Consolidated Bioprocessing of Lignocellulosic Feedstocksfor Ethanol Fuel Production

Brian G. Schuster & Mari S. Chinn

Published online: 4 December 2012# Springer Science+Business Media New York 2012

Abstract Ethanol fuel can be produced renewably from nu-merous plant and waste materials, but harnessing the energyof lignocellulosic feedstocks has been particularly challengingin the development of this alternative fuel as a substitute forpetroleum-based fuels. Consolidated bioprocessing has thepotential to make the conversion of biomass to fuel an eco-nomical process by combining enzyme production, polysac-charide hydrolysis, and sugar fermentation into a single unitoperation. This consolidation of steps takes advantage of thesynergistic nature of enzyme systems but requires the use ofone or a few organisms capable of producing highly efficientcellulolytic enzymes and fermenting most of the resultingsugars to ethanol with minimal byproduct formation whiletolerating high levels of ethanol. In this review, conventionalethanol production, consolidated bioprocessing, and simulta-neous saccharification and fermentation are described andcompared. Several wild-type and genetically engineeredmicroorganisms, including strains of Clostridium thermocel-lum, Saccharomyces cerevisiae, Klebsiella oxytoca, Escheri-chia coli, Flammulina velutipes, and Zymomonas mobilis,among others, are highlighted for their potential in consoli-dated bioprocessing. This review examines the favorable andundesirable qualities of these microorganisms and their en-zyme systems, process engineering considerations for partic-ular organisms, characteristics of cellulosomes, enzymeengineering strategies, progress in commercial development,and the impact of these topics on current and future research.

Keywords Bio-ethanol . Fuel . Consolidatedbioprocessing . Simultaneous saccharification andfermentation . Co-cultures . Enzyme hydrolysis .

Pretreatment

AbbreviationsCBP Consolidated bioprocessingSSF Simultaneous saccharification and fermentationEtOH Ethanol

Introduction

Ethanol has a variety of favorable qualities as an alternativefuel to gasoline. Combustion of ethanol in a conventionalgasoline engine results in lower emissions of harmful airpollutants including fine particulates, ozone-forming carbonmonoxide, and benzene, but volatile organic compoundemissions are increased by ethanol combustion [1]. Ethanolhas been shown to reduce net greenhouse gas emissionswhen it replaces gasoline, and it has the potential to becomenet carbon neutral or even carbon negative due to carbonbiomass sequestration, leading to the mitigation of globalclimate change [2]. Although it reduces greenhouse emis-sions, is it actually renewable? Life cycle analyses havedemonstrated that ethanol from corn, wheat, and sugar beetsis renewable and offers a net energy gain [3, 4]. Despite itslower energy content than gasoline, ethanol’s high octanerating reduces engine knock, improving engine perfor-mance, even in dilute ethanol–gasoline blends [5]. Mostimportantly, ethanol and other biomass-derived fuels havethe potential to replace petroleum-based fuels as renewable,climate-neutral alternatives. As society faces rising gas pri-ces, new, fundamentally sustainable transportation fuel sour-ces must take the place of diminishing fossil fuel reserves.Research and development on the entire alternative fuelproduction supply chain will be critical to these efforts.

B. G. SchusterDepartment of Chemical & Biomolecular Engineering,North Carolina State University,Raleigh, NC 27695, USA

M. S. Chinn (*)Department of Biological & Agricultural Engineering,North Carolina State University,Raleigh, NC 27695-7625, USAe-mail: mschinn@ncsu.edu

Bioenerg. Res. (2013) 6:416–435DOI 10.1007/s12155-012-9278-z

For decades, ethanol fuel from simple sugars and starchhas been produced by a conventional method that consists ofmultiple, distinct steps involving the use of industrial yeaststrains to ferment certain sugars to ethanol. Brazil’s abun-dant sugarcane crops are a great feedstock for this process,and in the USA, starch-rich corn is the primary feedstock forethanol production [6]. With some extra processing, starchescan be enzymatically cleaved into simple sugars and fer-mented with relatively little difficulty. The major difficultiesarise when lignocellulosic feedstocks are used to produceethanol due to the structural complexity and polysaccharideheterogeneity [7]. Despite the challenges of using lignocel-lulose, there is a vast supply of this biomass available acrossmany climates. Crops intended for lignocellulosic ethanolproduction are sometimes cheaper to grow and harvest thansugar- or starch-rich crops [8]. In other cases, lignocelluloseis considered a waste material with no value at all [9–12].

Consolidated bioprocessing (CBP) is an alternative methodthat combines certain steps of the biomass conversion processin order to reduce the cost of ethanol production from plentifullignocellulosic feedstocks. Contrary to conventional ethanolproduction, CBP combines the steps of enzyme production,polysaccharide hydrolysis, and sugar fermentation into oneunit operation performed by one or several organisms [13,14]. In addition to offering an economical platform for ethanolproduction, CBP also has the potential to improve the produc-tion of other chemicals and fuels, such as butanol, from rawbiomass [15, 16]. In this research review, we discuss opportu-nities for the consolidated production of ethanol as well asseveral candidate organisms for CBP.While past reviews, suchas those by Lynd et al., Olson et al., and van Zyl et al. [13,17–19], have discussed consolidated bioprocessing progress,we comprehensively present specific prospects and challengesassociated with consolidated ethanol production and makerecommendations for future research. In particular, we providevaluable insight into some of the most promising microorgan-isms for fuel production from lignocellulosic biomass.

Lignocellulosic Feedstocks

One of the major incentives for utilizing lignocellulose as afeedstock for ethanol production is that it is ubiquitous in fast-growing perennial grasses, forestry residues, and crop residues[20]. Lignocellulose, consisting of cellulose, hemicellulose,and lignin, is a fundamental component of all plants. Approx-imately 70 million dry metric tons of crops are wasted everyyear, but much of this can be utilized for ethanol production[20]. Collecting crop residues can, however, increase cropprices by reducing organic matter available to the next seasonof crops and by causing higher rates of erosion if properagricultural management practices are not utilized [20, 21].Some examples of byproduct crop residues and waste biomass

that could be converted to ethanol are corn stover, barleystraw, oat straw, rice straw, sorghum bagasse, wheat straw,sugarcane bagasse, nut shells, corn cobs, leaves, and paper[20, 22]. Ethanol production also offers a profitable and re-newable alternative use of disposed paper, which could re-place a significant proportion of North American gasolinedemand as an ethanol feedstock [23]. Additionally, there arenumerous plants that could be grown specifically for ethanolproduction as dedicated energy crops, including switchgrass,Bermuda grass, poplar trees, and sweet sorghum. One analysisindicated that the conversion of nearly 42 million acres of USfarmland to switchgrass crops could offer a $6 billion increasein annual farm income compared to existing agricultural uses[24]. However, competition between energy and food cropsshould be avoided to prevent major increases in food prices.Figure 1 displays the cellulose and hemicellulose composi-tions of several potential lignocellulosic ethanol feedstocks.

Lignocellulose Composition and Hydrolysis

Among the three main carbohydrate components in ligno-cellulose, cellulose, the longest component of most plantcell walls, is a polymer of D-glucose connected by glyco-sidic bonds. Cellulose chains are bound in a microfibrilarrangement, where individual polysaccharides are

Fig. 1 Cellulose and hemicellulose constituent compositions (%mass). Lignin, not shown, is another major component of lignocellu-losic feedstocks [9–12, 22, 112]. Asterisk Algal cell wall is derivedfrom the freshwater green alga, Chlorella pyrenoidosa

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connected in parallel by hydrogen bonds and other intermo-lecular forces [25]. Hemicellulose, a component that comesin a variety of forms, is a polymer of pentose and hexosesugars, as well as sugar acids [26, 27]. The most commonhemicelluloses are xylan, mannan, and arabinofuranosyl,which differ in their composition and arrangement, butprimarily consist of xylose, arabinose, and glucose [28,29]. Xylan is one of the most abundant renewable polysac-charide in nature [30, 31]. Based on scanning electronmicrographs, hemicellulose has been hypothesized to pro-vide a matrix for attachment of cellulolytic microbes, suchas those used in CBP [32]. Lignin, the most obstructivecomponent, is a complex, heterogeneous polymer connect-ing hemicellulose to cellulose, reinforcing the structuralintegrity of the cell wall. Despite its structural inconsistency,lignin is constructed principally from three p-hydroxycin-namyl precursors: p-coumaryl alcohol, coniferyl alcohol,and sinapyl alcohol [33–35]. Because of lignin’s complexityand extreme heterogeneity, the chemical interactions be-tween lignin, cellulose, and hemicellulose are not fullyunderstood, though it has been proposed that covalent link-ages exist between these polymers [33].

Figure 2 illustrates various polysaccharides withinplant cell walls and the sugars resulting from the cleav-age of these carbohydrates, as well as the hydrolyticenzymes responsible. A number of different enzymesare used to achieve the end goal of obtaining smallermono- and disaccharides by hydrolysis, or saccharifica-tion. Cellulases and hemicellulases are a class ofenzymes that cleave glycosidic bonds linking sugarswithin polysaccharides. Hemicellulases, including xyla-nase, mannanase, arabinanase, galactanase, arabinofura-nosidase, and certain esterases, form a subgroup ofhemicellulose-degrading enzymes [29]. Endoglucanasescleave bonds internally along cellulose polymers, where-as exoglucanases (cellobiohydrolases) processively cleavecellobiose and other oligosaccharides from the termini. Thesetwo cellulases act synergistically to degrade cellulose [36–38].The third key enzyme class, β-glucosidases, is responsible forthe hydrolysis of cellodextrins, such as cellobiose, to glucose.

It is critical that a feedstock can be hydrolyzed to simplesugars, but the ease with which plant materials are decom-posed varies greatly. The digestibility of a particular plantfeedstock [39] depends on the biomass particle size [40],cell wall porosity [41], cellulose crystallinity (structuralorder) [42], hemicellulose side-chain branching [43], poly-saccharide cross-linking [44, 45], and lignin content [44].Changes in these factors have a considerable effect ondigestibility because they affect the ability of enzymes andchemical reagents to access functional sites on the carbohy-drates. These aspects of the substrate can be altered todiffering extents by a variety of pretreatment methods,reviewed in more depth by Sousa et al. [39].

Conventional Bioprocessing

Conventional bioprocessing as a means of manufacturingethanol consists of the individual processes of pretreatment,enzymatic hydrolysis, fermentation, and product recovery(typically ethanol distillation). However, this form of bio-processing requires that the hydrolytic enzymes be producedexogenously, adding significant costs due to enzyme purifi-cation. A few ways of improving the cost effectiveness ofthe ethanol supply chain while maintaining conventionalmeans of production are under development, such as im-proving biomass collection and storage, reducing the size offeedstocks before transport, and genetic modification ofplants to alter their structure and composition [14]. The goalof consolidated bioprocessing is to replace conventionalmethods as a more cost-effective and efficient approach tofuel ethanol production.

Pretreatment

After biomass has been harvested and collected, the rigidlignocellulose structure, in particular the recalcitrant andhighly variable lignin structure, of the plant material mustbe partially disrupted so that enzymes can access the carbo-hydrates present in order to hydrolyze them before they arefermented. Pretreatment has been estimated to represent 16–19 % of the capital investment of ethanol production today[46]. During pretreatment, it is favorable to avoid degradinglignin completely because it has uses in other commodities,such as binders for bricks and feed pellets; dispersants foroil, pesticides, and concrete, as well as water treatmentadditives [47]. Another constraint of pretreatment is theformation of fermentation-inhibiting toxic compounds, suchas vanillin, acetate, syringaldehyde, hydroxybenaldehyde,hydroxymethylfuraldehyde, and furaldehyde. Vanillin isthe most inhibitive, mainly affecting xylose fermentationby some yeast species, while acetate is the least toxic [48].Physical, chemical, and biological pretreatment as well assolvent fractionation, can be used in conjunction (i.e., com-bined physical and chemical methods) or individually, asdescribed below [39]:

1. Physical pretreatment uses mechanical stress togrind the feedstock into small particles, increasingthe surface area/volume ratio, which facilitates ele-vated enzyme activity. For example, ball millinguses tumbling spheres to grind the substrate[49–51].

2. Solvent fractionation takes advantage of the differentialdissolution of lignocellulose components by varioussolvents. These components are partitioned by disrup-tions to the hydrogen bonds between cellulose micro-fibrils [52]. The Organosolv process removes lignin,

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making cellulose more accessible, and recovers the al-cohol solvent, but requires large solvent inputs [53, 54].Phosphoric acid fractionation decomposes the cell wallinto its components and requires moderate heat, butrecovery of the phosphoric acid is difficult [55, 56].Ionic liquid fractionation yields highly digestible cellu-lose using moderate heat, but the chemical cost is veryhigh without recovery of the ionic liquid [57, 58].

3. Chemical pretreatment employs the use of acidic, alka-line, or oxidative compounds to degrade covalentbonds. Steam explosion, involving an abrupt transitionfrom high to low pressure in the presence of an acidiccatalyst, is an example of acidic pretreatment [59, 60].Similarly, ammonia fiber expansion utilizes ammonia tosolubilize and extract lignin and decrystallize cellulose[61]. Oxidative pretreatment uses oxidants, such as hy-drogen peroxide, to remove lignin and hemicellulose,but it yields inhibitory byproducts [62]. Ultraviolet ra-diation and other forms of radiation have also been usedto degrade and remove lignin [63, 64].

4. Biological pretreatment involves the use of organisms,such as basidiomycetes and actinomycetes fungi, toremove lignin enzymatically rather than using harshchemicals. Although enzymes are often an effectivemeans of hydrolyzing these carbohydrates, the organ-isms consume some of the cellulosic substrate, reducingyields, and are relatively expensive to maintain [39].Phanaerochaete chrysosporium is an example of a fun-gus with ligninolytic enzymes. In one experiment, afungal culture was used to pretreat dried, ground ricestraw, and inhibitory byproducts were not an issue;however, this biological approach required much moretime than acidic pretreatment [65]. Trametes versicolorand Flammulina velutipes (discussed further in

“Organisms of Interest”) have also been investigatedfor lignin removal [66]. Heterologous expression ofthese organisms’ enzymes in combination with cellu-lases and hemicellulases may offer better results.

Enzymatic Hydrolysis (Saccharification)

There are many variations of enzymes responsible for poly-saccharide hydrolysis, most of which are produced commer-cially by genetically modified strains of Trichoderma reesei,Fusarium venenatum, Aspergillus oryzae, and Aspergillusniger [67]. Catalytic rate constants of these enzymes aresignificant, but adsorption of cellulases to feedstock sub-strate is also a major factor affecting the efficiency ofhydrolysis [68]. The noncatalytic polypeptide sequences atthe ends of some cellulases, known as carbohydrate bindingmodules, enhance adsorption of enzymes to cellulose, im-proving degradation [69]. Oxidative compounds, such asfree hydroxyl radicals, cleave covalent linkages, allowinglarge enzymes or enzyme complexes access into lignocellu-lose pores [70, 71]. Not only is it important to cheaply massproduce enzymes with high specificity, but it is also valu-able to consider process engineering as a means of reducinginputs. For example, enzyme immobilization and cell teth-ering could reduce enzyme inputs [72, 73]. In addition, pHrange, temperature range, time, substrate loading, enzymeconcentration, synergistic enzyme cooperation, and sub-strate loading must be manipulated to achieve optimal yields[74, 75]. Enzyme recycling has also received attention forthe potential to reduce inputs [76, 77], although efforts arelimited by enzyme adsorption to lignin. Adsorption is oftenprevented by altering pH, adding a surfactant, or removinglignin prior to hydrolysis [77]. Further process improve-ments have involved combining ligninolytic and cellulolytic

Fig. 2 Flow diagram ofpolysaccharides and thehydrolytic enzymes used tocleave them into shorterpolymers and simple sugars[29, 105]. Endoglucanase andexoglucanase are cellulases,while xylanase, arabinase,arabinofuranosidase,mannanase, and galactanase arehemicellulases [29].

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enzymes to achieve pretreatment and saccharification in asingle step [75], though the economics of such an approachremain indeterminate.

Enzyme Engineering

A large effort has been made to engineer enzymes withimproved activity and stability by both rational design anddirected evolution. Rational design involves specific en-zyme modifications that require a detailed understandingof the relationship between amino acid sequences and func-tion. Unfortunately, this sequence–function relationshipremains an enigma for the vast majority of enzymes, sodirected evolution is often preferred [78, 79]. In particular,the most challenging rational design approaches are thosethat involve intentional modification of specific amino acidssince it is very difficult to predict how small molecularchanges affect the desired function [78]. Many enzymaticproperties are not confined to one or a few amino acid sites,but are usually distributed far across a protein [80]. None-theless, a successful enzyme engineering approach has beenachieved in creating a multifunctional hemicellulase bycombining catalytic domains of two enzymes via a flexiblepeptide linker. The resulting chimera contained xylanase,endoglucanase, arabinofuranosidase, and xylosidase activityderived from a Clostridium thermocellum β-xylanase geneand an α-arabinofuranosidase gene from a previously un-cultured bacterium in a compost starter mix. The engineeredhemicellulase performed better than both parental enzymesin degrading arabinoxylans and corn stover [81].

Directed evolution, the primary approach to enzyme en-gineering, is a group of techniques used to screen mutantenzymes for those with the most favorable characteristics. Itis most effective when the enzyme of interest initially hasmost of the desirable properties. With only a few mutations,directed evolution can yield enzymes with substantialimprovements. Random mutagenesis, the most basic direct-ed evolution strategy, is a way to uncover arbitrary enzymeenhancements through random mutations to a parent gene,often using error-prone PCR. Site-directed (targeted) muta-genesis can be more effective because only a few keyresidues are selected for mutation [79]. The technique hasalso been a critical tool in characterizing cellulases, espe-cially the catalytic and binding residues [82]. Alternatively,genetic recombination by DNA shuffling may be utilized tocombine structurally similar enzymes, sometimes resultingin a greater degree of structural alterations than mutagenesis.The best results are often achieved with a combination ofmutagenesis and DNA shuffling since multiple iterations ofmutagenesis without shuffling may lead to diminished per-formance [79]. For each technique, it is pivotal to establish adirect connection between enzyme performance duringscreening and actual performance on the desired feedstock.

However, quantitative cellulase and hemicellulase assaysare difficult to develop due to the heterogeneity of plant cellwalls, making it a challenge to identify the best saccharo-lytic enzymes. Zhang et al. [25] emphasize that the key toefficient cellulase selection is to utilize a continuous cultureof mutants with cell-surface enzyme displays acting on aninsoluble cellulosic substrate.

One common application of directed evolution has beenthermostability enhancement to match enzyme temperatureoptima to process temperatures, especially using hyperther-mostable parent enzymes [83, 84]. Thermostability is im-portant not only because it allows for higher processtemperatures but also because it reduces the rate of enzymedenaturation at moderate temperatures, making enzymerecycling more economical [77]. In order to bring optimaltemperatures of archaeal β-glycosidases to industrially ap-plicable levels, Kaper et al. reduced temperature optima byshuffling two genes: CelB from Pyrococcus furiosus andLacS from Sulfolobus solfataricus [83]. Xylanase geneshave also been shuffled to improve thermal stability andpH optima, but an attempt by Gibbs et al. [85] did notyield improved enzymes due to the low number ofvariants screened. On the contrary, a sevenfold increasein endoglucanase thermostability has been achieved viarecombination of two genes from Clostridium cellulo-vorans [86]. The thermostability of a family 5 glycosi-dase from Clostridium phytofermentans has also beenimproved [87]. Excessive stability could result in dimin-ished catalytic activity, but there may be methods ofworking around such a limitation [88].

Other alternatives have also been investigated to reducethe cost of enzymatic hydrolysis, such as the formation ofcellulases by transgenic tobacco, which have been reportedto produce enzymes with higher thermostability and lowerpH specificity than Escherichia coli [89]. Not only is en-zyme engineering critical to the economic viability of CBP,but enzyme expression must also be optimized to achievesuccessful saccharification processes [86]. Enzyme en-hancement has been the center of many efforts, but thereremains much work to be done in optimizing the metabolicregulation of enzyme expression.

Fermentation

During fermentation, ethanol, carbon dioxide, acetic acid,hydrogen, and other organic products are typically producedfrom simple sugars under anaerobic conditions. Formationof acetic acid and other organic acids is suppressed as muchas possible. A range of organisms, both natural and genet-ically engineered, have varying efficiencies of fermentationand yield products of differing compositions, some withhigher ethanol concentrations than others due to the utiliza-tion of different metabolic pathways [90]. Raising bioreactor

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temperature above ambient levels can improve fermentationto an extent by increasing the organism’s growth rate, in-creasing cellulase activity, and decreasing the risk of con-tamination [91]. Despite its overall value, ethanol is aninhibitor of the process of fermentation itself, interferingwith the cell membrane and initiating negative feedbackcell signaling pathways. In respiratory microbes, thisinhibition is linear with respect to concentration, but inanaerobes, inhibition is exponentially correlated [92].Inhibition is less severe when the ethanol is addedexternally than when it is produced by the organism’sown fermentation due to its higher concentration withinthe cell during fermentation [92].

Product Recovery

Ethanol recovery is commonly achieved by distillation,which involves heating the product solution in order tocreate a vapor enriched with the more volatile species,ethanol; however, the energy requirement for distillation ishigh [93, 94]. Pervaporation is an alternative or addition todistillation that uses a selectively permeable membrane toallow only ethanol to pass through and evaporate. Thisprocess is especially valuable when combined with thefermentation step because it removes ethanol as it is formedin order to avoid inhibition [95].

Metabolic Engineering and Genetic Modification

A range of techniques have been used to manipulate geneexpression and alter metabolic pathways. Metabolic engi-neering has served as a means of expanding opportunitiesfor microorganisms to create new products or to increaseefficiency by shutting down unnecessary pathways or favor-ing useful pathways [96]. Efforts to improve the field con-sist not only of specific experiments designed to alterorganisms but also investigations of ATP allocation withinan organism’s array of metabolic pathways [97] and trackingof metabolites by flux analysis [98]. Genetic engineering isan essential tool for obtaining organisms with the ability tocreate products on an industrial scale, and researchers in-vestigating ethanol bioprocessing have used various meth-ods to obtain desirable organisms: gene knockout [99], geneknockdown (RNA interference) [100], overexpression [14],and genetic recombination by viral transduction and trans-formation [101, 102]. Genes targeted for manipulation havebeen those involved with substrate attachment, substrateuptake, enzyme secretion, intracellular metabolite transport,cell growth and maintenance, feedback systems controllingsugar consumption, and fermentation [96]. Strain breeding,an alternative to active genome manipulation, has been usedto select for the best organisms to degrade lignocellulose or

produce ethanol under a certain set of conditions [103].Mutagenesis, or directed evolution, has been utilized toachieve similar goals on a larger scale by creating randommutations among many cells and screening for the bestmutants [103, 104].

One of the largest barriers to progress in genetic engi-neering microbes for utilization in CBP has been the sheerquantity of different genes to manipulate in a variety of hostorganisms. Furthermore, once the best genes are selected,the genes must be correctly expressed. Not only is propertranslation necessary, but posttranslational steps must alsoremain functional in order to avoid improper folding andsubsequent impairment of enzymes caused by endoplasmic-reticulum-associated protein degradation mechanisms andunfolded protein response [103]. It must be noted that evenwith a perfect metabolic pathway, successful genetic engi-neering requires that protein transport remains uninterruptedwithin the cell and its membrane [103]. There is still muchwork to be done in fine tuning microbes’ metabolism andgenetic expression, but the potential rewards for such re-search are great.

Consolidated Bioprocessing

Consolidated bioprocessing is the combination of enzymeproduction, polysaccharide hydrolysis (i.e., saccharifica-tion), and multisugar fermentation into one step, ideallyachieved by a single organism [14]. The cost of this formof ethanol production compared to conventional bioprocess-ing has been projected to be lower than the traditionalprocess once the technology matures [13], although theprocess still faces hurdles in becoming industrially viable.The following are a few of the fundamental benefits associ-ated with CBP:

1. Saccharification is limited by product inhibition. InCBP, fermentation transforms these products beforethey become inhibitive to hydrolysis [105].

2. In contrast to the use of exogenous saccharifyingenzymes, CBP organisms generate their own cellulolyt-ic and hemicellulolytic enzymes for lignocellulose de-composition, offering large cost savings [19, 106].

3. Consolidated bioprocessing requires fewer unit opera-tions (i.e., fewer reactor vessels), reducing maintenanceand capital costs [14, 19].

4. Pretreatment could be avoided partially or entirely ifCBP microbes achieve sufficient yields without this step[19, 105].

However, consolidated bioprocessing is also potentiallylimited by process optimization. For example, the tempera-ture in a single unit may not be optimal for both saccharifi-cation and fermentation [105].

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Consolidated bioprocessing is designed to achieve severalfunctions, depending on the organisms utilized. FundamentalCBP involves the use of a single organism to complete allsubstrate hydrolysis and ethanol fermentation without the aidof exogenous enzymes or pretreatment. Cocultures may alsobe utilized such that (1) one organism is saccharolytic whilethe other is ethanologenic, or (2) both organisms are ethano-logenic, but each provides different key saccharifyingenzymes. SSF is similar to CBP, but enzymes produced in aseparate process are added to the fermentation step to aid inhydrolyzing the lignocellulosic substrate.

Cellulosomes are a natural means of harnessing the syn-ergistic nature of certain hydrolytic enzymes in order todegrade lignocellulose. A cellulosome is an exocellularenzymatic complex associated with some anaerobic fungi(e.g., Neocallimastix frontalis) and bacteria (e.g., Clostridi-um thermocellum, C. cellulovorans, C. papyrosolvens, andC. cellulolyticum). The complex attaches to lignocellulosicsubstrates via carbohydrate-binding modules or polypeptideregions on each enzyme with an affinity for polysacchar-ides. The saccharolytic enzymes are anchored to scaffoldingproteins, the scaffoldins (CipA), by the dockerin domains oneach enzyme. At least 71 genes code for enzymes capable ofintegrating with the cellulosome in C. thermocellum [69,107]. Cellulosomes are typically more complete in theirhydrolysis of polysaccharides than free enzymes becauseenzymes contained within cellulosomes are synergistic. Asthe endoglucanases create new termini, the exoglucanaseshave more substrate to cleave. In contrast to free enzymes,cellulosomes fixed to cells concentrate hydrolysis productsnear the cell, which is favorable for fermentation of theresulting sugars [106, 108]. Similar to a cellulosome, arosettazyme is a synthetic enzyme complex structuredaround an 18-subunit, self-assembling protein formation,the rosettasome. The structure was designed by Mitsu-zawa et al. [107] to dock four cellulases, which can beselected specifically for a particular feedstock. Cellulo-somes and rosettazymes have significant potential bene-fits for CBP due to their ability to greatly improvesaccharification yields.

The qualities and outputs of various cellulolytic organ-isms, some of which have been genetically engineered toproduce ethanol, are summarized in Table 1. Over 14,000fungal species [105], in addition to a number of other micro-organisms, are capable of cellulose degradation, but only aselect few are presented. General goals for ethanologenic(i.e., ethanol-producing) organisms are to increase theirethanol yield, eliminate byproducts, improve ethanol toler-ance, and to find new metabolic pathways to simultaneouslyferment multiple types of sugars [14]. Favorable features ofsaccharolytic organisms are adherence to lignocellulose,cleavage of oligosaccharides (esp. cellobiose), high specificactivity of cellulase systems [13], and enzyme stability.

Summary of Consolidated Bioprocessing CandidateOrganisms’ Performance

Ideally, an industrial CBP organism could grow directly onuntreated plant material, though some of the wild-typeorganisms detailed in Table 2 are unable to hydrolyze cel-lulose. Nonetheless, many of these microbes have beengenetically modified to be capable of saccharification. Inall studies reviewed, these ethanologens were either grownon simple sugars, artificial cellulose, or actual biomass.Some were cultured concurrently with Spezyme CP, a mix-ture of commercial exogenous hydrolytic enzymes.

If a microbe required genetic modification or exogenouscellulases/hemicellulases, this information is supplied in the“Hydrolysis” column, where Cel 0 cellulase, Xyl 0 xyla-nase, Pec 0 pectinase, and Clm 0 cellulosome (cell-bound).Absolute ethanol yield (“Abs. EtOH Yield”), measured asgram EtOH/gram substrate, indicates an organism’s abilityto produce ethanol from a particular feedstock. Fermentativeethanol yield (“Ferm. EtOH Yield”), measured as gramEtOH/gram sugars reflects how well an organism fermentssugars. A comparison of absolute yield with fermentativeyield sheds light on an organism’s ability to saccharifysubstrate and transport sugars. The theoretical yield is basedon either the absolute yield (abs) or fermentative yield(ferm) and is based on a maximum theoretical yield of0.51 g/g. The value for ethanol concentration gives anindication of the cost and energy-intensiveness of the prod-uct recovery step.

Organisms with Combined Saccharolyticand Ethanologenic Capabilities

Clostridium thermocellum

This thermophilic anaerobic bacterium is an example of aCBP organism that naturally degrades cellulose and is alsoable to ferment the resulting sugars primarily into ethanol,lactate, acetate, carbon dioxide, and hydrogen [74, 109]. C.thermocellum has been isolated from such sources as horsemanure, human feces, soil, marine mud, elephant droppings,and goat droppings [110–112] and was first reported to havebeen isolated and used to convert cellulose to ethanol in1925, when it was found that the bacterium could fermentpolysaccharides (cellulose, starch), oligosaccharides (su-crose, maltose, raffinose, and lactose), and monosaccharides(galactose, mannose, fructose, xylose, and arabinose) [113].

Because of its preferential fermentation of certain sugars,C. thermocellum has been cocultured with other bacteriathat are able to convert pentose sugars to ethanol. Reddyet al. [112] combined the abilities of two other anaerobicthermophiles, Thermoanaerobacter ethanolicus ATCC

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31937 and Clostridium thermosaccharolyticum HG8, withC. thermocellum CT2 in order to better utilize pentosesresulting from hydrolysis of banana agricultural residue,which is composed of roughly equal masses of hemicellu-lose and cellulose. This transgenic strain, among manyothers, represents an enhancement of wild-type strains.

The bacterium is well known for its exocellular cellulo-lytic enzyme complex, the cellulosome. In C. thermocellum,this consists of endoglucanase (CelA), cellobiohydrolase(CelK), and xylanolytic (XynC) subunits, as well as themost common processive exoglucanase (CelS) subunit[114]. When the cellulosome was separated and used tohydrolyze cellulose in the presence of a different ethanolo-genic bacterium, hydrolytic activity was greatly diminished,indicating the significance of the synergistic enzyme-cellrelationship [106].

The efficacy of the cellulases themselves is another fun-damental factor of hydrolysis, so it is important to compareactivity with enzymes derived from commercial microor-ganisms. In a comparison of C. thermocellum’s isolatedcellulases to Trichoderma reesei’s, C. thermocellum’senzymes hydrolyzed cotton faster than T. reesei’s. It wasalso found that 37 °C was the optimal temperature to main-tain residual cellulase activity, even though the bacteriumitself thrives in hotter conditions [108]. Also differing fromthe organism’s optimum environment, the optimal condi-tions for endoglucanase activity are 65 °C and pH 5.2, and64 °C and pH 5.4 for exoglucanases [115]. It is important toconsider that cellulase synthesis not only affects hydrolysis,but it also requires large quantities of energy from a cell,markedly affecting growth rates and fermentation [114].

A good amount of research has involved engineeringprocesses to be more productive and less costly, especiallywith C. thermocellum. One example is solid substrate culti-vation (SSC), an alternative to submerged fermentation(SmF) that uses less water to grow the organism. Theprimary advantages are higher product concentrations, low-er downstream processing costs, and less energy input [116].However, higher product concentrations are typically fleet-ing since liquid is needed to extract the products [117].Furthermore, SSC initially offers more rapid end productformation, but after a short period, product concentrationsequilibrate at levels below those achieved in submergedfermentation due to product inhibition [117, 118].

Another process engineering approach has been to alterhydrogen concentration in the process in order to increasethe ratio of ethanol-to-byproduct formation. It has beenshown that stirring cultures leads to decreased dissolvedhydrogen, shifting C. thermocellum’s equilibrium to pro-duce more hydrogen and acetate [119–122]. On the con-trary, adding hydrogen leads to a higher ratio of ethanol toacetate [120]. According to Henry’s Law, increasing thevessel pressure could also increase ethanol/acetate ratiosby minimizing hydrogen transfer from the aqueous to gas-eous phase.

While process design has the potential to improve ethanolproduction from C. thermocellum, genetic engineering canalso increase ethanol yields. Computationally modeled met-abolic processes for the entire genome are able to facilitate agenetic engineering approach to improving the organism’sethanol production capabilities [109]. Stevenson andWeimer [123] noted that transcriptional regulation is not

Table 1 Overview of the cellulolytic and fermentative abilities of several wild-type microorganisms

Wild-type organism Sugars fermented to ethanol

Cellulolytic Hexoses Pentoses Cellobiose Ethanol tolerance Sources

Clostridium thermocellum Yes Yes No Yes 14 g/L 180

Thermoanaerobacterium saccharolyticum Xylanolytic Yes Yes – – 101, 125

Clostridium cellulolyticum Yes Yes No Yes – 127

Fusarium oxysporum Yes Yes Yes Yes 15 g/L 129, 130

Saccharomyces cerevisiae No Yes No No Higha 103

Klebsiella oxytoca No Yes Yes Yes Higha 138

Zymomonas mobilis No Yes No No 100 g/L 141, 142

Pichia stipitis No Yes Yes Yes 46 g/L 92, 116

Candida shehatae No Yes Yes No 48 g/L 92, 104

Flammulina velutipes Ligninolytic Yes No Yes 120 g/L 144

Escherichia coli No No No No 60 g/L 73, 147

Trichoderma reesei Yes No No No – 151

Thermoascus aurantiacus Yes, and xylanolytic No No No – 152, 153

Thermomyces lanuginosus Xylanolytic No No No – 153

a High ethanol tolerance indicates that the value is not tabulated and is well above tolerance levels for other organisms

Bioenerg. Res. (2013) 6:416–435 423

Tab

le2

Sum

maryof

consolidated

biop

rocessingcand

idateorganism

s’performance

Organism

species

Strain

Substrate

Tim

e(h)

Hydrolysis

Abs.EtOH

yield(g/g)

Ferm.EtOH

yield(g/g)

Theor.yield

Final

EtOH

Conc.(g/L)

pHTemp.

(°C)

Source

Therm

ophilic

anaerobes

C.thermocellum

ATCC27405

Alkali-pretreated

bagasse

100

None

0.109a

0.19

21%

a(abs)

1.09

7.0

60180

C.thermocellum

SS19

Filter

paper

120

None

–0.41

81%

(ferm)

––

60110

C.thermocellum+C.

thermosaccharolyticum

+T.

ethanolicus

CT2,

HG8,

ATCC31937

Alkali-pretreated

banana

waste

192

None

0.22

a0.41

43%

(abs)

227.5

60112

C.thermocellum

ATCC27405

Crystallin

necellu

lose

240

None

0.17

0.27

33%

a(abs)

2.66

6.7

60117

C.thermocellum

ATCC27405

Sugarcane

bagasse—

solid

240

None

––

–3.5

–60

116

C.thermocellum

ATCC27405

Paper

pulp

sludge—

solid

240

None

––

–14.1

–60

116

T.saccharolyticum

B6A

-RI

Xylan

24None

––

–1.75

–60

125

T.saccharolyticum

ALK2

Xylose—

continuous

10None

N/A

0.46

90%

(frem)

3.22

5.2−

5.5

55101

T.saccharolyticum

ALK2

Crystallin

ecellu

lose

90Cel

0.42

a−

82%

(abs)

215.0

50101

Facultativ

eAnaerobes

S.cerevisiae

424A

(LNH-ST)

AFEX-pretreatedcorn

stover

96Cel,Xyl,Pec

0.192

0.46

61%

(abs)

404.8

50181

S.cerevisiae

EBY100(m

odified)

Phosphoricacid

swollen

cellu

lose

48Clm

–0.49

95%

(ferm)

3.5

–30

137

K.oxytoca

P2

Mixed

waste

office

paper

80Cel

0.426

–83

%(abs)

39.6

5.0−

5.2

35138

K.oxytoca+K.

marxianus

P2,

CBS12

Crystallin

ecellu

lose

168

Cel

0.405

–79

%(abs)

40.5

5.0

37140

K.oxytoca+Z.mobilis

P2,

ZM4

Crystallin

ecellu

lose

168

Cel

0.381

–75

%(abs)

38.1

5.0

37140

K.oxytoca

P2

Acid-pretreated

sugarcane

bagasse

144

Cel

0.39

–69

%(abs)

19.2

5.2

3532

F.veluptipes

Fv-1

Glucose

144

N/A

N/A

0.45

88%

(ferm)

4.5

–25

144

F.veluptipes

Fv-1

Cellobiose

192

N/A

N/A

0.45

83%

(ferm)

4.5

–25

144

P.stipitis

NRRY-7124

Acid-pretreated

wheat

straw

60H2SO4

0.36

–70.6

%(ferm)

12.9

6.5

2828

P.stipitis

CSIR-Y

633

Glucose,mannose,

galactose,xylose

48N/A

0.37

––

18.6

5.0

30104

C.shehatae

CSIR-Y

492

Glucose,mannose,

galactose,xylose

38N/A

0.27

––

13.4

5.0

30104

E.coli

KO12

Glucose

48N/A

N/A

0.57

112%

b(ferm)

54.4

6.0

30146

E.coli

KO11

Xylose

48N/A

N/A

0.53

103%

b(ferm)

41.6

6.0

30146

F.oxysporum

F3

Wheat

straw

144

None

0.48

a–

89%

(abs)

9.6

6.0

34129

Mesophiles

Z.mobilis

ZM4

Glucose

32N/A

N/A

0.5

98%

(ferm)

305.0

30141

Z.mobilis

CP4(pZB5)

Xylose

46N/A

N/A

0.44

86%

(ferm)

11–

30142

C.cellu

lolyticum

H10

Crystallin

ecellu

lose

240

None

~0.14a

––

0.18

7.0

37127

aValue

was

calculated

from

prim

arydata

bValuesareabov

e10

0%

becausethemicrobe

ferm

entedvariou

smedia

compo

nentsforwhich

theauthor

didno

ttake

accoun

t

424 Bioenerg. Res. (2013) 6:416–435

the only factor responsible for changes in ethanol produc-tion and end product distribution by C. thermocellum; intra-cellular substrate concentration and protein activation alsoplay a role in metabolism and genetic expression. Theseconsiderations are applicable not only to C. thermocellumbut also to a number of other CBP candidate organisms.

Thermoanaerobacterium saccharolyticum

Isolated from thermal springs in Wyoming, USA, thisthermophilic anaerobe is recognized for its xylanolyticqualities and its ability to ferment xylose, mannose,galactose, and glucose [101, 124], but it is not cellulo-lytic. The bacterium produces endoxylanase, whichbreaks down xylan chains primarily to xylobiose andxylotriose, and β-xylosidase, which breaks the xylooli-gosaccharides into xylose, as well as other xylanolyticenzymes with minor roles [125].

Like most bacteria, T. saccharolyticum adjusts itsprotein production, including enzymes and substrate-binding proteins, to accommodate a particular substrate.When concentrations of xylan are higher, xylanasesmore often remain bound to the cell rather than beingreleased as free enzymes. When endoxylanase was pu-rified from cell cultures by gel filtration and affinitychromatography, it was found that the optimal condi-tions are pH 6.0 and 70 °C [125].

Because of its natural ability to effectively hydrolyzexylan, T. saccharolyticum has been genetically engineeredto produce higher levels of ethanol. Genes that produceorganic acid byproducts during fermentation have beenknocked out, leading to ethanol formation as the majorproduct. Compared to the wild-type strain, one modifiedstrain produced about 60 % more ethanol on xylose [101].This was a substantial improvement upon work by Desai etal. [99] involving gene knockout in T. saccharolyticum thateliminated lactic acid production. Plus, research done on thisbacterium has applications to C. thermocellum because thetwo bacteria have similar metabolic pathways [101]. Be-cause of its strong xylose fermentation abilities, geneticallymodified T. saccharolyticum could be cocultured with acellulolytic ethanologen for maximal biomass conversionin CBP.

Clostridium cellulolyticum

With a cellulosome similar to that of C. thermocellum andC. cellulovorans, this mesophilic bacterium is well-equipped to grow on cellulose and can grow on xylan aswell, but it has weak fermentative abilities [124, 126, 127].Its cellulolytic characteristics are not entirely unique, as it isclosely related to C. cellobioparum, C. termitidis, and C.papyrosolvens [124]. While it does produce some ethanol

when grown on cellulose, the primary end products areacetate and lactate. However, C. cellulolyticum’s mesophilictemperature preference, in contrast to C. thermocellum’sthermophilic optimum, allows it to be cocultured with othermore robust mesophiles, such as Zymomonas mobilis [127].

Due to its rapid cellulose hydrolysis, the cellulosome hasbeen the primary focus of studies on C. cellulolyticum. Fiveendoglucanases are present in the complex (CelA, CelD,CelC, CelG, and CelE), in addition to exoglucanase (CelF)and the scaffolding protein, also referred to as the noncata-lytic cellulosome integrating protein (CipC) [124, 126, 128].While these components are common to all cellulosomes ofC. cellulolyticum, the ratios of enzymes differ based onsubstrate and environmental conditions [126]. Comparedto crystalline cellulose, amorphous cellulose is a bettersubstrate for CelF exoglucanase activity [128] due to theaccessibility of polysaccharide termini. In addition to itshydrolytic properties, the cellulosome’s thermostability isanother important factor. At higher temperatures, calciumions have been shown to maintain the enzymatic activity ofthe cellulosome [126]. In combination with fermentativemicrobes, C. cellulolyticum’s cellulosome shows potentialin consolidated bioprocessing.

Fusarium oxysporum

This fungus is another true CBP candidate that secretes highlevels of cellulases and ferments both pentose and hexosesugars. F. oxysporum F3 has achieved high ethanol yields onwheat straw and is able to ferment glucose, xylose, andcellobiose [129]. In an experiment with the same straingrown on Cellulose 123, ethanol yield was lower due tohigher production of acetic acid. Cellobiose did not accu-mulate, indicating that β-glucosidase activity was sufficient;however, an accumulation of glucose meant fermentationlagged behind hydrolysis, possibly due to the organism’slow ethanol tolerance [130].

Despite a limited capacity for fermentation, F. oxysporumhas proved to have an efficient cellulolytic system. Cellu-lases purified and isolated from a culture on brewers’ spentgrain and corn cobs were most active at 40 °C and a pH of 6when applied to hydrothermally pretreated wheat straw.Hydrolysis was improved by removing glucose, demonstrat-ing that sugars are competitive inhibitors of the cellulases.Further improvement of hydrolysis was achieved by addingbovine serum albumin, a protein acting as a surfactant thatpreferentially binds to lignin, which prevents the enzymesfrom uselessly attaching to these sites [68]. Although F.oxysporum can ferment sugars to ethanol, the fungus is ofparticular interest because it offers a potential source ofgenetic information for transgenic CBP organisms withstronger fermentative abilities due to its diverse arsenal ofcellulolytic enzymes.

Bioenerg. Res. (2013) 6:416–435 425

Ethanologenic Organisms

In many of the studies of ethanologenic organisms, simul-taneous saccharification and fermentation has been utilizedas a stepping stone toward a more consolidated processwhere a single organism or cocultures could be utilized toachieve a true form of consolidated bioprocessing with noexogenous enzymes.

Saccharomyces cerevisiae

Commonly known as baker’s yeast, this fungus has longbeen used in beer- and wine-making as well as baking. Theadvantages of using S. cerevisiae in a CBP system includeits ability to naturally ferment hexoses with a high ethanolyield, high tolerance to end products, and high level ofheterologous gene expression. Evolutionarily, the reasonfor high ethanol yields and tolerance has been to kill com-petitors and consume them [103]. Despite its tolerance toethanol, growth and ethanol production are temporarilyinhibited by furaldehyde, hydroxymethylfuraldehyde, andvanillin, products that can result from substrate degradationduring pretreatment steps [48].

Due to their inability to ferment pentoses (i.e., xylose andarabinose), wild-type yeasts have been genetically engi-neered to express xylose isomerase genes from Piromycesspiralis that allow them to efficiently ferment xylose. In itsnatural form, S. cerevisiae is also unable to ferment cellobi-ose and other oligosaccharides that are often the products ofcellulose hydrolysis. Even in cases where it has beengenetically engineered, S. cerevisiae preferentially fer-ments glucose before other sugars, so certain genesmust be disrupted in order for it to utilize multiplesugars simultaneously [103]. Additionally, cells needsufficient transporter proteins that are not suppressedby other sugars in order to increase xylose uptake.Insertion of transporter genes from Pichia stipitis haveimproved xylose uptake at low concentrations [131].

Of course, the major drawback of S. cerevisiae as a CBPcandidate is its inability to hydrolyze cellulose. Manyattempts have been made to genetically recombine theyeast’s genome with fungal cellulase genes and, in fewercases, bacterial cellulase genes. Cellobiohydrolases havebeen expressed in many different experiments, but theirspecific activity and secretion has often been low [103].The specific activity of cellobiohydrolase produced by S.cerevisiae is less than when it is expressed in native organ-isms because of hyperglycosylation, the excessive additionof glucose radicals to the enzymes during cellular proteinprocessing [132]. Nonetheless, the fungus has the capacityto grow on cellulose with heterologous cellobiohydrolasesand endoglucanases. The microbe has also been geneticallyengineered to secrete hemicellulases, most successfully with

xylanolytic enzymes [103]. Patents recently filed byscientists at Mascoma Corporation protect a transgenicS. cerevisiae strain potentially capable of expressingover 300 heterologous genes that support cellulase pro-duction [19, 133, 134].

A thermostable endoglucanase from Thermoascus aur-antiacus was expressed in the yeast with optimal activity at50 °C, above the microbe’s optimum temperature [135].When genes encoding an endoglucanase of T. reesei andβ-glucosidase of Saccharomyces fibuligera were insertedinto the yeast genome, S. cerevisiae was able to produceethanol from phosphoric acid swollen cellulose (i.e., cellu-lose with low crystallinity), albeit at low concentrationscompared to growth on simple sugars [136]. Tsai et al.reason that low yields are a result of the small amount ofenzymes that can be secreted by the organism when sub-jected to energy-limiting anaerobic conditions. Cellulaseactivity is improved when the enzymes are in proximity toeach other. Thus, the goal of another project was to expressa mini-cellulosome on the surface of S. cerevisiae. Whengrown on swollen cellulose, the yeast produced ethanolconcentrations several times higher than those describedabove, but levels were still not anywhere near S. cerevisiae’sfermentative abilities when grown on simple sugars. Theauthors expressed three cellulases in this artificial cellulo-some, but the addition of more enzyme types could improvethe synergistic effect of catalysis [137].

Because there are so many possibilities for genetic recom-bination, directed evolution has been used to select for yeaststrains with improved cellulase properties, higher rates ofenzyme secretion, and synergistic ratios of cellulases [103,131]. In addition to producing saccharifying enzymes, S.cerevisiaewill also require the ability to adhere to its substratein order to efficiently hydrolyze it and utilize the resultingsugars. Other challenges that must be overcome are codonbias during translation, improper protein folding, interruptedprotein transport, and accumulation of large proteins atthe cell wall due to low permeability. The key tosuccessful genetic engineering of this yeast, like allorganisms, is to carefully manipulate its genes ratherthan simply overexpressing them [103].

Klebsiella oxytoca

Klebsiella oxytoca has native enzymes for the uptake andfermentation of cellooligosaccharides, xylooligosacchar-ides, hexoses, and pentoses. A recombinant strain of thiscellobiose-fermenting yeast, P2, was created with genesencoding Z. mobilis’ ethanol fermentation pathway. StrainP2 has exceeded the performance of both natural K. oxytocaand Z. mobilis [138]. Removal of cellobiose by fermentationis important because the disaccharide is a competitive in-hibitor of cellulolytic enzymes [139].

426 Bioenerg. Res. (2013) 6:416–435

In one simultaneous saccharification and fermentationexperiment, K. oxytoca P2 produced high yields of ethanolon mixed waste office paper when exogenous cellulaseswere added. In this case, the major barrier to SSF was theprohibitive cost of cellulases, which were required in rela-tively large amounts. Recycling the paper within the processreduced this cost and increased ethanol yield by 27 % due toenzyme retention on the substrate [138]. In another SSFexperiment, lower substrate and enzyme feed concentrationssupported more efficient utilization of inputs and decreasedbyproduct formation. Yields were also improved with asecond inoculation and temperature shift to favor sacchari-fication. Temperature was particularly important because itaffected the balance between endoglucanase and exogluca-nase activity such that neither enzyme was rate limiting [32].As these findings have indicated, improvements to processconditions are pivotal to successful fermentation.

Modified constructs of strain P2 were created from Z.mobilis’ fermentation genes as well as C. thermocellum’sendoglucanase genes. With just the Z. mobilis genes, therecombinant microbe rapidly converted cellobiose to etha-nol, but expression of the cellulases decreased ethanol yield.The best strain could not reach C. thermocellum’s ethanolproduction abilities on cellulose unless commercial cellulasewas added [139]. Cocultures of K. oxytoca with a morethermo-tolerant and ethanol tolerant organism, Z. mobilisor Klebsiella marxianus, improves yields and decreasesfermentation time. In this case, multistage or coculture fer-mentation could be a more cost effective process rather thanfermentation by a single organism [140].

Zymomonas mobilis

This facultative anaerobic, ethanologenic bacterium hasbeen isolated from such sources as pulque, the alcoholproduced from the sap of the maguey plant, as well astainted cider. It has also been found in palm juice, sugarcanejuice, ripening honey, and tainted beer. Natural strains arecapable of growing on raffinose and ferment glucose, fruc-tose, and sucrose [141]. One of the bacterium’s mainstrengths is that its high rates of sugar uptake and ethanolproduction exceed most yeasts’ capabilities, though indus-trial scale fermentation using Z. mobilis has not been asextensively developed as with yeast species [141, 142].Rapid sugar uptake is a result of its extensive sugar transportsystem that operates by facilitative diffusion. It is able toachieve higher ethanol yields than yeasts because it utilizesthe Entner–Doudoroff pathway, an alternative fermentationpathway that produces less ATP per mole of glucose, mean-ing that more of the substrate is utilized for ethanol produc-tion rather than supporting cell growth [96, 142].

High variation is present among the many strains of Z.mobilis. Depending on the strain, the optimal temperature

falls between 25 and 31 °C at a pH between 3.5 and 7.5, butmutant strains grew at higher temperatures. Ethanol toler-ance is at least 55 g/L for all strains, but is upwards of 100 g/L for others. Most strains tolerate at least 200 g/L of sugarwhile others can tolerate up to 400 g/L [141]. Z. mobilis, likeall potential CBP microbes, is susceptible to inhibition bybiomass pretreatment byproducts, but it has shown fortitudein resisting these toxic compounds. The cells eventuallyovercome inhibition by assimilating the toxins; however, itcannot assimilate vanillin, which inhibits xylose fermenta-tion. Glucose fermentation is inhibited primarily by hydrox-ybenzaldehyde [48].

Genetic engineering has made it possible for Z. mobilis toferment arabinose and xylose at 98 and 86 % of theoreticalyield, respectively [142, 143]. Five genes from E. coli wereexpressed in Z. mobilis in order for it to ferment arabinose[143]. Another recombinant ferments xylose, but has asomewhat diminished ability to ferment glucose. Glucosealso competitively inhibits xylose uptake and is preferen-tially utilized in sugar mixtures. Nonetheless, ethanol wasproduced from xylose with 86 % theoretical yield like in theaforementioned experiment [142].

Pichia stipitis and Candida shehatae

These ethanologenic yeasts ferment mannose, glucose, andgalactose, but they stand out in comparison to S. cerevisiaedue to their ability to efficiently utilize xylose. P. stipitis isalso able to ferment cellobiose, while neither can fermentarabinose [104]. Both yeast species experience optimal eth-anol tolerance from 11 to 22 °C, but these temperatures maynot coincide with the optimal growth temperatures [92].

Each yeast’s ability to utilize xylitol, an alcohol byprod-uct of xylose fermentation, for growth has also been mea-sured. In 120 h, P. stipitis was able to use 88 % of availablexylitol for growth while C. shehatae was only able to use6 %, but C. shehatae produced low levels of xylitol duringfermentation of xylose. Both preferentially ferment glucosewhen other sugars are present [104]. A modified strain of P.stipitis grown on acid-pretreated wheat straw containing46.4 % cellulose and 31 % hemicellulose produced a highyield of ethanol despite inhibition by toxic compoundsresulting from pretreatment [28]. C. shehatae is moreresistant to these byproducts than S. cerevisiae and P.stipitis [48]. This robustness is highly desirable forindustrial scale production. These organisms show po-tential for CBP or as a source of genes for modificationof other candidate organisms.

Flammulina velutipes

This basidiomycetes mushroom has been commercially cul-tivated for food, but its fermentative abilities and high

Bioenerg. Res. (2013) 6:416–435 427

ethanol tolerance also make it valuable as a potential CBPorganism. High conversion efficiencies of glucose, man-nose, sucrose, fructose, maltose, and cellobiose to ethanolhave been achieved. However, the fungus does not fermentgalactose and pentose sugars to ethanol in the absence ofother sugars, and fermentation times are relatively long,usually 6 days or more to reach maximum yield. Xyloseand galactose utilization was substantially improved whenthe sugars were mixed with glucose [144].

F. velutipes is not only suited for fermentation, but it canalso degrade lignin. Lignin degradation is the focus offeedstock pretreatment because lignin removal or dissolu-tion opens up the polysaccharides for saccharifyingenzymes to access. When grown on sugarcane bagasse at28 °C, the fungus decomposed 10 % of the substrate byweight after 40 days. Although it was less efficient atdegrading the bagasse than the white-rot fungus, T. versi-color, which converted 20 %, F. velutipes preferentiallydegraded lignin over other lignocellulose components. Acorrelation between laccase activity and lignin degradationwas found, suggesting laccase was the primary enzymeresponsible for lignin degradation under the given condi-tions [66]. Although F. velutipes has poor saccharificationabilities, cocultures of T. versicolor and F. velutipes are anattractive option due to their combined ability to degradeand ferment biomass.

Escherichia coli

Having been used extensively in the production of numer-ous commodities, E. coli is a valuable model organism forgenetic engineering. The ability to ferment sugars has beenconferred to E. coli by expressing fermentation genes fromZ. mobilis, consisting of pyruvate decarboxylase and alcoholdehydrogenases. A transgenic strain containing genes fromanother plasmid (pLOI295) produced ethanol as the majorfermentation product, an improvement over the insignificantquantities produced by the parent strain. Expression of thesegenes also resulted in a tenfold increase in cell density.Growth was further increased by the addition of magnesium,a limiting nutrient [145].

Ethanol yields were improved in a similar experimentutilizing genes from Z. mobilis. In order to improve stabilityof gene expression, genes were recombined with the chro-mosome rather than expressing them from a plasmid. For-mation of succinate, a competing fermentation product, waseliminated by the deletion of a gene. Yields were very high,but xylose and glucose concentrations must be kept low toavoid diminishing yields, likely resulting from E. coli’smoderate ethanol tolerance [146]. Ethanol tolerance hasbeen increased by mutagenesis in strain LY01, which pro-duces up to 60 g/L of ethanol. The improved tolerance is aresult of significant changes in metabolism, as well as

alterations to the cell envelope. Investigation of the strainshowed that osmotolerance may be associated with ethanoltolerance [147].

In order for E. coli to be truly capable of CBP, it mustalso be able to hydrolyze cellulose. Although there arecurrently no reported strains that can both degrade celluloseand ferment the resulting sugars, several experiments haveled to cellulolytic strains. Two cellulases and two xylanasesderived from the anaerobic thermophile, Anaerocellum ther-mophilum, have been expressed in E. coli [148], in additionto two cellulases from C. cellulolyticum [149]. Two cellu-lases from the gut of the wood-feeding termite, Coptotermesformosanus, were also expressed in E. coli. When secretedon carboxymethyl cellulose, the endoglucanases generatedprimarily oligosaccharides with some glucose [150].

Organisms with Saccharolytic Capabilities

Trichoderma reesei

This fungus is able to rapidly produce large quantities ofcellulases and is widely used to produce these enzymes com-mercially. Due to the low specific activity of these cellulaseson crystalline cellulose, large quantities are necessary forcomplete cellulase hydrolysis. Mutagenesis of parent strainQM 9414 has provided a strain (CL 847) with a fivefoldimprovement in cellulase production and tenfold improve-ment in β-glucosidase production. This strain was selectedthrough a multistep screening process primarily not only forits high productivity but also for favorable characteristics suchas stability, rapid growth, and reduced nutritional require-ments [151]. This is just one example of improvements incellulase production by T. reesei, whose capabilities havemuch to offer CBP research. Thorough investigation of T.reesei and its metabolic pathways could give insight intohow to integrate these strengths into ethanologenic organisms.

Thermoascus aurantiacus and Thermomyces lanuginosus

These thermophilic fungi, which have been isolated fromjute compost and potato, produce large quantities of xyla-nases, while T. aurantiacus also produces some cellulasesthat act synergistically with xylanases [152, 153] by reduc-ing crystallinity and opening up new sites for enzyme activ-ity. On wheat bran, T. lanuginosus secreted more xylanasesthan T. aurantiacus. Lower xylanase production by T. aur-antiacus is attributable to its diversion of energetic resourcesto cellulase production [152]. Regardless, both organisms’xylanases are most active at 70 °C, and in addition to beingthermostable, are stable across a broad pH range [152, 153].However, the temperature optimum does not correspond tothe fungus’ optimal growth temperature of 50 °C [146].

428 Bioenerg. Res. (2013) 6:416–435

Corn cobs and wheat straw are the most suitable sub-strates for the induction of xylanase activity as compared torice bran, rice straw, sugarcane bagasse, sulfite pulp, saw-dust, and jute dust [152, 153]. Xylanase activity was furtherimproved by the surfactant, Tween 80, likely due to ligninblocking, as described previously [68, 152]. While xyla-nases are the primary enzymes secreted by T. lanuginosus,this fungus also secretes some α-galactosidase, anotherhemicellulase. Other hemicellulases are integral to the com-plete degradation of xylan, so it is important for a CBPorganism to express a variety of hemicellulases. Some hemi-cellulases release side-chain substituents on the xylan back-bone to make the xylose polymers more accessible toxylanases [153]. Once again, the synergistic nature of hy-drolytic enzymes plays an important role in saccharification.

Progress Toward Commercialization

Although the technology is still under development, consol-idated bioprocessing has been the focus of a few companies’efforts to produce commercial-scale quantities of lignocel-lulosic ethanol. Qteros, a Massachusetts-based startup estab-lished in 2006, planned to build their platform on aproprietary strain of Clostridum phytofermentans, trade-marked as the “Q Microbe” [154–156]. The company raisedapproximately $52 million in funding to build a 15,000 ft2

demonstration facility in Chicopee, MA, USA. However,difficulties led to a lay-off of many of its employees in 2011,replacement of the original CEO, and closing of the Chic-opee facility in early 2012 [157, 158].

Mascoma, another competitor formed in New Hampshire(USA), has enjoyed better success in recent years as it con-tinues to form strategic partnerships with ethanol producersaround the country. The company was established in 2005 bytwo expert scientists in the field of CBP, Charles Wyman andLee Lynd [159]. In the short term, they plan to implement theirtechnology, known as Mascoma Grain Technology, in currentfirst-generation corn ethanol plants [160]. Patent literatureindicates that the enzyme substitute is likely a mixture ofyeasts including S. cerevisiae engineered for heterologousexpression of termite cellulases [133]. Like Qteros, however,Mascoma relies heavily on government funding rather thanactual sales and has not yet proven its technology on cellulosicfeedstocks. At the planned Kinross, Michigan pilot plant, theoperating cost for hardwood ethanol is estimated at $1.77/gallon, but Mascoma will need to drop costs significantly toreach commercial viability. Mascoma believes that they canreach $1.00/gallon by selling byproducts and scaling up pro-duction [161]. Despite challenges ahead, they have success-fully secured funding from Valero and will soon beginconstruction on the Kinross facility, expected to cost $232milliom including commission and start-up [162].

Although Mascoma is currently the only major industryplayer utilizing CBP technology, a number of other compa-nies are striving to produce commercial quantities of ethanolfrom lignocellulosic feedstocks. POET, the largest ethanolproducer in the world, has set up a pilot facility producingethanol at a rate of 20,000 gallons per year from corn cobs[163]. Using 315,000 dry tons of crop residue and cellulosiccrops per year, Abengoa, another promising competitor,anticipates producing 25 million gallons of ethanol eachyear in addition to 25 MW to power the plant [164]. RangeFuels, on the contrary, has gone out of business after spend-ing over $80 million in federal grants at its Soperton, Geor-gia plant [165]. Shortly after the shutdown, LanzaTech wasable to acquire the facility and its technology for develop-ment of their gas–liquid fermentation technologies intendedfor waste biomass conversion [166]. However, cellulosicethanol is not the only next-generation renewable fuel onthe horizon. A number of other companies are avidly pur-suing a variety of technologies to produce economical fuels.Bio Architecture Lab, for example, intends to utilize sea-weed as a fuel source due to its lack of lignin and potentialfor large-scale aquafarming in coastal waters [167]. Theprimary challenge facing researchers across the world is toidentify the most viable technologies before attempting tocommercialize them. Fortunately, recent business historyserves as a useful indicator of progress for the multitude ofmicrobial platforms under development.

Conclusion

Consolidated bioprocessing has the potential to not onlyenable efficient ethanol production, but also butanol synthe-sis. While this review focuses on ethanol-related systems,microbial strains capable of acetone–butanol–ethanol fer-mentation for advanced biofuels are also known to havecellulolytic properties useful in an ideal CBP process; how-ever, their application in a CBP system has not been fullyexplored.

An ideal CBP process would utilize a single substrate-tolerant, product-tolerant microorganism that concurrentlyutilizes multiple sugars for fermentation with metabolicpathways that produce minimal byproduct. However, thesequalities may be pragmatically achieved through the use ofcocultures. A major remaining challenge is to achieve near-optimal conditions within a single bioreactor for all steps inthe process: hydrolytic enzyme secretion, saccharification,and fermentation. Companies attempting to utilize consoli-dated bioprocessing for fuel production must overcome thisand many other challenges.

The lignocellulose-degrading abilities of CBP organismsare pivotal in enabling commercialization of the technology.Ideal process organisms make use of the synergistic effect of

Bioenerg. Res. (2013) 6:416–435 429

cellulases and hemicellulases together in order to efficientlydegrade lignocellulose to a greater extent. Depending on thefeedstock used, the ratio of endo- and exoglucanases mustbe altered in order to maximize polysaccharide degradationand prevent the accumulation of inhibitory intermediateoligosaccharides, such as cellobiose. Cellulases from F.oxysporum and xylanases from T. saccharolyticum arestrong candidates for enzyme engineering and heterologousexpression, although there are a wide range of saccharolyticenzymes produced by other microbes available for industrialapplication.

Cellulosomes are a promising means of achieving stringentspecifications for saccharification because enzyme ratios canbe altered, and substrate-specific carbohydrate-binding mod-ules are present. In addition, bound cellulosomes concentratesugars near the cells for maximal uptake. Rosettazymes cur-rently serve as good models for studying the kinetics ofsaccharification, but cellulosomes will likely remain a moreefficient means of grouping saccharifying enzymes.

Whereas enzyme grouping will likely be investigated ona case-by-case basis, high-throughput techniques are neces-sary for specific enzyme enhancements, such as increases inthermostability and pH stability. Directed evolution withcontinuous screening is likely to remain superior to rationaldesign for the majority of advancements, though enzymeshave been rationally grouped via peptide linkers to achieveimprovements in biomass depolymerization. Furthermore,enzyme recycling must be more extensively developed sincecells use a large portion of their energy to produce andsecrete enzymes. If enzymes are anchored to cells via boundcellulosomes, enzyme recycling could be simpler and moreeconomical to implement since cell recycling has alreadybeen implemented in a number of industrial applications. Ifenzyme recycling proves effective, genetic regulation willbe important in preventing an over-allocation of cellularenergy to enzyme synthesis so that cell growth and fermen-tation are not significantly impacted. In order to maximizeethanol fermentation, genetic alterations are imperative toknock out genes that produce organic acid byproducts. Thebest ethanol yields could also result from Z. mobilis, whichutilizes the more primitive Entner–Doudoroff pathway. Us-ing this pathway, less energy is provided to the cell in theform of ATP, and a feedstock’s chemical energy is insteadtransferred more extensively to ethanol formation [168].

While saccharification and fermentation are central toefforts to reduce costs of CBP, a number of process designimprovements are critical in enabling large-scale lignocellu-losic ethanol production. Using Le Chatelier’s principle, equi-librium should be shifted within a bioreactor to favor desirablefermentation pathways. Such metabolic shifts can be accom-plished by altering growth conditions (i.e., hydrogen additionor substrate limitations). Another key consideration is themode of cell culture. In comparison to solid substrate

cultivation, submerged fermentation has been more effectiveat facilitating adequate diffusion of cellular products into theculture medium, limiting product inhibition and enhancingethanol formation. Culture temperature must also be carefullyregulated to balance optima for enzyme activity, cell growth,and fermentation.

Other process improvements are achievable through pro-cess integration, where ethanol production is paired withother operations. For example, pulp and sugar mills generatewaste and byproduct streams that could lower feedstockexpenses and provide existing infrastructure to enable com-mercial scaling [169–171]. Simultaneous power generationduring ethanol production could also accelerate commercial-ization [170, 172–176]. Lignin-rich residues resulting frombiomass degradation can be used as a boiler fuel to produceheat and electricity, significantly lowering energy consump-tion and electricity expenses [17, 177–179]. Opportunitiesfor process integration, lignin-based power generation, andother suggestions for increased energy efficiency are reviewedin more depth by van Zyl et al. [17].

Regardless of process improvements, the success of bio-mass conversion platforms depends critically on the micro-organisms utilized. The most promising candidateorganisms for CBP are those that are robust enough totolerate the many stressors present in a bioreactor, especiallyethanol itself. Some microbes are better suited for industrial-scale production due to their productivity ranges, tolerancelevels, and generally higher yields. Although studies varywidely in process conditions and type of substrate, a fewmicrobes stand out for their high theoretical yields (fermen-tative or absolute) on various substrates:

& F. oxysporum (89 % abs) on wheat straw [144]& T. saccharolyticum (82 % abs) on crystalline cellulose

[101]& K. oxytoca and K. marxianus (79 % abs) on crystalline

cellulose [140]& Z. mobilis (98 % ferm) on glucose [141]& S. cerevisiae (95 % ferm) on phosphoric acid swollen

cellulose [137]& F. velutipes (88 % ferm) on glucose [144]& C. thermocellum (81 % ferm) on filter paper [180].

Note that each of these yields is not comparable becausesome studies used exogenous enzymes while others reliedon microbes’ genetic capabilities. Lab-scale ethanol produc-tion must be standardized in order for the research commu-nity to truly assess which candidates are the best organismsfor consolidated bioprocessing. Values of ethanol yield mustalso be reported in literature for accurate comparison. Fur-thermore, microbial conversion platforms must be designedfor a particular substrate, and a variety of pretreatmentmethods and microbial strains are likely to emerge as strongsuitors for commercial operations [17].

430 Bioenerg. Res. (2013) 6:416–435

To date, consolidated bioprocessing has not accom-plished a degree of economic viability that allows it tocompete with petroleum-based fuels, but the lessons learnedfrom research in the field are extremely valuable in design-ing improved ethanol production processes from widelyabundant lignocellulosic feedstocks. Successful commer-cialization of next-generation lignocellulosic ethanol tech-nology will require scientists and engineers to combine amultitude of individual successes into one high-yield, low-cost process.

Acknowledgments Support for this review was provided by theNorth Carolina Biotechnology Center (NCBC). Any opinions, find-ings, conclusions, or recommendations expressed in this publicationare those of the authors and do not necessarily reflect the views andpolicies of the NCBC.

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