhibited to a greater extentby ABE than is C. acetobutylicum grown on glucose.Among the end products of fermentation, butanol is themost potent inhibitor, and results obtained by Ounine et al.(1985) indicates that when glucose and xylose are used assubstrates in the C. acetobutylicum fermentation, growthinhibition on both substrates is correlated with the inhibitoryeffects of butanol on sugar transport. This phenomenon ismost pronounced when growth occurs on xylose rather thanon glucose. C. beijerinckii cells had enhanced glucoseutilization when the butanol concentration in the mediumwas 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 mediumeven though a large amount of glucose was fermented duringbatch and fed-batch fermentations (Ezeji et al. 2003, 2004a).

The cell membrane and the solventogenic switch Typically,in a batch-fermentation mode, the ABE fermentationproceeds in two phases: the first is called the acidogenicphase and is growth associated, and the second is asolventogenic phase characterized by the uptake of acids,ABE production, and is relatively non-growth associated.The shift in metabolic activity, which occurs when cellsswitch from the acid-producing phase to the solvent-producing phase, is accompanied by a corresponding shiftin cellular content of enzymes involved in acid- andsolvent-producing pathways (Yan et al. 1988). Organic acid

concentration increases as acids are produced during theacidogenic phase of ABE fermentation and decreases asacids are absorbed by the cells to produce ABE. Formationof organic acids during ABE fermentation, however, cannotbe avoided because the solventogenic clostridia obtain partof their metabolic energy (two ATP molecules from onemole of pyruvate) during organic acid formation (Fig. 1a).Excessive accumulation of undissociated acetic and butyricacid caused by poor buffering capacity of the fermentationmedium or addition of excess acetic and butyric acid tofermentation medium can be detrimental to solventogenicclostridia nutrient uptake and growth.

In cell membranes, proteins may be embedded in the outerlayer, the inner layer, or they may span the two layers asdepicted in Fig. 2. The individual phospholipids and proteinscan freely move around within the membrane layer similar tothat which would occur in a liquid phase, resulting in rapiddiffusion of molecules such as acetic and butyric acid in andout of microbial cells. The correlation between hydropho-bicity of aliphatic alcohols and inhibition of cellular growthand nutrient uptake suggests the membrane is the site ofinhibitory interactions (Linden and Moreira 1982). Moreiraet al. (1981) reported that addition of aliphatic alcohols to C.acetobutylicum ATCC 824 cultures resulted in an instanta-neous inhibition of membrane-bound ATPase activity.Membrane-bound ATPase is essential for maintenance ofinternal 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 undissociat-ed acetic and butyric acid across phospholipids bilayer of a Gram-positive (solventogenic clostridia) cell membrane. Acetic and butyricacid can destroy cell function by acidifying clostridia cells cytoplasmbelow the maximum pH of tolerance. Broken arrow and cross showthat dissociated acetic and butyric acid are non-membrane permeableand cannot freely diffuse across cell membranes (model is a modifiedform of Russell and Diez-Gonzalez (1997) model)

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increasing the activation energy of the enzyme and disrupt-ing phospholipids of the cell membrane (Bowles andEllefson 1985). The undissociated form of acetic and butyricacid are membrane permeable and can pass across bacterialcell membranes (Russell and Diez-Gonzalez 1998), unlikethe dissociated forms which are non-membrane permeable(Fig. 2). The intracellular acid concentration associated withthe solventogenic Clostridium cells is usually different fromthe measured concentration in the culture medium (Monot etal. 1984). Because butyric and acetic acid are produced andsecreted by solventogenic Clostridium cells in the undisso-ciated form (Terracciano and Kashket 1986), there will be aconcentration gradient across the cell membrane. Moreover,with growing clostridia, the internal pH is greater than theexternal pH due to a proton translocation process catalyzedby a membrane-bound ATPase (Booth and Morris 1975;Riebeling and Jugermann 1976; Herrero 1983; Monot et al.1984). When 104M N-N dicyclohexylcarbodiimide(DCCD), a known specific inhibitor of the membrane-bound ATPase, was added to the culture medium, DCCDcompletely inhibited the growth of C. acetobutylicum. Whena lesser concentration (105M) of DCCD was added at thebeginning of the growth phase, there was cellular growth, butmetabolic activity of the cell was modified due to inhibitionof the membrane-bound ATPase and decreased intracellularpH (Riebeling et al. 1975). Because butanol reduces theintracellular concentrations of ATP in solventogenic clostridia(Bowles and Ellefson 1985) and inhibits membrane-boundATPase, invariably intracellular pH maintenance and growthwill be negatively affected when there are greater concen-trations of butanol present.

Biochemistry of solvent production by solventogenicclostridia

Glucose is fermented via the EmdenMeyerhofParnaspathway to pyruvate (Fig. 1ac). Solventogenic clostridiaproduce two major types of products, solvents (ABE) andgases (carbon dioxide and hydrogen), and one major type offermentation intermediate product, organic acids (acetic andbutyric acid). Solventogenic clostridia are obligate anaerobesand generate only two net molecules of ATP duringglycolytic metabolism of glucose to pyruvate (Fig. 1ac).The acetic and butyric acid pathway reactions have importantroles in solventogenic clostridia metabolism because synthe-sis of these acids is accompanied by generation of ATP,which is important for cell growth and metabolism. Althoughgreater concentrations of total acetic acid (acetic acid fromP2 medium+acetic acid produced by C. beijerinckii BA101)than butyric acid are always measured in the bioreactorduring butanol fermentation by C. beijerinckii BA101 (Ezejiet al. 2003, 2005b, 2007a, b), more butyric acid than acetic

acid is produced both during the acidogenic (exponentialgrowth phase) and solventogenic phase. Butyric acid isquickly re-assimilated for butanol production during thesolventogenic phase. The formation of butyric acid duringthe acidogenic phase is important for maintenance of theredox equilibrium because nicotinamide adenine dinucleo-tides (NADHs) produced during glycolysis are only oxidizedin the butyric acid formation pathway, not in the acetic acidformation pathway, resulting in the regeneration of NAD+

(important for continuation of glycolysis and generation ofpyruvate by C. beijerinckii (Fig. 1a)). A mechanism thatinvolves metabolism of pyruvate for acetic and butyric acidproduction (Fig. 1a; two molecules of ATP produced) anduptake of acetic and butyric acid for ABE production(Fig. 1c) is energetically more attractive than a reactionscheme (no ATP produced) that results in direct productionof ABE (Fig. 1b). Under certain conditions, lactic acid maybe produced (Fig. 1a), and some of the two precursors ofbutyric acid, acetoacetyl-CoA, and butyryl-CoA are directlyconverted by solventogenic clostridia to the neutralproductsacetone and butanol, respectively (Fig. 1b). TheCoA moiety from acetoacetatyl-CoA is transferred to acetate.The resulting acetoacetate is decarboxylated to acetone by areaction catalyzed by acetoacetate decarboxylase (Fig. 1a, c).However, direct production of ABE from pyruvate occursbecause acids and small amounts of ABE are producedduring acidogenic phase growth of C. beijerinckii in batchfermentations (Ezeji et al. 2003; Shi and Blaschek 2008).Addition of reducing compounds such as viologen dyes orcarbon monoxide purging of the fermentation medium maycause metabolic shifts and result in a favorable carbon flowto butanol production instead of butyrate production (Meyeret al. 1986; Rao and Mutharasan 1987; Tashiro et al. 2007).Although part of the NADH produced by the EmdenMeyerhofParnas pathway is acted upon by NADH-ferredoxin reductase to produce reduced ferredoxin andhence molecular hydrogen (Peguin and Soucaille 1995), thereaction may shift from pyruvate to lactic acid formation tofacilitate rapid NADH and NAD+ regeneration (Fig. 1a). Areduced ferredoxin is the physiological electron donor ofhydrogenase in C. acetobutylicum, and bacterial-type ferre-doxins are low molecular weight carrier proteins containingtwo [4Fe4S] clusters involved in low-potential oxidation-reduction reactions (Guerrini et al. 2008; Quinkal et al.1994). Five open reading frames (ORFs) have beenannotated in the C. acetobutylicum genome for codingputative ferredoxins (Nolling et al. 2001), and ORFCAC0303 was shown to express the major ferredoxin inthe solventogenic C. acetobutylicum cells (Demuez et al.2007). Pyruvate oxidation to acetyl-CoA requires ferredoxin(Fd) reduction. Reduced Fd (FdH2) is oxidized by hydrog-enase (NADPH) or (NADH), which regenerates Fd, NAD+,or NADP+ and releases molecular hydrogen. The reaction is

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reversible, and depending on culture conditions, either FdH2or NADH may be formed. The reverse reaction (NADHformation from FdH2) is inhibited by NADH and proceedsonly after rapid removal of the NADH (Petitdemange et al.1976; Rao and Mutharasan 1987). Despite the critical role offerrodoxin reductase in the maintenance of a low redoxpotential during ABE fermentation in solventogenic clostrid-ia (Ezeji et al. 2007c), there is no information in the literaturethat describes how NADH-dependent or NADPH-dependentferrodoxin reductase is affected by presence of butanol andbutanol toxicity.

Autolysin production by the solventogenic clostridia andthe effect of butanol on clostridial cells during the solvento-genic phase of butanol fermentation is worthy of mention.Barber et al. (1979) reported the production of greaterconcentrations of autolysin toward the end of the exponentialgrowth phase of C. acetobutylicum (onset of solvento-genesis) and was accompanied by lysis of culture cells anddecrease in ABE-producing cells. This study was the firstwhere direct linkage of autolytic degradation in solvent-producing cells of C. acetobutylicum and presence of butanolduring growth was established, and that inhibitory concen-trations of butanol were involved in the induction of releaseof cell-free autolysin during the solventogenic phase (Barberet al. 1979; Jones and Woods 1986). Van Der Westhuizen etal. (1982) reported the effects of acetone and butanol on thegrowth and stability of swollen-phase bright-stationary-phasecells (clostridial forms) of C. acetobutylicum P262 and anautolytic-deficient mutant (lyt-1) strain where it was deter-mined that butanol concentrations between 7 and 16 g/L,which are within the range obtained in ABE fermentations,increased butanol-induced degeneration of strain P262clostridial forms but had no effect on the stability ofautolytic-deficient mutants that never underwent autolysis.The conclusion was that there is a relationship betweenbutanol tolerance and autolytic activity. Although theautolytic-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 produceABE when greater butanol concentrations existed wascompromised. Because greater concentrations of butanolinhibited butanol production by an autolytic-deficient mutant(lyt-1) but not growth of the mutant strain, examination ofglobal gene expression in lyt-1 alongside enzyme kineticsstudies 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 tolerancein acetone and butanol-producing microorganisms

The major challenge with the ABE fermentation is that itsuffers from a number of limitations (e.g., low concentra-

tion, yield, and productivity) due to butanol stress andtoxicity to the microbial cells (Ezeji et al. 2004a, b).Researchers have made several parallel observations be-tween butanol stress and other forms of stress that can beimposed on the microbial cell. Heat shock stress is probablythe most examined form of stress and results in therepression of synthesis of many cellular proteins, while aspecific set of about 20 heat shock proteins (hsps) areinduced in response to temperature increases. Theseproteins are involved in the cellular response and adaptationto stress, as many of these proteins are also induced byexposure of bacterial cells to other forms of stresses,including alcohols (Michel and Starka 1986; Terraccianoet al. 1988). Solventogenic clostridia respond to theperturbing effect of butanol as a result of expression ofseveral heat shock protein genes and modifying the fattyacid (saturation) composition of membrane lipids. Expres-sion of stress proteins was enhanced in C. acetobutylicumby increasing concentration of butanol by as little as 11 mM(about 6% of the total butanol produced by the cells) duringthe acidogenic phase (Terracciano et al. 1988). The de novosynthesis of similar proteins during the solventogenic phaseand in the absence of an added stress suggests a connectionbetween the stress response and solventogenesis (Terraccianoet al. 1988).

The toxicity of alcohols appears to increase with chainlength, with long-chain alcohols being more toxic at alesser concentration than short-chain alcohols (Lepage et al.1987). Alcohol toxicity to microbial cells has beensuggested to occur as a result of damage to the cellmembrane (Osman and Ingram 1985) and direct inhibitionof metabolism (Herrero et al. 1985).

Vollherbst-Schneck et al. (1984), during their evaluationof the effect of butanol on lipid composition and fluidity ofC. acetobutylicum ATCC824, found that when grown in thepresence of butanol, C. acetobutylicum synthesized greateramounts of saturated acyl chains of fatty acids at theexpense of unsaturated chains. The presence of addedbutanol affected the cellular acyl fatty acids chain compo-sition in the same way as did growth at the peak (stationaryphase) of butanol production (Vollherbst-Schneck et al.1984). This increase in saturation of cellular lipids mayhave occurred in order to compensate for the increase influidity of cell membranes that is induced by butanol (Baeret al. 1987). Increase in cell fluidity facilitates leakage ofcellular contents and subsequently cell death.

The observed membrane change makes it the principaltarget for cellular adaptation to potentially toxic amounts ofextracellular alcohol (Taylor et al. 2008). This assertion washighlighted in 1976 by Ingram, who reported that themembrane fatty acid composition of E. coli K-12 wasradically altered when the strain was grown in the presenceof butanol and other alcohols. The proportion of longer-

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chain (18:1) fatty acids was found to increase at theexpense of relatively shorter-chain (

butanol tolerance. This is noteworthy because it indicatesthe production of butanol is not triggered by, or directlycorrelated to, butanol concentration or tolerance limits.Strain pJC4BK(pTAAD) did not produce increased levelsof butanol relative to pJC4BK, indicating that butanolproduction is not limited by alcohol dehydrogenase activity.However, pJC4BK(pTAAD) did produce increasedamounts of ethanol as well, corresponding to a total alcoholtolerance of 21.2 g/L. Both ethanol and butanol arehypothesized to disrupt the cell membrane; however, theincreased alcohol toxicity threshold with no specificselection for alcohol tolerant strains suggests that this maynot be the primary effect (Green et al. 1996; Harris et al.2000). Another study by Harris et al. (2001) againinvestigated the effect of overexpression of alcohol dehy-drogenase in SolRH(ptAAD) compared to the engineeredstrain SolRH, both lacking the solvent formation repressorSolR. SolRH achieved final solvent concentrations whichwere 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 insolvent tolerance in connection with increased solventproduction without specific selection.

In 2003, a C. acetobutylicum strain 824(pGROE1) wasproduced by overexpressing genes in the class I stressresponse operon groESL (Tomas et al. 2003). This straindemonstrated 85% less growth inhibition from butanol thanthe control strain, resulting in 17.1 g/L butanol and 8.6 g/Lacetone. Furthermore, overexpressing groESL resulted inlonger active metabolism, increased expression of motilityand chemotaxis genes, and decreased expression of majorstress response genes. A follow-up study used DNA array-based 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 todifferentiate genes that are likely associated with generalbutanol stress response and those that are associated withincreased butanol tolerance (Tomas et al. 2004). Geneticchanges found to relate to general butanol stress responseincluded the overexpression of major stress protein genes,solvent and butyrate formation genes, and the butyryl-coenzyme A biosynthesis operon genes, as well asdecreased expression of the fatty acid synthesis operon,glycolytic genes and sporulation genes. Genes suspected tobe specifically involved in increased butanol toleranceinclude rlpA, artP, and a hemin permease encoding gene.A second transcriptional analysis study compared theexpression patterns of 824(pMSPOA)a strain with over-expression of the sporulation gene spo0Awith that of thespo0A knock-out strain SKO1 and a plasmid control(Alsaker et al. 2004). As with the previous study, mostdifferentially expressed genes were related to a generalstress response. The 824(pMSPOA) strain demonstrated

increased tolerance and prolonged metabolism when sub-jected to butanol stress, and genes associated with imme-diate butanol stress responsehence likely contributing toincreased tolerancewere identified. When 824(pMSPOA)was compared to 824(pGROE1), 160 total differentiallyexpressed genes were identified under butanol challengeconditions. The main difference seen between the twostrains was the time (during fermentation) at which genesshowed high expressionin one strain, increased expressionwas seen early on, but in the other, the increased expressionhad a more delayed response. Counterintuitively, bothstudies found that butanol stress appeared to overexpresssolvent formation genes and underexpress fatty acidsynthesis genes.

Borden and Papoutsakis expanded the search for genesto target C. acetobutylicum's ability to withstand greatersolvent concentrations using a genomic library. Plasmidswere inserted into wild type C. acetobutylicum cells viaelectroporation, and the cells were challenged with variousamounts of butanol (Borden and Papoutsakis 2007).Sixteen genes were identified as contributing to the cellsability to withstand greater concentrations of butanol;pCAC1869 in particular showed a 45% increase intolerance. Similarly, pCAC0003 was found to have a 24%increase in butanol tolerance. CAC1869 is suspected to be atranscriptional regulator (KEGG) and was found to havemaximal transcription preceding induction of the solvento-genic genesaad, ctfA, and ctfB. This gene is activelytranscribed throughout the transitional phase.

C. beijerinckii NCIMB 8052 C. beijerinckii is phenotypi-cally quite similar to C. acetobutylicum, but its genome(which is 50% larger than C. acetobutylicum) was recentlysequenced, and genetic strain modification efforts are notyet as advanced. However, in 1991, direct acting N-methyl-N-nitro-N-nitrosoguanidine was used as a mutagen to createthe C. beijerinckii mutant BA101capable of producinggreater amounts of solvent than any C. acetobutylicumstrain engineered thus far (Qureshi and Blaschek 2001;Chen and Blaschek 1999). C. beijerinckii BA101 is bothstable and has hyper-amylolytic and hyper-butanologeniccharacteristics (Annous and Blaschek 1991). When grownon semi-defined P2 medium and in batch fermentation, C.beijerinckii BA101 produces up to 19 g/L butanol and atotal solvent concentration of 29 g/L, over 100% improve-ment when compared to the wild type C. beijerinckiiNCIMB 8052. Even though specific selection for solventtolerance was not conducted, C. beijerinckii BA101exhibits increased tolerance to butanol, with 100% cellinhibition occurring at 23 g/L butanol rather than 11 g/Lcharacteristic of C. beijerinckii NCIMB 8052 (Qureshi andBlaschek 2001). The exact mechanism for the observedincrease in tolerance is unknown.

1704 Appl Microbiol Biotechnol (2010) 85:16971712

Engineered microorganisms for ABE production andresistance With the history of bio-butanol production bythe natural solvent-producing clostridia, there are someclear inherent advantages in improving and using thesenative clostridial systems for producing bio-butanol. How-ever, there are several challenges to using these bacteriarelative to model fermentation organisms such as E. coliand S. cerevisiae: (1) fewer tools are available for geneticmanipulation of Clostridium metabolism, (2) clostridia haveslower growth rates, and (3) clostridia are obligate anae-robes. For these reasons, an alternative model for econom-ical production of solvents using well-characterized hostssuch as S. cerevisiae and E. coli for ABE production hasbeen investigated.

E. coli The first efforts towards using E. coli to producesolvents was the expression of four C. acetobutylicumATCC 824 genes (adc, ctfA, ctfB, and thl) in E. coli forenhanced acetone production (Bermejo et al. 1998). StrainATCC 11303 (pACT) produced 5.4 g/L acetone whengrown in glucose-fed shake flasksa concentration com-parable to wild type C. acetobutylicum. Acetone's greatvolatility limits toxic effects, and thus tolerance was notfound to be a concern (Bermejo et al. 1998). With theaddition of a secondary alcohol dehydrogenase and subse-quent strain optimization, this acetone-producing strain wasadapted to produce 4.9 g/L of isopropanol (Hanai et al.2007). E. coli has also been engineered to produce 1-butanol using the pathway from C. acetobutylicum (con-taining genes thl, hbd, crt, bcd, etfAB, and adhE2; Atsumiet al. 2008a). The engineered strain produced 13.9 mg/Lbutanol, and by overexpressing E. coli's acetyl-CoAacetyltransferase instead of the clostridia acetoacetyl-CoAthiolase, butanol concentration was improved. By deletingpathways competing for acetyl-CoA and NADH (ldhA,adhE, and frdBC) as well as genes to increase pyruvatedehydrogenase activity and decrease acetate production (fnrand pta), butanol production reached 373 mg/L (strainJCL88); this value was increased to 552 mg/L when grownin rich media. Nielsen et al. (2009) later increasedproduction to 580 mg/L using individual expression ofpathway genes alongside co-expression of fdh1 from yeast(for cofactor regeneration) and overexpression of the gapAgene from E. coli to increase glycolytic flux and the acetyl-CoA pool. Without specific selection for butanol toleranceor engineering for increased toxicity thresholds, E. colitolerated up to 1.5% butanola value competitive withclostridia (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 higheralcohols (e.g., 1-butanol) that avoids CoA-mediated chem-istry used by native solvent-producing organisms. By

utilizing E. coli's amino acid biosynthesis pathway, iso-butanol (Atsumi et al. 2008b; Atsumi and Liao 2008a), 1-butanol (Atsumi and Liao 2008b), 2-methyl-1-butanol(Atsumi et al. 2008a; Atsumi and Liao 2008a; Cann andLiao 2008), 3-methyl-1-butanol (Atsumi et al. 2008b;Atsumi and Liao 2008b; Connor and Liao 2008), and 2phenylethanol (Atsumi et al. 2008a; Atsumi and Liao2008a) are made from the valine, norvaline, isoleucine,leucine, and phenylalanine biosynthesis pathways, respec-tively. By diverting 2-keto acid intermediates to alcoholsynthesis, this study demonstrated up to 22 g/L ofisobutanol (Atsumi et al. 2008b). To achieve this, onlygenes for two additional enzymes (2-keto acid decarboxyl-ase and alcohol dehydrogenase) were needed. To maximizethe production of each product in turn, genes to enhance theintermediate 2-keto acid of interest were overexpressed, andgenes corresponding to competing reactions were deleted.In some instances, native E. coli genes were replaced withmore active genes from other hosts. Native 1-butanol-producing organisms (clostridia) tolerate up to 2% w/v ofthe product, and E. coli was found to initially be intolerantto 1.5% w/v isobutanol (a less toxic solvent). Afterperforming serial transfers to enhance tolerance, the E. colistrain survived in up to 2% w/v isobutanol.

Later, the initial isobutanol response network of E. coliwas characterized using gene expression, transcriptionfactor-gene interaction data, gene knockouts, and networkcomponent analysis (Brynildsen and Liao 2009). Anadditional comparison showed similar response networksfor both isobutanol and n-butanol. Notably, this study alsofound that isobutanol stress leads to the disruption ofimportant membrane components (most importantly, qui-nones) that subsequently affected respiratory, phosphate,and iron control. Network component analysis determined67 transcription factors to be active as a result of isobutanolstress, 16 (relating to stress mitigation, metabolism regula-tion, and nucleoproteins) of which were significantlyperturbed. Most significantly perturbed were transcriptionfactors effecting respiration (in particular, the transcriptionfactors ARcA, PdhR, and FNR), and further verification ofthis supported the assumption that solvent toxicity leads tocell membrane malfunction. Analysis of the responsenetworks showed that conditions affecting isobutanolresistance apply to n-butanol as well, except that 1-butanol had a greater repression effect on amino acidsynthesis in microorganisms than did isobutanol. A deletionstudy removing arcA, fur, and phoB did not significantlyincrease tolerance, supporting the hypothesis that toleranceresponse is a complex result of multiple mutations.Previous studies with C. acetobutylicum, which showedthat stress by increased 1-butanol concentrations elicits aresponse similar to heat shock proteins, were supported bythe network analysis showing that E. coli displayed greatly

Appl Microbiol Biotechnol (2010) 85:16971712 1705

enhanced activation of a heat shock sigma factor inresponse to isobutanol.

S. cerevisiae S. cerevisiae naturally produces small amountsof isobutanol and 3-methyl-1-butanol as byproducts offermentation, giving it a basal ability to resist solventtoxicity. Recently, S. cerevisiae was engineered to produce2.5 mg/L of n-butanol through the cloning of the 1-butanolpathway and various isozymes chosen from C. beijerinckii,E. coli, and Ralstonia eutropha into S. cerevisiae (Steen etal. 2008)with C. beijerinckii genes showing the bestresults. This amount of solvent production, however, is stillfar less than concentrations achieved by the most effectiveClostridium strains for butanol production (approximately20 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 thatonly 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 tobutanol found that butanol inhibits translation at theinitiation step (Ashe et al. 2001).

Solvent-resistant microorganisms as potentialacetone-butanol production hosts

As an alternative approach, future efforts to geneticallyengineer microorganisms for increased tolerance to butanolmay focus on investigations of host microorganisms thathave a natural resistance to solvent but that have notdemonstrated significant solvent formation. This approachwas reported recently by investigators who screened 24hosts, spanning seven genera, with the aim of identifyingmicroorganisms that possess great butanol tolerance capa-bility (Knoshaug and Zhang 2009). Of the yeast strains, S.cerevisiae ATCC 26602 and ATCC 20252, as well asCandida sonorensis, were able to grow on 2% of 1-butanol.In all cases, growth was only 10% to 20% that of thecontrol (without butanol challenge) cultures. Of the non-yeast species, Lactobacillus delbrueckii and Lactobacillusbrevis grew on 2% butanol with relative growth rates of55% 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) foundtolerance to be dependent on temperature: at highertemperatures, strains grew faster but had lower butanoltolerance thresholds, while strains at lower temperaturehad increased butanol tolerance thresholds but grewslower.

A separate study investigated the butanol resistance ofthree solvent-tolerant and one solvent-sensitive P. putidastrains (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 wasperformed to investigate the strain response to butanolrelative to the untreated strain. For untreated strains, glucoseuptake rate increased while growth decreasedwithout acorresponding increase in byproductsand corresponded toa redistribution of intracellular flux. Specifically, carbon wasredirected to the tricarboxylic acid (TCA) cycle, whichincreased regeneration rates of redox cofactors. The authorstherefore hypothesized that butanol leads to an increasedneed for cell maintenance energy. The solvent-tolerant strainappeared to have an adapted cell membrane to lower cellmaintenance needsdemonstrating reduced glucose uptake,TCA cycle usage, and redox cofactor regeneration rates.Recently, Nielsen et al. (2009) explored the productionability of two butanol-tolerant organisms, including twostrains of P. putida, using polycistronic expression of butanolbiosynthetic genes. The two P. putida strains produced 44and 50 mg/L butanol, respectively, when grown on glucoseand 122 and 112 mg/L butanol, respectively, when grown onglycerol. The study by Ruhl et al. showing tolerancethresholds of up to 6% butanol, however, suggests potentialfor increasing production in the solvent-producing strain.

Advanced fermentation techniques and downstreamprocessing

To realize the full potential of the altered candidate genes withrespect to butanol concentration, tolerance, and productivity,developed strains may be used for simultaneous ABEfermentation and recovery systems to overcome low solvent(ABE) resistance of these strains. Various alternative in situbutanol removal techniques including membrane-based sys-tems, 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), liquidliquid extraction (Evans and Wang 1988), and gas stripping(Ezeji et al. 2004b, 2005a) have been examined. A summaryof in situ butanol removal techniques and limitations isprovided in Table 1. Although there has been significantprogress in integration of fermentation with product recoverytechniques, there are few commercial applications of thesetechnologies. To advance butanol fermentation from labora-tory scale production to industrial scale, the integratedprocess must be microbial friendly, scalable, non-fouling,and enhance butanol productivity.

Liquidliquid extraction

Liquidliquid extraction is a technique used to reducesolvent (ABE) toxicity of solventogenic clostridia. In thisprocess, a water-insoluble organic extractant is mixed with

1706 Appl Microbiol Biotechnol (2010) 85:16971712

the fermentation broth. Butanol is more soluble in theorganic (extractant) phase than in the aqueous (fermentationbroth) phase; therefore, butanol selectively concentrates inthe organic phase (Ezeji et al. 2007c). ABE are concentrat-ed in the extraction solvent and recovered by back-extraction into another extraction solvent or by distillation(Maddox 1989). The extractant of choice for in situ ABErecovery among researchers has been oleyl alcohol becauseit is relatively non-toxic as well as being a high qualityextractant (Evans and Wang 1988; Ezeji et al. 2006;Karcher et al. 2005). Dibutyl phthalate, an importantextraction solvent, was found to be non-toxic to C.beijerinckii BA101 during butanol fermentation and recov-ery by liquidliquid extraction (Karcher et al. 2005). In adetailed investigation conducted by Roffler et al. (1987),six solvents or solvent mixtures were tested in batchextractive fermentations: kerosene, 30 wt.% tetradecanolin 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 mixtureof oleyl alcohol and benzyl benzoate provided the mostdesirable results and improved volumetric butanol produc-tivity by as much as 60%. Common extractants, toxicitydata, and partition coefficients used in ethanol and butanolfermentation are published elsewhere (Karcher et al. 2005).

There are some challenges associated with a liquidliquid extraction system including extraction solvent toxic-

ity to butanol-producing cells, formation of an emulsion,loss of extraction solvent, and the accumulation ofmicrobial cells at the extractant and fermentation brothinterphase (formation of rag layer; Ezeji et al. 2007c).

Perstraction

To avoid the toxicity problem brought about by theextraction solvent, investigators have used perstractivefermentation and recovery that employs a membranecontactor (Roffler et al. 1987; Traxler et al. 1985; Qureshiet al. 1992). Perstraction is an extractive fermentationprocess designed to reduce extraction solvent toxicity andbutanol toxicity to the producing culture, while improvingbutanol concentration, productivity, and selectivity. Themembrane contactor in perstractive process provides sur-face area where the two immiscible phases can exchangethe butanol. The total mass transport of butanol from thefermentation broth to the organic side depends on the rateof diffusion of butanol across the membrane (Ezeji et al.2007c). Because there is no direct contact between the twophases, extractant toxicity, phase dispersion, emulsion, andrag layer formation are drastically reduced or eliminated(Ezeji et al. 2006).

The major drawback of ABE fermentation and recoveryby perstraction is fouling of the membrane (Ezeji and Li2009). Even without membrane fouling, the membrane

Table 1 Summary of the integrated butanol fermentation and in situ butanol removal techniques

Process Relievesbutanol toxicity

Increasesyield

Increasesproductivity

Limitations

Adsorptiona,b Yes No Yes Loss of nutrients to adsorbent, clogging, loss of fermentation(acetic and butyric acid) intermediate products

Gas strippingc,d Yes Yes Yes Low butanol stripping rate

Liquidliquid extractione Yes No Yes Extractant toxicity to cells, formation of rag layer, emulsion,loss of fermentation intermediate products to extractant

Perstractionf Yes No Yes Loss of fermentation intermediate product to extractant,expensive to operate, membrane fouling, and lack simplicity

Pervaporationf,g,h,i Yes No Yes Loss of fermentation intermediate products to extractant dueto diffusion across membrane, membrane fouling

Reverse osmosisj Yes No Yes Fouling, loss of nutrients, and lack simplicity

a Ennis et al. (1987)b Nielson et al. (1988)c Ezeji et al. (2004b)d Ezeji et al. (2005a)e Evans and Wang (1988)f Qureshi et al. (1992)g Groot et al. (1984)h Qureshi and Blaschek (1999)i Qureshi and Blaschek (2000)j Garcia et al. (1986)

Appl Microbiol Biotechnol (2010) 85:16971712 1707

does, however, present a physical barrier that can limit therate of butanol extraction. In addition, the concentration ofmineral salts in the aqueous phase, brought about by saltaccumulation due to long fermentation times (alkali frommedia feed that reacts with acids or other components) canlead to cessation of fermentation.

Gas stripping

Gas stripping is a simple technique that can be integratedwith ABE fermentation to simultaneously recover ABEduring fermentation. This technique is useful for keepingbutanol concentration in the bioreactor below the thresholdof butanol toxicity to the culture and allowing fermentationto go on unimpeded until all the sugars present in thebioreactor are utilized and converted to ABE (Ezeji et al.2003, 2004a). Fermentation and recovery by gas strippinginvolves bubbling oxygen free nitrogen or fermentationgases (CO2 and H2) through the fermentation brothfollowed by cooling the enriched gas (or gases) with ABEin a condenser (Ezeji et al. 2003, 2004a, 2006). As the gasis bubbled through the fermentor, it captures ABE, which iscondensed in the condenser followed by collection in areceiver. After the solvents are condensed, the gas isrecycled back to the fermentor to capture more ABE asdepicted in Fig. 3. Application of gas stripping to batch andfed-batch butanol fermentation systems have been reviewedelsewhere (Ezeji et al. 2004b, 2006, 2007c).

Pervaporation

Pervaporation is a technique which allows selectiveremoval of volatiles from broths during fermentation usinga membrane. A pervaporation system is generally com-posed of feed pump, membrane module, condenser, and

vacuum pump (Fig. 4). This technique has been usedextensively to keep butanol concentration in the bioreactorbelow the threshold of butanol toxicity to solventogenicclostridia during ABE fermentation (Qureshi et al. 1992;Ezeji et al. 2004b, 2006, 2007c). The volatile or organiccomponent diffuses through the membrane as a vaporfollowed by recovery by condensation. Membranes used inpervaporation are either hydrophilic or hydrophobic. Thereare three steps involved in mass transfer of permeates inpervaporation: (1) adsorption of solvents into upstreamsurface of membrane, (2) diffusion of dissolved solventsthrough the membrane, and (3) absorption of dissolvedsolvents into permeate vapor at the downstream surface ofmembrane (Shao and Huang 2007). Generally, a vacuum oran inert sweep gas such as N2 are applied on the permeateside of the membrane to maintain a partial pressuredifference across the membrane which facilitates volatili-zation of permeates for subsequent condensation andrecovery (Fig. 4). The current benchmark pervaporationmembrane material is polydimethylsiloxane (PDMS). Thereported ethanolwater separation factor for PDMS mem-branes ranged from 4.4 to 10.8, while the reported butanolwater 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 membraneshave also been studied for ethanolwater or butanol waterseparation in a pervaporation system (Ezeji and Li 2009).The ethanolwater separation factors are largely ranked inthe following order: PDMS

Concluding remarks

Solvent production by microorganismsin particular, theproduction of butanolholds great interest as a means forgenerating sustainable transportation fuel and commoditychemicals. Current research efforts seek to better understandthe effects of solvent toxicity on the metabolism of micro-organisms, as well as ameliorate tolerance limitations throughstrain design and product recovery techniques. The solvento-genic clostridia are commonly studied organisms that producesolvents (ABE) from a variety of carbon sources using a two-phase fermentation process (acidogenesis and solventogene-sis). Butanol produced during solventogenesis in clostridia hasbeen shown to affect the PTS sugar transport system, iontransport, and the integrity of the cell membrane. Themetabolicchanges that occur in the presence of butanol have beenimplicated 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-expressionof genes related to solvent stress in organisms of interest, aswell broader approaches including the use of mutagens tocreate C. beijerinckii BA101. More recently, research effortsin ABE production have turned towards both industriallywell-characterized organisms (e.g., E. coli and S. cerevisiae)and organisms with known tolerance robustness to varioussolvents (e.g., P. putida). These strategies often make use ofnatural ABE production pathways from clostridia, thoughthey have not yet been able to match the production levels ofthese native ABE-producing microorganisms. In situ recoverytechniques such as liquidliquid extraction, perstraction, gasstripping, and pervaporation are being used alongside straindesign to maintain a low enough solvent concentration duringfermentation to sustain cell growth and product formation.While significant progress has been made towards improvingtolerance and increasing ABE yield, it is clear that toxicity isnot the only factor limiting ABE production. Furtheradvancements must be achieved in order to make the butanolproduction process economically competitive and deliver onthis highly promising avenue for biofuel production.

Acknowledgements This work was supported by funding fromNortheast Sungrant (Cornell University) Award/Contract numberGRT00012344, National Research Initiative of the USDA CooperativeState Research, Education and Extension Service, grant number 2006-35504-17419, NSF CAREER award (NDP) to Nathan Price, and Seedgrant from Ohio Agricultural Research and Development Center(OARDC), Wooster.

References

Aguilera F, Peinado RA, Millan C, Ortega JM, Mauricio JC (2006)Relationship between ethanol tolerance, H+-ATPase activity and thelipid composition of the plasma membrane in different wine yeaststrains. Int J Food Microbiol 110:3442

Alsaker KV, Spitzer TR, Papoutsakis ET (2004) Transcriptionalanalysis of spo0A over expression in Clostridium acetobutylicumand its effect on the cell's response to butanol stress. J Bacteriol186:19591971

Annous BA, Blaschek HP (1991) Isolation and characterization ofClostridium acetobutylicum mutants with enhanced amylolyticactivity. Appl Environ Microbiol 57:25442548

Ashe MP, Slaven JW, De Long SK, Ibrahimo S, Sachs AB (2001) Anovel eIF2B-dependent mechanism of translational control inyeast as a response to fuel alcohols. EMBO J 20:64646474

Atsumi S, Liao JC (2008a) Directed evolution of Methanococcusjannaschii citramalate synthase for biosynthesis of 1-propanol and1-butanol by Escherichia coli. Appl Environ Microbiol 74:78027808

Atsumi S, Liao JC (2008b) Metabolic engineering for advancedbiofuels production from Escherichia coli. Curr Opin Biotechnol19:414419

Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP,Chou KJY, Hanai T, Liao JC (2008a) Metabolic engineering ofEscherichia coli for 1-butanol production. Metab Eng 10:305311

Atsumi S, Hanai T, Liao JC (2008b) Non-fermentative pathways forsynthesis of branched-chain higher alcohols as biofuels. Nature451:8689

Baer SH, Blaschek HP, Smith TL (1987) Effect of butanol challengeand temperature on lipid composition and membrane fluidity ofbutanol-tolerant Clostridium acetobutylicum. Appl EnvironMicrobiol 53:28542861

Barber JM, Robb FT, Webster JR, Woods DR (1979) Bacteriocinproduction by Clostridium acetobutylicum in an industrialfermentation process. Appl Environ Microbiol 37:433437

Bermejo LL, Welker NE, Papoutsakis ET (1998) Expression ofClostridium acetobutylicum ATCC 824 genes in Escherichia colifor acetone production and acetate detoxification. Appl EnvironMicrobiol 64:10791085

Booth I, Morris JG (1975) Proton motive force in the obligatelyanaerobic bacterium Clostridium pasteurianum- a role in galac-tose and gluconate uptake. FEBS Letters 59:153157

Borden JR, Papoutsakis ET (2007) Dynamics of genomic-libraryenrichment and identification of solvent tolerance genes forClostridium acetobutylicum. Appl EnvironMicrobiol 73:30613068

Bowles LK, Ellefson WL (1985) Effects of butanol on Clostridiumacetobutylicum. Appl Environ Microbiol 50:11651170

Brynildsen MP, Liao JC (2009) An integrated network approachidentifies the isobutanol response network of Escherichia coli.Mol Syst Biol 5:277

Cann AF, Liao JC (2008) Production of 2-methyl-1-butanol inengineered Escherichia coli. Appl Microbiol Biotechnol 81:8998

Chen CK, Blaschek HP (1999) Acetate enhances solvent productionand prevents degeneration in Clostridium beijerinckii BA101.Appl Microbiol Biotechnol 52:170173

ConnorMR, Liao JC (2008) Engineering of anEscherichia coli strain forthe production of 3-methyl-1-butanol. Appl Environ Microbiol74:57695775

Demuez M, Cournac L, Guerrini O, Soucaille P, Girbal L (2007)Complete activity profile of Clostridium acetobutylicum [FeFe]-hydrogenase and kinetic parameters for endogenous redoxpartners. FEMS Microbiol Lett 275:113121

Durre P (2008) Fermentative butanol production: bulk chemical andbiofuel. Ann NY Acad Sci 1125:353362

Ennis BM, Qureshi N, Maddox IS (1987) Inline toxic product removalduring solvent production by continuous fermentation usingimmobilized Clostridium acetobutylicum. Enzyme Microb Technol9:672675

Evans PJ, Wang HW (1988) Enhancement of butanol fermentation byClostridium acetobutylicum in the presence of decanol-oleylalcohol mixed extractants. Appl Environ Microbiol 54:16621667

Appl Microbiol Biotechnol (2010) 85:16971712 1709

Ezeji TC, Blaschek HP (2007) Biofuel from butanol: advances ingenetic and physiological manipulation of clostridia. BioWorldEurope 2:1215

Ezeji TC, Li Y (2009) Advanced product recovery technologies. In:Vertes A, Qureshi N, Yukawa H, Blaschek H (eds) Biomass tobiofuel. Wiley, Hoboken, in press

Ezeji TC, Qureshi N, Blaschek (2003) Production of butanol byClostridium beijerinckii BA101 and in-situ recovery by gasstripping. World J Microbiol Biotechnol 19:595603

Ezeji TC, Qureshi N, Blaschek HP (2004a) Acetone-butanol-ethanol(ABE) production from concentrated substrate: reduction insubstrate inhibition by fed-batch technique and product inhibitionby gas stripping. Appl Microbiol Biotechnol 63:653658

Ezeji TC, Qureshi N, Blaschek HP (2004b) Butanol fermentationresearch: upstream and downstream manipulations. Chem Rec4:305314

Ezeji TC, Qureshi N, Karcher PM, Blaschek HP (2005a) Improvingthe performance of a gas stripping-based recovery system toremove butanol from Clostridium beijerinckii fermentation.Bioprocess Biosystems Eng 27:207214

Ezeji TC, Qureshi N, Blaschek HP (2005b) Continuous butanolfermentation and feed starch retrogradation: butanol fermentationsustainability using Clostridium beijerinckii BA101. J Biotechnol115:179187

Ezeji TC, Qureshi N, Karcher P, Blaschek HP (2006) Butanolproduction from corn. In: Minteer SD (ed) Chapter in AlcoholicFuels: Fuels for Today and Tomorrow. Taylor & Francis (Taylor& Francis Group), New York, pp 99122

Ezeji TC, Qureshi N, Blaschek HP (2007a) Butanol production fromagricultural residues: impact of degradation products on Clos-tridium beijerinckii growth and butanol fermentation. BiotechnolBioeng 97:14601469

Ezeji TC, Qureshi N, Blaschek HP (2007b) Production of acetone butanolethanol (ABE) in a continuous flow bioreactor using degermed cornand Clostridium beijerinckii. Process Biochem 42:3439

Ezeji TC, Qureshi N, Blaschek HP (2007c) Bioproduction of butanolfrom biomass: from genes to bioreactors. Curr Opin Biotechnol18:220227

Fischer CR, Klein-Marcuschamer D, Stephanopoulos G (2008)Selection and optimization of microbial hosts for biofuelsproduction. Metab Eng 10:295304

Garcia A, Iannotti EL, Fischer JL (1986) Butanol fermentation liquorproduction and separation by reverse osmosis. Biotechnol Bioeng28:785791

Green EM, Boynton ZL, Harris LM, Ruldolph FB, Papoutsakis ET,Bennett GN (1996) Genetic manipulation of acid formationpathways by gene inactivation in Clostridium acetobutylicumATCC 824. Microbiology 142:20792086

Groot WJ, van den Oever CE, Kossen NWF (1984) Pervaporation forsimultaneous product recovery in the butanol/isopropanol batchfermentation. Biotechnol Lett 6:709714

Guerrini O, Burlat B, Lger C, Guigliarelli B, Soucaille P, Girbal L(2008) Characterization of Two 2[4Fe4S] Ferredoxins fromClostridium acetobutylicum. Curr Microbiol 56:261267

Hanai T, Atsumi S, Liao JC (2007) Engineered synthetic pathway forisopropanol production in Escherichia coli. Appl Environ Micro-biol 73:78147818

Harris LM, Desai RP, Welker NE, Papoutsakis ET (2000) Characteriza-tion of recombinant strains of the Clostridium acetobutylicumbutyrate kinase inactivation mutant: need for new phenomenolog-ical models for solventogenesis and butanol inhibition. BiotechnolBioeng 67:111

Harris LM, Blank L, Desai RP, Welker NE, Papoutsakis ET (2001)Fermentation characterization and flux analysis of recombinantstrains of Clostridium acetobutylicum with an inactivated solRgene. J Ind Microbiol Biotechnol 27:322328

Hermann M, Fayolle F, Marchal R, Podvin L, Sebald M, VandecasteeleJP (1985) Isolation and characterization of butanol-resistant mutantsof Clostridium acetobutylicum. Appl Environ Microbiol 50:12381243

Herrero AA (1983) End-product inhibition in anaerobic fermentation.Trends Biotechnol 1:4953

Herrero AA, Gomez RF, Roberts MF (1985) 3lP NMR studies ofClostridium thermocellum. Mechanism of end product inhibitionby ethanol. J Biol Chem 260:74427451

Hu XH, Wang MH, Tan T, Li JR, Yang H, Leach L, Zhang RM, LuoZW (2007) Genetic dissection of ethanol tolerance in the buddingyeast Saccharomyces cervisiae. Genetics 175:14791487

Huang H.-J, Ramaswamy S, Tschirner UW, Ramarao BV (2008) Areview of separation technologies in current and future biorefi-neries. Separations Purifications Technol 62:121

Hutkins RW, Kashket ER (1986) Phosphotransferase activity inClostridium acetobutylicum from acedogenic and solvetogenicphases of growth. Appl Environ Microbiol 51:11211123

Ingram LO (1976) Adaptation of membrane lipids to alcohols. JBacteriol 125:670678

Ingram LO (1990) Ethanol tolerance in bacteria. Crit Rev Biotechnol9:305319

Jones DT, Woods DR (1986) Acetone-butanol fermentation revisited.Microbiol Rev 50:484524

Karcher P, Ezeji TC, Qureshi N, Blaschek HP (2005) Microbialproduction of butanol: product recovery by extraction. In:Satyanarayana T, Johri BN (eds) Chapter in Microbial Diversity:current perspectives and potential applications. IK InternationalPublishing House Pvt Ltd, New Delhi, pp 865880

Knoshaug EP, Zhang M (2009) Butanol tolerance in a selection ofmicroorganisms. Appl Biochem Biotechnol 153(13):1320

Lee J, Mitchell WJ, Blashek HP (2001) Glucose uptake in Clostridiumbeijerinkii NIMB 8052 and the solvent-hyperproducing mutantBA101. Appl Environ Microbiol 67:50255031

Lee J, Mitchell WJ, Tangney M, Blaschek HP (2005) Evidence forthe presence of an alternative glucose transport system inClostridium beijerinckii NCIMB 8052 and the solvent-hyperproducing mutant BA101. Appl Environ Microbiol71:33843387

Lepage C, Fayolle F, Hermann M, Vandecasteele JP (1987) Changesin membrane lipid composition of Clostridium acetobutylicumduring acetone-butanol fermentation: effects of solvents, growthtemperature and pH. J Gen Microbiol 133:103110

Lin YL, Blaschek HP (1983) Butanol production by a butanol-tolerantstrain of Clostridium acetobutylicum in extruded corn broth.Appl Environ Microbiol 45:966973

Lin YL, Blaschek HP (1993) butanol production by a butanol-tolerantstrain of Clostridium acetobutylicum in extruded corn broth.Appl Environ Microbiol 45:966973

Linden JC, Moreira A (1982) Anaerobic production of chemicals. In:Hollaender A (ed) Chapter in Basic biology of new develop-ments in biotechnology (Basic Life Sciences 25). Plenum Press,New York, pp 377403

Liyanage H, Young M, Kashket ER (2000) Butanol tolerance ofClostridium beijerinckii NCIMB 8052 associated with down-regulation of gldA by antisense RNA. J Mol Microbiol Biotechnol2:8793

Lovitt RW, Longin R, Zeikus JG (1984) Ethanol production bythermophilic bacteria: physiological comparison of solventeffects on parent and alcohol-tolerant strains of Clostridiumthermohydrosulfuricum. Appl Environ Microbiol 48:171177

Maddox IS (1989) The acetonebutanolethanol fermentation: recentprogress in technology. Biotechnol Genetic Eng Rev 7:190220

Mermelstein LD, Welker NE, Petersen DJ, Bennett GN, PapoutsakisET (1994) Genetic and metabolic engineering of Clostridiumacetobutylicum ATCC 824. Ann NY Acad Sci 721:5468

1710 Appl Microbiol Biotechnol (2010) 85:16971712

Meyer CL, Roos JW, Papoutsakis ET (1986) Carbon monoxidegassing leads to alcohol production and butyrate uptake withoutacetone formation in continuous cultures of Clostridium aceto-butylicum. Appl Microbiol Biotechnol 24:159167

Michel GP, Starka J (1986) Effect of ethanol and heat stresses on theprotein pattern of Zymolnonas mobilis. J Bacteriol 165:10401042

Mitchell WJ (1998) Physiology of carbohydrate to solvent conversionby clostridia. Adv Microb Physiol 39:31130

Mitchell WJ, Booth IR (1984) Characterization of the Clostridiumpasteurianum phophotransferase system. J Gen Microbiol130:21932200

Mitchell WJ, Tangney M (2005) Carbohydrate uptake by the phospho-transferase system and other mechanisms. In: Durre P (ed)Handbook on Clostridia Chapter 8. Taylor and Francis, New York,pp 155175

Mitchell WJ, Shaw JE, Andrews L (1991) Properties of glucosephophotransferase system of Clostridium acetobutylicumNCIMB 8052. Appl Environ Microbiol 57:23542539

Monot F, Engasser JM, Petitdemage H (1984) Influence of pH andundissociated butyric acid on the production of acetone andbutanol in batch cultures of Clostridium acetobutylicum. ApplMicrobiol Biotechnol 19:422426

Moreira AR, Ulmer D, Linden JC (1981) Butanol toxicity in thebutylic fermentation. Biotechnol Bioeng Symp 11:567579

Nielsen DR, Yoon LE, S-H TH-C, Yuan C, Prather KLJ (2009)Engineering alternative butanol production platforms in heterol-ogous bacteria. Metab Eng 11:262273

Nielson L, Larsson M, Holst O, Mattiasson B (1988) Adsorbents forextractive bioconversion applied to the acetone butanol fermen-tation. Appl Microbiol Biotechnol 28:335339

Nolling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q, GibsonR, Lee HM, Dubois J, Qiu D, Hitti J, Wolf YI, Tatusov RL,Sabathe F, Doucette-Stamm L, Soucaille P, Daly MJ, BennettGN, Koonin EV, Smith DR (2001) Genome sequence andcomparative analysis of the solvent-producing bacterium Clos-tridium acetobutylicum. J Bacteriol 183:48234838

Okamoto H, Sone N, Hirata H, Yoshida M, Kagawa Y (1977) Purifiedproton conductor in proton translocating adenosine triphospha-tase of a thermophilic bacterium. J Biol Chem 252:61256131

Osman YA, Ingram LO (1985) Mechanism of ethanol inhibitionof fermentation in Zymomonas mobilis CP4. J Bacteriol164:173180

Ounine K, Petitdamage H, Raval G, Gay R (1985) Regulation andbutanol inhibition of D-Xylose and D-Glucose uptake in Clostrid-ium acetobutylicum. Appl Environ Microbiol 49:874878

Peguin S, Soucaille P (1995) Modulation of carbon and electron flowin Clostridium acetobutylicum by iron limitation and methylviologen addition. Appl Environ Microbiol 61:403405

Petitdemange H, Cherrier C, Raval G, Gay R (1976) Regulation of theNADH and NADPH-ferredoxin oxidoreductases in clostridia ofthe butyric group. Biochim Biophys Acta 421:334347

Phillips JA, Humphrey AE (1983) An overview of process technologyfor the production of liquid fuels and chemical feedstocks viafermentation. In: Wise DL (ed) Organic chemicals from biomass.Benjamins/Cummings Publishing, Menlo Park, Calif, pp 249304

Quinkal I, Davasse V, Gaillard J, Moulis JM (1994) On the role ofconserved proline residues in the structure and function ofClostridium pasteurianum 2[4Fe-4S] ferredoxin. Protein Eng7:681687

Qureshi N, Blaschek HP (1999) Butanol recovery from modelsolution/fermentation broth by pervaporation: Evaluation ofmembrane performance. Biomass Bioenergy 17:175184

Qureshi N, Blaschek HP (2000) Economics of butanol fermentationusing hyper-butanol producing Clostridium beijerinckii BA101.Trans IChemE 78(Part C):139144

Qureshi N, Blaschek HP (2001) Recent advances in ABE fermenta-tion: hyper-butanol producing Clostridium beijerinckii BA101. JInd Microbiol Biotechnol 27:287291

Qureshi N, Ezeji TC (2008) Butanol (a superior biofuel) productionfrom agricultural residues (renewable biomass): Recent progressin technology. Biofuels Bioprod Bioref 2:319330

Qureshi N, Maddox IS, Friedl A (1992) Application of continuoussubstrate feeding to the ABE fermentation: relief of productinhibition using extraction, perstraction, stripping and pervapora-tion. Biotechnol Prog 8:382390

Qureshi N, Meagher MM, Hutkins RW (1999) Recovery of butanolfrom model solutions and fermentation broth using a silicalite/silicone membrane. J Memb Sci 158:115125

Ramos JL, Duque E, Gallegos M-T, Godoy P, Ramos-Gonzlez MI,Rojas A, Tern W, Segura A (2002) Mechanisms of solventtolerance in gram-negative bacteria. Annu RevMicrobiol 56:743768

Rao G, Mutharasan R (1987) Altered electron flow in continuouscultures of Clostridium acetobutylicum Induced by ViologenDyes. Appl Environ Microbiol 53:12321235

Riebeling V, Jungermann K (1976) Properties and function ofClostridial membrane ATPase. Biochim Biophys Acta 430:434444

Riebeling V, Thauer RK, Jungermann K (1975) The internal alkalinepH gradient, sensitive to uncoupler and ATP ase inhibitor, ingrowing Clostridium pasteurianum. Eur J Biochem 55:445453

Roffler SR, Blanch HW, Wilke CR (1987) In-situ recovery of butanolduring fermentation: Part 2. Fed-batch extractive fermentation.Bioproc Eng 2:181190

Ruhl J, Schmid A, Blank ML (2009) Selected Pseudomonas putidastrains able to grow in the presence of high butanol concen-trations. Appl Environ Microbiol 75:46534656

Russell JB, Diez-Gonzalez F (1998) The effects of fermentation acidson bacterial growth. Adv Microbial Physiol 39:205234

Saier MH, Stiles CD (1975) Molecular dynamics in biologicalmembranes. Springer-Verlag, New York

Schwarz WH, Slattery M, Gapes RJ (2007) The ABC of ABE.BioWorld Europe 2:810

Shao P, Huang RYM (2007) Polymeric membrane pervaporation. JMembr Sci 287:162179

Shi S, Blaschek HP (2008) Transcriptional analysis of Clostridiumbeijerinckii NCIMB 8052 and the hyper-butanol-producingmutant BA101 during the shift from acidogenesis to solvento-genesis. Appl Environ Microbiol 2008(74):77097714

Steen EJ, Chan R, Prasad N, Myers S, Petzold CJ, Redding A, OuelletM, Keasling JD (2008) Metabolic engineering of Saccharomycescerevisiae for the production of n-butanol. Microb Cell Fact 7:36

Tashiro Y, Shinto H, Hayashi M, Baba S, Kobayashi G, Sonomoto K(2007) Novel high-efficient butanol production from butyrate bynon-growing Clostridium saccharoperbutyl acetonicum N14(ATCC 13564) with methyl viologen. J Biosci Bioeng 104:238240

Taylor M, Tuffin M, Burton S, Eley K, Cowan D (2008) Microbialresponses to solvent and alcohol stress. Biotechnol J 3:13881397

Terracciano JS, Kashket ER (1986) Intracellular conditions requiredfor initiation of solvent production by Clostridium acetobutyli-cum. Appl Environ Microbiol 52:9691

Terracciano JS, Rapaport E, Kashket ER (1988) Stress- and growthphase-associated proteins of Clostridium acetobutylicum. ApplEnviron Microbiol 54:19891995

Tomas CA, Welker NE, Papoutsakis ET (2003) Overexpression ofgroESL in Clostridium acetobutylicum results in increasedsolvent production and tolerance, prolonged metabolism, andchanges in the cell's transcriptional program. Appl EnvironMicrobiol 69:49514965

Tomas CA, Beamish J, Papoutsakis ET (2004) Transcriptionalanalysis of butanol stress and tolerance in Clostridium acetobu-tylicum. J Bacteriol 186:20062018

Appl Microbiol Biotechnol (2010) 85:16971712 1711

Traxler RW, Wood EM, Mayer J, Wilson MP (1985) Extractivefermentation for the production of butanol. Dev Ind Microbiol36:519525

Van Der Westhuizen A, Jones DT, Woods DR (1982) Autolyticactivity and butanol tolerance of Clostridium acetobutylicum.Appl Environ Microbiol 44:12771281

Vane A (2005) Review of pervaporation for product recovery frombiomass fermentation processes. J Chem Technol Biotechnol80:603629

Vollherbst-Schneck K, Sands JA, Montenecourtv (1984) Effect ofbutanol on lipid composition and fluidity of Clostridium

acetobutylicum ATCC 824. Appl Environ Microbiol 47:193194

Yan RT, Zhu CX, Golemboski C, Chen JS (1988) Expression ofsolvent-forming enzymes and onset of solvent production in batchcultures of Clostridium beijerinckii (Clostridium butylicum).Appl Environ Microbiol 54:642648

Zhao Y, Hindorff LA, Chuang A, Monroe AM, Lyristis M, HarrisonML, Rudolph FB, Bennett GN (2003) Expression of a clonedcyclopropane fatty acid synthase gene reduces solvent formation inClostridium acetobutylicum ATCC 824. Appl Environ Microbiol69:28312841

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Achievements and perspectives to overcome the poor solvent resistance in acetone and butanol-producing microorganismsAbstractIntroductionTransport of substrates and organic acids and their effect on solventogenic clostridia cell metabolismBiochemistry of solvent production by solventogenic clostridiaCell membrane fluidity, cell death, and solvent tolerance in acetone and butanol-producing microorganismsAmelioration of solvent toxicity in acetone and butanol-producing microorganismsGenetic strain improvementSolvent-resistant microorganisms as potential acetone-butanol production hosts

Advanced fermentation techniques and downstream processingLiquidliquid extractionPerstractionGas strippingPervaporation

Concluding remarksReferences

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