Biotechnol. J. 2008, 3 DOI 10.1002/biot.200800158 www.biotechnology-journal.com
1 Introduction
The pursuit of suitable renewable alternatives tooil-based fuels has become a socio-economic pri-ority, starkly pertinent in South Africa at both thelevel of government and consumer because of dra-matic increases in the price of petrol at the pumpand increases in the price of basic foodstuffs. Thenegative impacts of fossil fuel combustion on hu-man health and the local and global environmentsare well established [1, 2] and include the emissionof sulphur oxide, nitrous oxide, carbon monoxideand carbon dioxide, potentially contributing to glo-bal warming [3, 4].
In consequence, there is significant interest inblending alcohols, particularly anhydrous ethanoland butanol, with gasoline, having the twofold ef-fect of limiting CO2 and particulate emissions andreducing national dependency on fossil fuels. Thebest example of successful national transition fromfossil fuel dependency to renewable fuel depend-ency in the transport sector is in Brazil where thenational alcohol programme was responsible, inthe 1996/97 season, for 273 million tons of har-vested (wet weight) sugar cane, leading to 13.7 mil-lion m3 ethanol destined for transportation [5–7].
Internationally, the biological production of al-cohol for fuel supplementation has always focusedon conventional fermentation using established or-ganisms (1st generation processes) such as Saccha-romyces cerevisiae [8, 9] and Zymomonas mobilis[10–12]. Such organisms have distinct advantagesin terms of ethanol yields, high solvent toleranceand very well understood fermentations. However,a major drawback to these processes is that they
Review
Microbial responses to solvent and alcohol stress
Mark Taylor1,2, Marla Tuffin1, Stephanie Burton3, Kirstin Eley2 and Don Cowan1
1 Institute for Microbial Biotechnology and Metagenomics (IMBM), University of the Western Cape, Cape Town, South Africa2 TMO Renewables Ltd, Guildford, Surrey, UK3 Department of Chemical Engineering, University of Cape Town, Cape Town, South Africa
Increasing fuel prices and doubts over the long-term availability of oil are currently major globalconcerns. Such concerns have led to national policies and objectives to develop microbially pro-duced alcohols as fuel additives or substitutes. However, in South Africa this solution poses thefurther dilemma of sourcing a suitable fermentative carbohydrate that will not impact negativelyon the availability of staple foods. The solution lies in the use of lignocellulosic materials, currentlya waste product of the food and agriculture industries, which could be used in conjunction with acatabolically suitable production strain. In the pursuit of lignocellulosic biofuel production, con-ventional fermentation strains have been shown to have limited catabolic versatility. However,catabolically versatile engineered strains and novel isolates engineered with ethanologenic path-ways have subsequently been shown to exhibit limitations in solvent tolerance, hindering their fullpotential as economically viable production strains. A considerable volume of research has beenreported on the general cellular mechanisms and physiological responses to solvent shock as wellas adaptive changes responsible for solvent tolerant phenotypes in mutant progeny. Here we re-view a number of the more common cell responses to solvents with particular focus on alcoholtolerance.
Correspondence: Professor Donald Cowan, Institute for Microbial Biotech-nology and Metagenomics, University of the Western Cape, Bellville 7535, Cape Town, South AfricaE-mail: [email protected]
Received 1 August 2008Revised 2 September 2008Accepted 3 September 2008
rely on readily fermentable C6 sugars (starch, su-crose and glucose) making such processes viableonly in countries with the agricultural capacitiesfor growing large quantities of carbohydrate-richcrops in excess of those required for consumption.
Dramatic increases in global populations andfood demand have motivated some researchers todevelop novel production strains with broadercatabolic properties [13–17]. The aim is that suchstrains will efficiently ferment sugars (and othercarbon fractions) derived from the hydrolysis ofhemicellulosic biomass to generate ethanol or bu-tanol and concurrently conserve food grade carbo-hydrate supplies for human consumption.
2 Fuel alcohol
The general observation can be made that many ofthe original process issues that stood in the way ofthe development of economically viable biofuel inthe latter half of the 20th century have re-emergedwith the resurgence of interest in fuel alcohol pro-duction [18–20]. Other than economically viable al-cohol yields, two principal requirements, seen bymany researchers as crucial in the development ofa process strain are: (i) catabolic versatility andhence the ability to ferment the carbohydrate frac-tion of biomass hydrolysates and (ii) the adaptationor development of process strains to grow and tol-erate high (>40 g/L) exogenous ethanol concentra-tions [21, 22].
The principal anaerobic metabolic pathwaysthat result in alcohol production have been de-scribed in some detail [9, 11, 12, 18]. One of the keyintermediary enzymes involved in ethanol synthe-sis is pyruvate decarboxylase (PDC), variants ofwhich have been described from a number ofmesophilic yeasts [23–29] and a limited number ofmesophilic bacterial species [30–34]. PDC catalysesthe Mg2+- and thiamine pyrophosphate (TPP)-de-pendent decarboxylation of pyruvate to acetalde-hyde and CO2. The subsequent conversion of ac-etaldehyde to ethanol is catalysed by a specific al-cohol dehydrogenase with the concurrent regener-ation of the cofactor NAD+, thus helping maintain aphysiological redox balance within the cell. Otheranaerobic metabolic routes to ethanol have beendescribed in both mesophilic and thermophilicspecies, principally via pyruvate catabolism catal-ysed by pyruvate-formate lyase or variants of pyru-vate dehydrogenase [35–38].
The anaerobic production of butanol forms partof the acetone, butanol and ethanol fermentation(ABE) process of anaerobic mesophiles, principal-ly characterised in Clostridium acetobutylicum and
Clostridium beijerinckii [39, 40]. Butanol productionresults from of a number of sequential reductionsof metabolic intermediates derived from acetylCoA, its production occurring concurrently withthe production of acetone, ethanol and other minorend products such as butyric acid [41].
Major advances have been made in the devel-opment of a number of catabolically versatile mu-tants in the established ethanologenic strains, Sac-charomyces cerevisiae [15, 16, 42] and Zymomonasmobilis [11, 43, 44] and in a number of other yeastand bacterial species that possess the Entner-Doudoroff metabolic pathway. This pathway in-cludes the key enzyme directing carbon flow toethanol, PDC [45, 46]. An alternative approach hasalso been the expression of the pdc gene and a suit-able alcohol dehydrogenase (adh) in metabolicallyversatile hosts such as E. coli [47–51].
For butanol production, some degree of catabol-ic versatility is inherent in the strains that haveformed the major focus in this research (C. aceto-butylicum and C. beijerinckii) [39]. However, solventtolerance has been cited as a major constraint inthe attainment of high production yields [52]. Thesensitivity of many microorganisms to high exoge-nous alcohol concentrations thus appears to be one(if not the most) important limiting factor in the de-velopment of new non-yeast biofuel productionstrategies.
Numerous cellular mechanisms have been re-ported to be involved in the responses to high ex-ogenous alcohol concentrations. Such mechanismsinclude responses at the site of solvent contact (i.e.compositional alterations of the cell membrane),enhanced capacity for membrane repair and theup-regulation or de novo expression of solvent ef-flux systems [10–12, 53–55].
3 Aspects of solvent tolerance in yeasts
Although not the primary focus of this review, in-trinsic alcohol and general stress tolerance is acharacteristic of many yeast species and has beenstudied extensively with general aim of improvingtheir applicability to many biotechnological pro-cesses [56, 57]. An understanding of the responsemechanism in these yeast strains has led to a morefocused and rational approach to similar studies inbacteria. Most notably, ethanol tolerance in S. cere-visiae has been well documented to correlate withan increased degree of fatty acid unsaturation inmembrane lipids [58, 59].
Ethanol tolerance in yeasts has also been attrib-uted to a number of other factors involved in gen-eral stress response, such as plasma membrane
composition/change [60] and accumulation of in-tracellular ethanol and osomoprotectants [61].Tol-erance has also been shown to vary with environ-mental changes such as medium composition [62],temperature [63] and osmotic influences [64].
Specific examples of yeast response mecha-nisms include two yeast strains (S. cerevisiae andKloeckera apiculata), which were initially reportedto respond to increasing ethanol by up-regulatingthe proportion of ergosterol and unsaturated fattyacid levels in their lipid membranes as well asmaintaining phospholipid biosynthesis [60]. In an-other unsaturated fatty acid auxotrophic yeastspecies, the role of ergosterol in tolerance was su-perseded by specific membrane lipid compositionchanges particularly with respect to oleic, linoleicand linolenic acid composition [59, 65].
More recently several groups examining globalgene expression in ethanol-tolerant yeast strainshave shown that multiple synergistic responses areresponsible for the desired phenotype, i.e. im-proved alcohol tolerance. Over 250 genes have beenimplicated in tolerance [66], including a number ofATPases [67–69] and genes encoding heat shockproteins. A summary of ethanol-elicited responsesfrom a variety of yeasts is given in Table 1.
4 General solvent responses in mesophilicbacteria
The general physiological responses to solventshock have been previously reviewed and are mod-erately understood in a number of Pseudomonasspp. [70–72], E. coli [55, 71] and Oenococcus oeni[73–75]. Toluene exposure in Pseudomonas putidaelicits both outer and cytoplasmic membrane adap-tations, regarded as physical mechanisms that pre-vent solvent penetrance [55, 70]. It has been re-ported that in strain DOT-T1 exposure to solventsmediates both short-term [increased membranerigidity achieved through rapid transformation ofthe cis-9,10-methylene hexadecanoic fatty acid tothe unsaturated 9-cis-hexadecenoic acid (C16:1,9cis) fatty acid and subsequently the trans isomer]and long-term responses (proportion of cardiolipinincreases and phosphatidylethanolamine decreas-es in the phospholipid polar head groups) [70, 72].
Several well-characterised solvent responsemechanisms involve solvent exclusion systems andenergy-dependent efflux pumps of the resistance-nodulation-cell division (RND) family, which ex-port toxic organic solvents [71]. The best under-stood efflux pump responses to solvent stress arethose involved in toluene exposure in E. coli,
Pseudomonas putida, and Ps. aeruginosa [71]. In Ps.putida three efflux pumps (TtgABC, TtgDEF, andTtgGHI), coupled to a proton motive force via theTonB system, are thought to be involved [71]. Theefflux pumps AcrAB-TolC and AcrEF-TolC per-form similar roles in E. coli in imparting n-hexanetolerance [71, 76] and are regulated at the tran-scriptional level by both specific and global regula-tors.
Solvent tolerance in E. coli is strain specific anddetermined genetically. Mutants with increasedtolerance have been constructed that were defec-tive in the marR gene that encodes a repressor pro-tein for the mar operon (responsible for environ-mental stress factors) [76]. In addition, increasingthe expression of the stress-response genes (soxS,marA, and robA encoding DNA-binding proteins/transcriptional activators) was shown to increasetolerance in several strains of E. coli through regu-lation of the AcrAB-TolC system [76, 77]. Overex-pression of the manXYZ genes, which code for asugar transporter of the phosphotransferase sys-tem, have also been implicated in solvent resist-ance [78]. A number of other adaptive responseshave been observed in various species, as shown inTable 1. Surprisingly, little has been reported onethanol tolerance responses in the ethanologen,Zymomonas mobilis, apart from the general induc-tion of expression of heat shock proteins [79].
It has been suggested that high exogenous alco-hol concentrations would elicit deleterious effectson bacterial growth, viability, and metabolism dueto the stimulation of leakage within the membrane[53]. This proposal is consistent with the knownbiophysical interactions of phospholipid bilayerswith alcohols. It was also suggested that increasedethanol tolerance phenotypes could result from ac-quired adaptive and evolutionary changes in cellmembrane rigidity, a mechanistic response illus-trated by Oenococcus oeni in response to ethanoland monitored by carboxyfluorescein retention(indicative of membrane integrity) [74, 80].
The principal target for cellular adaptation topotentially toxic levels of extracellular alcohol isthe cell membrane.This was recognised as early as1976 by Ingram [81], who reported that the mem-brane fatty acid composition of E. coli K-12 alteredradically when the strain was grown in the pres-ence of ethanol and that the proportion of 18:1 fat-ty acids increases at the expense of saturated fattyacids.
More recently, proteomic analysis of this strainunder ethanol stress has shown that inosine-5’-monophosphate dehydrogenase, phosphoglucona-te dehydrogenase and glutathione reductase werestrongly regulated, suggesting a key role of redox
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balance maintenance in ethanol adaptation [73]. Inaddition, significant increases in the expression ofcell wall biosynthetic proteins were demonstrated.
Ethanol tolerant cells of Oenococcus oeni havebeen reported to contain a higher proportion of un-saturated fatty acids and a decreased proportion of
total cell lipid content, decreasing membrane fluid-ity [74, 75] (Interestingly the opposite mechanismoccurs in some Staphylococcus spp which increasemembrane fluidity in response to solvent stress).However, in a subsequent report ethanol shock(14%) was found to elicit immediate fluidisation
Table 1. Solvent response processes in microorganisms
Species Solvent Genes implicated Phenotypic response Reference
Oceanomonas baumannii Phenol Fatty acid Induced increases in fatty acid saturation [104, 105]synthase genes and decreases in the mean chain length
of the fatty acids.
Staphylococcus haemolyticus Toluene Cis-trans isomerise, Induced increase in membrane fluidity [106]various fatty acid through increases in the anteiso-fatty acids synthase genes and decreases in straight-chain fatty acids.
Staphylococcus Benzene None specified Tolerance up to 40% benzene. [107]saprophyticus M36
(attaining rigidity gradually after shock) in O. oenias monitored by fluorescence anisotropy withdiphenyl-1,3,5-hexatriene [75].
One of the few investigations to improve solventtolerance in E. coli was reported in 1998 by Aono[76], who isolated several E. coli strains with im-proved tolerance to solvents. He reported that themutants were defective in the marR gene (encod-ing a repressor protein for the mar operon, respon-sible for an environmental stress factor). The highexpression of the stress-response genes, soxS,marA, and robA, also increased organic solvent tol-erance in several strains of E. coli.
Butanol tolerance in several Clostridium spp.has been reported to correlate with the decreasedexpression of glycerol dehydrogenase as demon-strated by transposon mutational analysis and tol-erance studies in C. beijerinckii [82]. Overexpres-sion of the groESL operons in C. acetobutylicumATCC 824 has resulted in higher yields and bu-tanol-tolerant variants with increased expressionof motility and chemotaxis genes and decreasedexpression of other major stress response genes[83, 84]. These researchers have also shown thatSpo0A overexpression increases butanol toleranceas part of a general stress response that includesthe regulation of genes involved in DNA synthesis,cell division, glycolysis and butanol synthesis andvarious heat shock proteins [85].
Many physiological responses, including growth,survival and reproductive ability have been shownto respond to the presence of increased concentra-tions of alcohol. In E. coli, glycolysis was found to beparticularly tolerant to alcohol shock, probably dueto its exergonic nature, which permits glycolyticflux to proceed even in the absence of active growthand cell division [53]. Subsequently, ethanol toler-ant E. coli variants of strain KO11, capable of pro-ducing more than 60 g/L ethanol on xylose, havebeen isolated.These isolates apparently exceed thetolerance threshold of their engineered S. cerevisi-ae and Z. mobilis counterparts. Ethanol tolerantvariants were subsequently found to possess anon-functional fnr gene [86, 87].
Most recently the expression levels of manX,manY, and manZ genes (encoding a sugar trans-porter of the phosphotransferase system) weremonitored in alcohol-shocked E. coli and found tobe strongly up-regulated [78]. The gcv, betIBA andbetT genes and the compounds glycine and betainehave also been linked to increased ethanol toler-ance, the latter as protective osmolytes [86].
5 Solvent responses in thermophilic bacteria
Because of significant interest in the discovery anddevelopment of second generation biofuel process-es designed around strains possessing broad cata-bolic ranges and enhanced alcohol production ca-pacities, several research groups have sought newmetabolically diverse and adaptable isolates frommore extreme environments.There is particular fo-cus on thermophilic species from the generaClostridium [21], Thermoanaerobacter [88–90] andGeobacillus [91, 92]. Thermophilic fermentationsoffer process advantages in product removal by application of a gas stream or low-grade vacuum[52, 93].
In an early attempt to develop an ethanol toler-ant strain of C. thermocellum, it was concluded thatadaptive responses included control of the alcoholproduction rate, yield and concentration [94]. Sub-sequently, it was reported that an increase in num-bers of shortened, unsaturated and anteio-bran-ched fatty acids was found to be associated withethanol shock resulting in increased membrane“fluidity” in C. thermocellum [95]. In C. thermohy-drosulfuricum low ethanol tolerance has beenshown to be due to membrane disruption or inhibi-tion of glycolysis [96].
In 2002, it was reported that a mutant strain ofThermoanaerobacter ethanolicus (39E H8) that wastolerant of 8% ethanol, lacked the primary adh (as-sociated with ethanol consumption) and conse-quently increased the percentage of transmem-brane fatty acids (long chain C30 fatty acids) in re-sponse to the increasing levels of ethanol generat-ed from the functional secondary ADH enzyme(ethanol producing) [97]. It was suggested that thebiochemical basis for alcohol tolerance in ther-mophilic ethanologens varied from species tospecies. For example, C. thermocellum alcohol de-hydrogenase is inhibited by 1% ethanol while thereversible activity T. brockii and C. thermohydrosul-furicum homologues is stimulated by 5% solvent[38]. Further studies of tolerance in C. thermohy-drosulfuricum showed that ethanol tolerance wastemperature dependant and not an artefact ofmembrane disruption or glycolytic inhibition. In asubsequent report it was shown that tolerance toethanol in a tolerant mutant of C. thermohydrosul-furicum was linked to maintenance of a redox bal-ance and reductions in the activities of the reducedferredoxin-linked NAD reductase and NAD-linkedalcohol dehydrogenase [96, 98].
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To advance any production scale biofuel process,solvent tolerance must be exhibited in the fermen-tative strain and the biochemical and adaptivemechanisms of tolerance must be understood. Sig-nificant advances have been made in the develop-ment of a number of strains with the ability to fer-ment the carbohydrate fraction of biomass hy-drolysate. It has long been recognised that alcoholtolerance would be as important, if not more so,than catabolic flexibility in strain developmentprogrammes. However, although many alcohol tol-erant strains of various species were developed orisolated and several reports made on the mecha-nisms of tolerance in these strains, it would be fairto say that an intimate understanding of solventtolerance in some key ethanologenic organismshas taken second place in lieu of engineering cata-bolic versatility. This is a trend that is now beingreaddressed by exploiting some of the tools of the‘-omics’ era. High coverage and high qualitygenome sequencing is becoming increasingly ac-cessible at an affordable price, allowing re-searchers to compare, clone, express and deletespecific genes more readily and with a higher de-gree of confidence. Coupling genomic data withDNA microarray technology also allows globalgene expression studies (transcriptomics) to be un-dertaken. In a recent study investigating short-term ethanol stress in S. cerevisiae, ethanol shockwas found to have a significant impact on gene ex-pression over a range of physiological areas in-cluding ionic homeostasis, heat protection and en-ergy management [99]. Similar approaches havebeen applied to help understand global butanolstress responses in C. acetobutylicum [100] and toquantify differential mRNA expression in a set of17 cellulose degrading genes of C. thermocellum[101].
Proteomics technologies have been extensivelyapplied to the study of physiological responses toalcohol stress in various microbial strains. Recentapplications of this technology have been reportedfor O. oeni [73] and C. thermocellum [102], bothshowing significant alterations in cell membraneprotein profiles.
An understanding of the physiological process-es effected by environmental change cannot onlyuncover potential gene targets for mutational stud-ies but may also increase our appreciation of theglobal physiological impact that single mutationsmay have on the expression of genes other thanthose targeted. However, it is only when genomicanalysis is combined with transcriptomic and pro-teomic data that a true understanding of global reg-
ulatory machinery is obtained, particularly at thephenotypic level which is so important in anyprocess with a biotechnological application [103].
By combining -omics data with techniques suchas genome shuffling, global transciptome machin-ery engineering and directed evolution, new toolsand integrative platforms can be developed thatwill allow facilitated engineering of industriallysignificant strains. In the context of biofuels, suchmethods would be part of a process engineeringand development strategy to extend beyond firstgeneration biofuels processes towards second gen-eration and cellulosic alcohol [103].
The authors have declared no conflict of interest.
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Don Cowan was born in New Zealand
in 1954 and underwent his undergradu-
ate and graduate training at the Univer-
sity of Waikato in Hamilton, NZ. After
several years post doctoral research
with the Thermophile Research Unit,
led by Professor Roy Daniel, he moved
to London to take up a Lectureship in
the Department of Biochemistry and
Molecular Biology at University College
London. In 2001, he moved from a Readership in the same depart-
ment to the Chair of Microbiology at the University of the Western
Cape (UWC) in Cape Town, South Africa. In 2006 he established and
is currently Director of the Institute for Microbial Biotechnology and
Metagenomics (IMBM) at UWC. The Institute, with a team of some 30
researchers, is involved in a range of studies from microbial ecology
and phylogenetic, gene discovery to enzyme structure-function and en-
gineering and applied microbiology. The Biofuels Research Program in
IMBM is one of the larger such programs in South Africa.
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