INVITED REVIEW

Using microorganisms to brew biofuels

Reeta Prusty Rao & Nicholas Dufour & Jeffrey Swana

Received: 30 November 2009 /Accepted: 21 May 2011 /Published online: 22 September 2011 / Editor: P. Lakshmanan# The Society for In Vitro Biology 2011

Abstract Interest in alternative fuel sources has grown inrecent years in response to a confluence of factors, includingconcerns over our reliance on and increasing demand forfossil fuels as well as the deleterious environmental effects offossil fuel extraction and utilization. The use of microbe-derived fuel alcohols is a viable alternative, as they arerenewable, emit fewer greenhouse gasses, and require littleaugmentation of current energy infrastructure as compared toother sustainable transportation options such as electricvehicles and fuel cells. Here, we present a brief overviewof candidate substrates for alcohol production with a focuson lignocellulosic sources, relevant microorganisms underresearch for industrialization and the biotechnologicaltechniques used to improve alcohol production phenotypes.

Keywords Bioethanol . Alternative energy .Metabolicengineering .Microbial fermentation

Introduction

Clean, renewable energy has become a research priorityamid increasing global energy demands, reliance onpetroleum, and concerns over global warming. Amongrenewable energy technologies, biofuels, fuels derived frombiomass (Giampietro et al. 1997; Lin and Tanaka 2006;Cardona and Sanchez 2007) have come to the forefront.One such biofuel under active research is biologically

derived ethanol. Ethanol is a simple two-carbon alcohol andis advantageous because it is a product of microbialfermentation.

There are a number of obstacles that must be overcomebefore any serious move to an ethanol fuel economy can beaccomplished. Among the greatest of these is obtainingsubstrate for microbial conversion to ethanol. Currentindustrial production relies on crop-based materials, likesugarcane and corn starch, which are also used to createfood as well as livestock fodder. This competition forsubstrate drives up the price of both food and ethanol; up to40% of the price of ethanol produced in this way is deriveddirectly from the cost of raw materials.

These raw materials contain glucose, a monosaccharidealdohexose sugar, in a form that is readily usable byorganisms from bacteria to humans, making them attractiveboth for use in biofuel and food production. Besides thereadily accessible glucose, all plants are composed ofpolymers that are composed primarily of sugars but arediscarded as wastes in agricultural processes because of therigidity of the polymer structures. These polymers (cellu-lose, hemicellulose, and lignin) are collectively referred toas lignocellulose. Cellulose is composed almost entirely ofglucose while hemicellulose is amorphous and contains avariety of sugars, including glucose and the pentoses,xylose, and arabinose. Lignin, like hemicellulose, is anamorphous polymer but contains no readily fermentablesugars (Ragauskas et al. 2006). Lignocellulose is discardeddespite being composed of sugars because the polymerizedform of the sugars makes it impossible for humans toutilize, and livestock must rely on microbes to digest thematerial for them.

Although agricultural by-products that contain lignocel-lulose are otherwise discarded, it is an attractive potentialfeedstock for industrial biofuels because it is possible for

R. P. Rao (*) :N. Dufour : J. SwanaDepartment of Biology and Biotechnology,Worcester Polytechnic Institute,100 Institute Road,Worcester, MA, USA 01609e-mail: rpr@wpi.edu

In Vitro Cell.Dev.Biol.—Plant (2011) 47:637–649DOI 10.1007/s11627-011-9374-3

some microorganisms to ferment lignocellulose to ethanol.Lignocellulose is astonishingly abundant since it makes upthe cell wall in nearly all plant life. Cellulose is the mostcommon natural polymer, the most common organicmolecule (Peters 2006), and makes up more than half ofglobal biomass. Table 1 details the lignocellulosic content,by percentage, of common waste products, both agriculturaland otherwise. Economic calculations further bolster thecase for using lignocellulose as the substrate of industrialethanol production. A study by Hinman determined that itis necessary to improve upon the industrial processing oflignocellulose for production of ethanol for bioethanol tocompete viably with fossil fuels (Hinman et al. 1989).

Production of lignocellulosic alcohol, the collective termfor any alcohol (including ethanol as well as other potentialbiofuels, like butanol) is problematic. Eukaryotes generallydo not readily utilize xylose or arabinose anaerobicallybut can be engineered to do so (Shi et al. 1999). Whilesome bacteria and fungi can naturally utilize such sugarsanaerobically, the primary means of growth is non-fermentative (Sonderegger and Sauer 2003). Nevertheless,identifying organisms naturally capable of efficientlyconverting lignocellulose as well as component sugarsxylose and arabinose into ethanol or other alcohols is anarea of active research (Jeffries and Shi 1999b; Jeffries2006; Rao et al. 2008). Similarly, much work is focusedon artificially producing an organism with these capacitiesby genetically modifying one already known to produceethanol from other sources (Koskinen et al. 2008; Menonet al. 2010).

Bioprocessing

Several microbes are particularly relevant to industrialprocesses, though each organism is suited more specificallyfor different steps or forms of the bioprocessing ofbioethanol. To fully appreciate the relevance of eachmicroorganism listed, a brief overview of each is provided.

Conventionally, there are three main steps in the process:pretreatment, hydrolysis, and fermentation. Typical pretreat-

ment involves a thermo-mechano-chemical step to removethe structural rigidity of the plant material and theindigestible lignin from the sugars. Microbes are beingconsidered for pretreatment, but the biological degradationof lignin is slow and inefficient as compared to the otheroptions (Sun and Cheng 2002). Common treatments for thisstep involve acid hydrolysis, steam explosion, ammoniafiber expansion, and sulfite pretreatment to overcomerecalcitrance of lignocellulose (Himmel et al. 2007). Thisstep is a crucial one, especially if subsequent steps involvemicrobial populations. The choice of the method ultimatelydepends on the feed material and type of downstreamprocessing. A thorough evaluation on the different pretreat-ment options and their advantages and disadvantages waspublished in Hendriks and Zeeman (2009).

The second and third steps, hydrolysis and fermentation,result in the depolymerization of cellulose and hemicellu-lose into fermentable sugar products (i.e., cellulose depo-lymerizes to glucose monomers) and conversion of thosesugars into ethanol. The hydrolysis step can be performedchemically or enzymatically. Here we focus on thebiological process of enzymatic hydrolysis, which requiresthe activities of hemicellulases, cellulases, and glucosidases(Chandel et al. 2007). For this hydrolysis step, there arethree approaches that may be used. The first two requireisolation and purification of the enzymes. In the first case,called separate hydrolysis and fermentation (SHF), theenzymes are added to the pretreated material to allowdigestion of polymers in a step called saccharification (orsweetening). The microorganism for fermentation is thenadded later to convert the monomeric sugars into ethanol.The drawback of SHF processing is that the inhibition ofthe hydrolysis enzymes by the monomeric sugars theyproduce significantly reduces the yield. To overcome this, aconstant removal of sugars after the hydrolysis is necessary.The second case is referred to as simultaneous saccharifi-cation and fermentation (SSF). The limitation of thisprocedure is in maintaining the ideal temperatures of thetwo steps. Generally, the enzymes for hydrolysis have ahigher ideal temperature (above 50°C) than the microbesthat are able to ferment at temperatures between 20°C and35°C (for most yeast; Weber et al. 2010). Hence, muchresearch has been invested in isolating microbes, which arenaturally thermotolerant or engineered to be so (Shaw et al.2008). The third approach is simplified even further.Referred to as consolidated bioprocessing (CBP), thehydrolytic enzymes are produced at the same time asfermentation either by a single microbe or with a co-culture(Weber et al. 2010). Below, we discuss the microbialspecies that are currently used or being considered forindustrial production of lignocellulosic biofuels. We alsooutline steps and areas in which further improvements maybe considered.

Table 1. Lignocellulosic content of common biological wastes(adapted from Sun and Cheng 2002)

Agricultural waste Lignocellulosic content (%)

Wheat straw 95

Discarded newspapers ∼100Leaves and lawn refuse 90

Swine waste >30

Cattle manure 15

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Major Species

Eukaryotes. Trichoderma. Trichoderma is a genus ofprevalent soil-dwelling fungi, notable for the speciesTrichoderma reesei, which readily degrades biomass andis widely used in industry to produce hemicellulose andcellulose degrading enzymes. Research efforts have focusedon improving the strain to harness the organism directly forbioethanol production. Genetic engineering has producedsome promising results; however, the lack of knowledge onthe T. reesei genome, which is threefold more complex thanand contains twice as many genes as Saccharomycescerevisiae (Martinez et al. 2008), has hampered progress.Nevertheless T. reesei remains one of the best producers ofextracellular enzymes for degradation of plant-derivedfeedstocks, producing up to 100 g of extracellular enzymeper liter (Cherry and Fidantsef 2003).

Three classes of cellulases are required for efficientbreakdown of lignocellulosic material: endoglucanases(EG), cellobiohydrolases (CBH; also known as exocellu-lases), and β-glucosidases (BG). EGs are responsible forbreaking the intramolecular bonds between glucose resi-dues, CBHs primarily act on the ends of cellulose chains,and BGs act on the products of the CBHs and EGs,cellobiose and short oligosaccharides respectively. T. reeseiis only capable of producing sufficient quantities of theCBH and EG groups of enzymes, leading to a buildup ofcellobiose, which is inhibitory to the hydrolysis process.Generally, supplementation of BG enzymes from otherspecies (such as Aspergillus niger) is required (Dashtbanet al. 2009). The recent release of the T. reesei genomesequence, while of great value to industrial biofuelsproduction, revealed little about T. reesei’s capacity forefficient enzyme production (Martinez et al. 2008).Surprisingly, the organism contained fewer carbohydratemetabolizing genes than any other known fungi capable ofdegrading plant cell walls. Indeed, in some enzymaticcategories, the organism had the fewest genes of any otherorganism in its lineage. Availability of the genomesequence did however facilitate genetic manipulation andsubsequent upregulation of the endogenous β-glucosidasegene (Rahman et al. 2009).

Research has now turned to investigating the nonrandomclustering of carbohydrate metabolizing genes observed inthe T. reesei genome, which mimics that of its relatives. It isinteresting to note that this species is able to compete withother fungi capable of degrading cellulose and hemicellu-lose even though it is lacking many polysaccharide-degrading genes compared to other species. The clusteringmay result in a more efficient system of regulation as someof the clusters are also in proximity to secondary signalingpathways (Martinez et al. 2008). It would be interesting toassess the clustering of cellulolytic enzymes expressed by

non-native hosts such as S. cerevisiae where there isprecedence for some amino acid biosynthesis genes tobe clustered.

Aspergillus. A. niger is a soil-dwelling black fungus thathas been used since the early twentieth century for itsproduction of citric acid (Schuster et al. 2002), but morerecently it has been exploited for the production ofcellulases for industrial hydrolysis of lignocellulosic mate-rial (Kang et al. 2004). The species is able to undergogenetic manipulation through transformation (Kelly andHynes 1985), has recently been sequenced (Pel et al. 2007)and is listed as generally regarded as safe (GRAS) by theFood and Drug Administration (Schuster et al. 2002). A.niger was one of the first organisms to be used in co-culturewith S. cerevisiae for the production of ethanol using SSFtechniques obtaining 96% of the theoretical yield of ethanolin 2 d from potato starch (Abouzied and Reddy 1986). Theuse of A. niger for lignocellulosic ethanol production isbeing explored. More specifically, BGs (Kang et al. 1999),a class of cellulases not abundant in T. reesei, are harvestedfrom A. niger (Gutierrez-Correa et al. 1999).

Candida. The Candida genus is composed of more than200 species, including the human pathogen Candidaalbicans (Odds 1988). Aside from this pathogen, the genusalso contains species, which have potential for theproduction of biofuels. A number of species have theability to metabolize xylose (Candida lusitaniae, Candidashehatae, Candida tenuis, and Candida tropicalis; Toivolaet al. 1984b).

C. shehatae was evaluated with 200 other yeasts(Chandel et al. 2007) for xylose to ethanol conversion,and it was chosen along with Pichia stipitis to be one of themost productive (Toivola et al. 1984b). One drawback ofC. shehatae is its inability to produce ethanol in thepresence of oxygen. Current studies are focused onmeasuring the optimal oxygen uptake rate required formaximum ethanol production from xylose (Fromanger et al.2010). These studies reveal that while C. shehatae is able toproduce ethanol from xylose, the yield is rather low. Futureefforts are aimed at increasing yield using genetic tools orby process engineering.

C. tropicalis was one of the first yeasts discovered, in1959, that could directly convert xylose into ethanol byKarczewska (Karczewska 1959; Jeffries and Shi 1999a;Rattanachomsri et al. 2009). It is presently of interestbecause of its thermotolerance and applicability to SSFoperations. It has been used successfully in co-culturefermentations. A recent study (Patle and Lal 2008) showedthat the ethanol production from a waste product of tapiocaprocessing, thippi, using Zymomonas mobilis and C.tropicalis independently, produced 83% and 77% ethanol,

MICROBIAL BIOETHANOL PRODUCTION 639

respectively. Using a mixed culture, the production increasedto 93% at a concentration of 72.8 g/L in 48 h.

Kluyveromyces. A genus of budding yeast, Kluyveromyceswas initially noted for its ability to produce ethanol (VanDer Walt 1956) from a wide range of substrates, includingwhey (Aaron et al. 1958) and xylose (Margaritis and Bajpai1982). Kluyveromyces marxianus can produce up to 5.6 g/Lethanol when grown on xylose. Furthermore, K. marxianushas been granted GRAS status (Kostova et al. 2008)making it an attractive candidate for industrial lignocellu-losic ethanol production. However, the slow growth ofK. marxianus on xylose makes this problematic.

Current research on this species focuses on itsthermophilic nature (the species is capable of ethanolproduction at 45°C); this, along with its ability toferment pentose sugars makes it an interesting candidatefor SSF processing. Although genetic manipulation withthis species has been demonstrated, only a handful of geneshave been sequenced, and little is known about this microbe(Fonseca et al. 2008). An SSF process using K. marxianuswas recently described in which the strain was capable ofproducing ~12g/L ethanol in 2 d using olive pulp(Ballesteros et al. 2002). K. marxianus grows on cellobiose,xylose, xylitol, arabinose, glycerol, and lactose but does notferment xylose. Another drawback to the species is thelower tolerance to ethanol as compared to S. cerevisiae(Hacking et al. 1984). Future research is geared towardameliorating the growth defects associated with thisorganism.

Pichia. The genus Pichia is of interest primarily because amember species P. stipitis, recently renamed Scheffersomycesstipitis (Kurtzman and Suzuki 2010), can ferment xyloseproducing 67% of the theoretical maximum amount ofethanol (Schneider et al. 1981; Toivola et al. 1984a).Additionally, P. stipitis has a tolerance to high ethanolconcentrations and is capable of generating ethanol fromglucose, mannose, galactose, and cellobiose (Parekh andWayman 1986). The ability to ferment cellobiose is ofparticular relevance since the model yeast S. cerevisiae isunable to do so, and the enzyme β-glucosidase that isresponsible for breakdown of cellobiose is not producedwith high activity by T. reesei. Present research isfocused on increasing ethanol production using novelpretreatment procedures such as boiling and overliming(Nigam 2001) and altering culture conditions (Skoog andHahn-Hagerdal 1990).

Saccharomyces. Saccharomyces is a genus of buddingyeast, which includes the model eukaryote S. cerevisiae.S. cerevisiae has been cultivated and used by mankind inbread making for at least five millennia (Maksoud et al.

1994), and its use as a means to produce ethanol infermented beverages is even older, dating to the Neolithicperiod (McGovern et al. 2004).

A number of the characteristics, which include a plethoraof genetic, genomic, biochemical, and molecular biologicaltools, make it ideal for industrial alcohol production.Species in the genus can ferment glucose, fructose,galactose, maltose, sucrose, xylulose, dextrin, raffinose,and starch (Wickerham 1951; Rose and Harrison 1969;Lodder 1970; Wang et al. 1980), and S. cerevisiae, amongother species, has been engineered to ferment xylose andarabinose (Kotter and Ciriacy 1992; Hahn-Hagerdal 2001;Becker and Boles 2003; Sonderegger et al. 2004;Karhumaa 2005; Kuyper et al. 2005; Wisselink et al.2007; Tian et al. 2008).

S. cerevisiae tolerates concentrations of ethanol in excessof 20% (v/v) in liquid culture (Kodama 1993; Morais et al.1996) and can produce 19% (v/v) ethanol after 2 d withsufficient substrate (Alfenore et al. 2002), and even moreover a 20-d period (Hayashida and Ohta 1981). S. cerevisiaeis the subject of considerable metabolic engineering(Nevoigt 2008) to improve its biofuel production capacities.Novel techniques such as global transcription machineryengineering (gTME) have been employed to systematicallyimprove ethanol tolerance to 7% w/v ethanol (see “Hybridapproaches” in “Strain improvement approaches” sectionon later in this review; Alper et al. 2006).

Beyond numerous attempts, some successful, at extendingthe substrate range of S. cerevisiae the species has even beenengineered to degrade lignocellulosic polymers with trans-genic enzymes from T. reesei (Lynd et al. 2005). A drawbackof S. cerevisiae is its inability to degrade and fermentcellulose and pentoses naturally but through transgenicapplications ethanol production from xylose is possible(Sedlak and Ho 2004; Brat et al. 2009). Modified strainsof S. cerevisiae are able to ferment corn stover throughdiversification sources of cellulases (Lau et al. 2010).

Prokaryotes. Bacillus. Bacillus is a remarkably diversegenus of largely saprophytic bacteria. The species ischaracterized by Gram-positive rod-shaped cells (Har-wood 1989) representing both facultative and obligateanaerobes. Widely known species include the highlyvirulent B. anthracis (the causative agent of anthrax) andthe model organism B. subtilis. Bacillus species arecapable of direct growth on arabinose, xylose, starch,raffinose, and trehalose in addition to the more commonhexose substrates (Slapack et al. 1987; Cruz Ramos et al.2000). Various products like ethanol, lactate, acetic acid,acetate, and 2,3-butanediol are all generated (Nakano et al.1997).

The genus also contains a number of thermophiles ofinterest to industry because their heat resistance allows

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them to endure enzymatic substrate hydrolysis duringfermentation. The thermophilic nature of this bacteriumwould allow for reduction in production costs as comparedto S. cerevisiae or Z. mobilis because the later microbesrequire cooling of the pretreated material before fermenta-tion can begin. Maintaining the feedstock at such a hightemperature would also reduce the risk of contamination(Fig. 1).

These strains are also resistant to general stressors suchas growth inhibitors and heavy metals (Wiegel et al. 1979;Wiegel and Ljungdahl 1981). For example, Bacillusstearothermophilus, also known as Geobacillus stearother-mophilus, produces lignocellulose-degrading enzymes(Nanmori et al. 1990), allowing it to ferment starch,arabinose, xylose, and sorbitol (Sharp et al. 1980; Payton1984; Pennock and Tempest 1988) at 70°C. This strain isbeing sequenced and has been identified as a goodcandidate for CBP (Taylor et al. 2009).

B. caldolyticus tolerates even greater temperatures, beingcapable of growth at 105°C, and doubles every 15 min at75°C (Heinen and Heinen 1972). This organism is underinvestigation for its production of thermostable extracellularamylases for use in pretreatment.

Clostridium. Species of the genus Clostridium are Gram-positive rod-shaped obligate anaerobes (Minton and Clarke1989). They contain some pathogenic species, notably theagents of botulism and tetanus. Nevertheless, the Clostridiacan use a wide range of substrates, including arabinose,xylose and cellobiose that constitute lignocellulosic hydro-lysate, and degrade xylan (Hyun and Zeikus 1985a,1985b),starch, hemicellulose, and cellulose (Mitchell 1998).

Most species of Clostridium that are of interest toindustry are those that participate in acetone-butanol-

ethanol (ABE) fermentations (Maddox 1989; Mousedale2008). During ABE fermentation, acetone, butanol, andethanol are obtained in varying proportions, depending onthe species, substrate, and culture conditions (Mishra andSingh 1993). A significant drawback is that Clostridiumproduces all three products in equal or comparablequantities; therefore, solvent purification is a difficult yetnecessary and expensive last step.

Current research has focused on improving ABEfermentation. “Hyper-producing” strains of Clostridiumbeijerinckii have been obtained that yield 42% (w/w)solvent, of which 34% (w/w) is butanol (Qureshi andBlaschek 1999). Process engineering tools have been usedto develop novel fermentation systems to further increasesolvent production to 90% (w/w; Qureshi and Blaschek1999). In addition to their broad substrate range, somespecies of Clostridia are thermophiles, which is an attractivetrait. For example, Clostridium thermocellum is capable ofgrowth and of crystalline cellulose at 65°C, due to theexpression of an endogenous cellulases (Demain et al. 2005).

The primary drawback of this species is the production oflactate and acetate as byproducts of fermentation, so researchhas focused on genetic modification of the species to increaseethanol production by mutating key enzymes in lactatemetabolism. For example, mutants lacking lactate dehydro-genase (Ldh) activity were identified in a screen for strainsresistant to a compound (fluoropyruvate), which is convertedto a toxic intermediate by Ldh (Tailliez et al. 1989b).

This procedure allowed for an increase in ethanolproduction to 89% of the theoretical yield starting fromcellulose. This strain has been primarily used for cellulaseproduction because of its relative ethanol sensitivity(Taylor et al. 2009), which if overcome would make theorganism a candidate for CBP fermentation. Clostridia areof particular interest because they are amenable to geneticmanipulation. One specie, C. thermocellum (ATCC 27405),have been sequenced and evaluated for its ability toproduce ethanol (Roberts et al. 2010) at an industrial scale.The species also has the ability to undergo specified geneticmodification through transformation of plasmid DNA(Demain et al. 2005).

Clostridia phytofermentans is another intriguing memberof the Clostridia family that is capable of directly fermentinga wide spectrum of sugar sources such as cellulose, xylan,pectin, cellobiose, glucose, fructose, galactose, mannose,arabinose, and xylose (Weber et al. 2010). Unlike C.thermocellum and other thermophilic Clostridia, its optimalgrowth temperature is between 35°C and 37°C, thus not anideal candidate for CPB without further strain improvement.This species has also been recently sequenced.

Escherichia. Escherichia coli, a gut-dwelling rod-shapedbacterium of the genus Escherichia, is better known as a

Figure 1. The components of different feedstocks used for biofuelproduction, including agricultural residues and energy crops (bothhardwoods and grasses). These substances are considered the leadingsubstrate candidates for biofuels production. As indicated by thegraphs, glucose is the most abundant component of lignocellulosicmaterials making up between 32% and 42% of the total material. The“other” lignocellulose portion includes ash, uronic acids and otherextracts that are not integral to the cellular structure of the material(USDOE).

MICROBIAL BIOETHANOL PRODUCTION 641

molecular biology tool (Kellogg and Shaffer 1993;Dworkin 2006). While E. coli can utilize a wide range ofsubstrates, including all major sugars present in plants(Alterthum and Ingram 1989), the primary reason E. coliis being considered for biofuels production is the easewith which it can be genetically manipulated and theenormous body of knowledge that has been accumulatedabout gene and protein function.

“Mixed-acids,” including lactic, acetic, formic, andsuccinic acid and small amounts of ethanol are producedduring anaerobic fermentation (Moat et al. 2002). Trans-genic E. coli harboring genes from Z. mobilis showedincreased ethanol production from xylose and glucose(Ohta et al. 1991).

E. coli ethanol tolerance has been improved by artificialselection by increasing ethanol concentration (Yomano etal. 1998), as well as immobilizing the bacterial cells onglass spheres (Zhou et al. 2008). A novel area of researchfocuses on increasing the unsaturated fatty acid levels in thelipid membrane to improve ethanol tolerance. Overexpres-sion of b-hydroxydecanoyl thio-ester dehydratase, which isinvolved in fatty acid synthesis, resulted in increased toleranceto ethanol (Luo et al. 2009).

One disadvantage is that glucose inhibits xylose uptakeand fermentation. A strain of E. coli expressing a mutatedxylose transporter from Z. mobilis is able to transportxylose in the presence of glucose, though no co-fermentation was observed (Ren et al. 2009). This strainwas further manipulated by deleting the methylglyoxalsynthase gene allowed for co-fermentation of 2% each ofglucose, mannose, arabinose, xylose, and galactose within72 h of fermentation, as compared to a control strain thattook more than 125 h to process all available sugars(Yomano et al. 2009).

Klebsiella. Klebsiella species are non-motile, Gram-negative, and rod-shaped bacteria. The genus containsorganisms that can ferment both pentoses and hexoses,including lignocellulose hydrolysate-derived xylose, arab-inose, cellobiose, and cellotriose (Dien et al. 2003).Because the primary product of fermentation is organicacids, much current research is focused on inducingethanol production.

For example, a transgenic Klebsiella oxytoca strainexpressing Z. mobilis genes was able to produce ethanolfrom glucose as well as the complex carbohydratecellobiose (Wood and Ingram 1992). Because K. oxytocacan transport cellobiose and cellotriose directly, there is noneed for additional enzymatic treatment to further depoly-merize the lignocellulosic hydrolysate into glucose mono-mers. The poor ethanol tolerance of K. oxytoca is theprimary limitation to its commercialization as an ethanolproducer (Golias et al. 2002).

Zymomonas. The genus Zymomonas contains an efficientethanol producer, Z. mobilis, which converts carbohydratesat 98% of the theoretical maximum efficiency (Skotnickiet al. 1981). The bacteria rivals and even exceeds theproduction efficiency of S. cerevisiae in some cases(Karsch et al. 1983). It is the first anaerobe found to usethe Entner–Duodoroff mechanism for glucose catabolism(Swings and De Ley 1977).

Z. mobilis produces ethanol from glucose and fructose,although some strains are capable of fermenting sucrose,sorbitol, and raffinose to varying degrees (Swings and DeLey 1977). Recombinant strains given xylose assimilationand pentose phosphate pathway genes are capable ofutilizing xylose, producing ethanol at 86% efficiency(Zhang et al. 1995). Importantly, when given glucose, therecombinant strain was nearly as effective as the controlstrain, producing ethanol at 97% the efficiency of thecontrol.

Current research has focused on developing recombinantZ. mobilis strains that can ferment pentoses (Deanda et al.1996; Mohagheghi et al. 2002). The greatest challenge tocommercialization of Z. mobilis is that it is fragile, beinghypersensitive to small changes in pH and salinity (Rogerset al. 2007).

Strain Improvement Approaches

While many microorganisms generate compounds that arebiofuels themselves (e.g., ethanol) or are of substantial useto biofuels production processes (e.g., feedstocks) (Zeikus1980), the metabolism of the organism is rarely optimizedto industrial processes as a consequence of naturalevolution (Bailey et al. 1990; Bailey 1991). Fortunately, ahost of techniques has been developed that permit optimi-zation of microbial strains so they are better suited toindustry. The process of modifying microorganisms toincrease the expression of beneficial or desired traits anddecreasing the expression of undesirable ones is termedstrain improvement (Shetty et al. 1999). Strain improve-ment has been carried out by humans for some time(Parekh 2004); for instance, in the brewer’s yeast S.cerevisiae, which has been used since prehistory toproduce alcohol (Maksoud et al. 1994). Modern techno-logy has transformed strain improvement, and today itconstitutes the following three distinct approaches: classi-cal approaches (such as artificial selection and iterativemutagenesis) metabolic engineering, and more recent“hybrid” approaches (which combine metabolic engineer-ing and mutagenesis).

Classical approaches. Classical approaches to strain improve-ment are possible with minimal technology, and as such

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have been available to mankind for some time. Artificialselection is the iterative process of selecting the individualor set of individual organisms in a population with thegreatest expression of a desired trait and expanding them(Fig. 2), thereby driving “natural” selection artificially. In2009, Huang et al. used “directed evolution” to adapt astrain of P. stipitis for improved tolerance to inhibitorsformed during the acid-based hydrolysis of rice straw. Theresearchers used sequentially increasing percentages ofhydrosylate, beginning at 20% and moving up to 100%.This process provided a strain of yeast capable oftolerating the inhibitors in the hydrosylate. The adaptedstrain was then compared to the parent strain in bothtreated and untreated hydrosylate; the researchers usedover-liming as a technique to treat the hydrolysate andremove some of the inhibitory compounds. In the testing,the adapted strain performed better than the parent in allmedia types, producing ethanol equating to nearly 87% ofthe theoretical amount based on the amount of fermentablesugars present after hydrolysis after 72 h. A similartechnique was used on E. coli to construct the commonlyused ethanologenic strain of E. coli KO11 (Yomano et al.1998) that simultaneously increased ethanol production aswell as tolerance to high concentrations of ethanol.

This process has been improved through the use ofmutagenesis, a technique that accelerates the accumulationof genetic changes through the use of mutagens. Mutagen-esis has been used successfully to engineer improvedbiofuel production phenotypes (Rowlands 1984). Mutagen-esis does not increase the chances that beneficial geneticchanges will occur; rather, it simply speeds up the rate atwhich all (i.e., phenotypically silent or detrimental)changes take place. However, since mutagens often producespecific types of mutation, it is possible to maximize thefrequency of mutations of the same type as desirablemutations (Rowlands 1984). For instance, ethyl methane-sulfonate produces only transition mutations, and of them,96% are G-C to A-T (Kohalmi and Kunz 1988). A classicexample of this used two rounds of UV mutagenesis toproduce a strain of C. thermocellum capable of producingnearly 15 g of ethanol from 63 g of cellulose substratecompared to approximately 5 g of ethanol for the controlstrain (Tailliez et al. 1989a).

Metabolic engineering. With the rise of recombinant DNAtechnology, a new form of strain improvement becamepossible. Termed “metabolic engineering” (Bailey et al.1990), the approach is defined as “the improvement of

Figure 2. Rounds of cultures are produced and the individualorganism expressing the trait maximally is expanded for anotherround of selection.

Figure 3. Metabolic bottlenecks are isolated, the genes responsibleoverexpressed to increase flux. Competing pathways are downregu-lated, further allocating metabolites through the desired pathway. Graycircle metabolite, red circle undesired end product, green circle desiredend product, red X downregulation, green arrow upregulation.

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cellular activities by manipulation of enzymatic, transportand regulatory functions of the cell with the use ofrecombinant DNA technology” (Bailey 1991). Metabolicengineering differs fundamentally from classical methodsof strain improvement in that it is directed and rational,whereas classical approaches relied on the stochasticprocess of mutation to elicit beneficial changes.

Metabolic engineering has since become further dividedinto two distinct subfields, constructive and inversemetabolic engineering (Bailey et al. 1996). In constructivemetabolic engineering, a genetic change that would producea beneficial phenotype is hypothesized, based on theexpected perturbation it produces (Fig. 3) (Bailey et al.1996). Inverse metabolic engineering, on the other hand,consists of identifying the desired phenotype in a differentorganism and then conferring that phenotype on the hostorganism (Fig. 4).

Metabolic engineering is not without certain pitfalls. Theperturbations produced by genetic changes are complex,unpredictable, and often counterintuitive (Alper et al. 2006;Bailey 1991; Bailey et al. 1990, 1996; Stephanopoulos1998). These effects can take place on the genetic level

(e.g., genetic stability and gene expression), protein level(e.g., unanticipated posttranslational modifications orproteolysis), and metabolic level. However, improvementsin metabolic modeling (Klamt and Stelling 2002), advancesin computer technology (Stephanopoulos et al. 2004;Bonchev and Rouvray 2005), and the combination ofmetabolic engineering with artificial selection approaches(Cameron and Chaplen 1997) can mitigate this. Further,constructing a cell may require as little as 256 genes(Mushegian and Koonin 1996); thus, it is possible thatmany confounding factors could be eliminated entirely byreducing the genetic complexity of industrial organisms.The majority of references discussed in the descriptions ofmajor species section allude to examples of these types ofmetabolic engineering. A representative example ofinverse metabolic engineering would be the integrationof xylose metabolizing genes from organisms like P.stipitis into S. cerevisiae to enhance its fermentative abilitythrough using both xylose and glucose for ethanolproduction (Chu and Lee 2007).

Figure 4. The desired phenotype is observed in another organism.Genetic basis for phenotype is elucidated via reverse genetics. Gene(s)is transferred to the host industrial organism, conferring desiredphenotype.

Figure 5. The gene coding for a transcription factor is isolated (in thiscase, the gene coding for the TATA Binding Protein, TBP). Numerousmutated copies are generated via mutagenic PCR, then transferred backinto host cells either against a background of the wild-type gene orreplacing it via recombination. Mutated transcription factor alters hosttranscriptome generating great phenotypic diversity. High throughputscreening isolates the best-performing individual organisms.

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Hybrid approaches Hybrid approaches utilize both thecombinatorial, stochastic processes of “classical” strainimprovement methods in conjunction with the modernrecombinant DNA technology found in metabolic engineer-ing. For instance, one technique developed by Alper utilizesdirected random mutagenesis of transcription factors toglobally alter gene expression and thus generate enormousphenotypic diversity more rapidly than traditional mutagen-esis (Alper et al. 2006; Alper and Stephanopoulos 2007)(Fig. 5). The technique, gTME, is advantageous in that it caninduce expression changes in multiple genes simultaneously.Under a traditional metabolic engineering framework, suchmodifications would need to be made sequentially, a lengthy

process that is difficult if not outright impossible. gTME hasbeen used successfully to improve the production oflycopene (when used in conjunction with metabolic genemanipulation) in E. coli as well as increase ethanol tolerance(Alper and Stephanopoulos 2007). Others have reportedsimilar successes, specifically in improving utilization ofxylose for ethanol production by S. cerevisiae (Liu et al.2008). Liu et al. used the gTME process and mutated theTATA-binding protein (spt15 in S. cerevisiae) to generatemutants capable of growth and ethanol production in 50 g/Lxylose, metabolizing nearly 94% of the xylose after 102 h.

More recently,Wang et al. (2009) developed an automatedtechnique for introducing and accumulating genetic change

Figure 6. Representational diagram of the effects of constructive genetic engineering (top panel), inverse metabolic engineering (middle panel)and gTME (bottom panel). Green is used to denote wild-type function, red indicates modulated function.

MICROBIAL BIOETHANOL PRODUCTION 645

from a pool of premade variant sequences. These variantsequences undergo iterated homologous recombination inthe host organisms, forcing rapid genetic change. Thistechnique, termed multiplex automated genome engineering(MAGE) was successfully used to increase lycopeneproduction in E. coli. By targeting up to 24 genessimultaneously, MAGE is able to overcome the simultaneousmodification barrier and generate, in the developers’estimation, some 4.3 billion mutants in a 24-h period. After3 d of being subject to MAGE cycles, variants were isolatedcapable of lycopene production five times that of theprogenitor strain (Fig. 6).

Neither gTME nor MAGE rely on genetic technologythat was previously unavailable; rather, they combineclassical strain improvement with metabolic engineering.gTME and MAGE are both targeted: gTME mutatesregulatory genes, MAGE produces variant oligomers ofgenes involved in the target metabolic pathway. Further,both introduce these genetic variants by means of recom-binant DNA technology. Thus gTME and MAGE can beconsidered types of metabolic engineering under Bailey’sdefinition. While targeted, the precise nature of the geneticchange in any individual organism subjected to gTME andMAGE is unknown; in gTME, this is because of theprocess of random mutagenesis, while in MAGE it isbecause of the enormous pool of variant oligomers andstochastic nature of their recombination. In this sense,they are related to the mutagenize-and-select approach ofclassical strain improvement that appeals not to rationaldesign but to combinatorics to elicit an optimizedphenotype.

Conclusion

The overall goal, globally, is to develop a process capableof competing with fossil fuels economically. This requiresrobust and streamlined fermentation technology minimizingwaste and time. Therefore, to streamline the process,finding or engineering organisms for high-temperature,high-yield ethanol fermentation would significantly reducecosts (Sun and Cheng 2002). In this regard, consolidatedbioprocessing is an attractive route for advancement, butfinding or making modifications to a single organism fordirect hydrolysis and fermentation is challenging.

Our analysis outlines potential microbes under consider-ation for production of lignocellulosic ethanol. It is unlikelythat any one species in particular will be used as theprimary producer; rather, a combination of several microbeswill be used in a number of different processes. Further-more, the number of published works cited in this reviewshowed evidence that mixed culture fermentations resultedin higher ethanol production and yield. In nature, there is a

vast array of lignocellulosic material available for fermen-tation, each posing its unique challenge in pretreatment andhydrolysis, and each organism used has its own specificadvantages and disadvantages for fermentation. Ultimately,the choice of microbial species will likely depend on thegeographic location and available feedstock and pretreat-ment procedures.

Whether the fermentation involves one or more micro-organisms, the following desirable characteristics shouldbe considered: (1) the microbes should be able, ideally, toutilize a broad range of substrates (i.e., hexoses andpentoses); (2) the microbial fermentation process shouldbe optimized to maximize ethanol yield and productivity;(3) the microbe must be tolerant to ethanol and othercompounds formed during pretreatment or fermentation;and (4) a general hardiness in terms of industrial processesis required for effective scale-up. Currently, no singlemicroorganism contains all these traits in such a mannerto be a decisive favorite. However, there is a globalcommitment to developing the next generation of biofuels.While there is no clear answer as to when the process willbecome economically viable, several research and engi-neering efforts are being employed and the process willcontinue to mature.

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