Integrating White Biotechnology in Lignocellulosic Biomass ...download.xuebalib.com/9fuyfvyZwcBI.pdf · demand, biofuels just being a part of the solution [1]. In contrast, chemical

The Role of Catalysis for the Sustainable Product

of Bio-fuels and Bio-chemicals

C H A P T E R

13

Integrating White Biotechnologyin Lignocellulosic Biomass

Transformations: From Enzyme-Catalysis to Metabolic Engineering

Fabrizio Sibilla*, Pablo Domı́nguez de Marı́a†*Biomaterials and Resources Management, nova-Institut GmbH. Chemiepark Knapsack,

Industriestraße 300. D-50354 Hürth, Köln, Germany†Institut für Technische und Makromolekulare Chemie (ITMC), RWTH Aachen University.

Worringerweg 1, 52074 Aachen, Germany

ion

O U T L I N E

13.1 Motivation for the Implementationof White Biotechnology inBiorefineries 445

13.2 Biocatalysis for LignocelluloseProcessing: Free, Isolated Enzymes 449

445

13.3 Fermentation and MetabolicEngineering for the Production of Bio-Based Commodities 458

13.4 Concluding Remarks 463

13.1 MOTIVATION FOR THE IMPLEMENTATION OF WHITEBIOTECHNOLOGY IN BIOREFINERIES

Research on biomass has been gaining momentum in the last decades with the ultimategoal of providing a sustainable—and virtually inexhaustible—source of chemicals and fuelsfor future societies [1–7]. Albeit much research focusing on biomass processing wasconducted decades ago, for the recent renaissance several concomitant facts have occurred.First, the perception that the “petroleum peak” has been already passed, or will do in the next

# 2013 Elsevier B.V. All rights reserved.

446 13. INTEGRATING WHITE BIOTECHNOLOGY IN LIGNOCELLULOSIC BIOMASS TRANSFORMATIONS

years, together with the geopolitically unsustainable external energetic dependency on fossilsources. Second, environmental and climate change concerns, pressuring on the need of novelprocesses for fuel and material supply. This book focuses on different catalytic technologiesthat in all cases pursue the same common final goal.

Biofuels can be divided into three different categories—first-, second-, and third-generationbiofuels—according to the kind of biomass used, the technique involved in the processing andthe final target molecule. The first generation typically refers to ethanol obtained from glucoseor saccharose. In this generation, the fermentable sugars are obtained from starchy biomass—corn, cassava, potatoes—aswell as from sugar obtained from sugar cane or sugar beets. Glucoseand saccharose are fermented by yeasts to obtained ethanol, which is subsequently distilled,dehydrated, and used as drop-in solution for gasoline. The term first generation may also referto biodiesel when obtained from edible vegetable oils [1–11]. On the other hand, the term sec-ond generation refers to ethanol obtained via fermentation of glucose or other sugars, when thefermentable sugars are obtained from the saccharification of thewhole biomass, comprising theso-called lignocellulose ethanol [1–11]. Many different industrial processes have beenestablished in the last years, where the full vegetable crops are saccharified to a pool of ferment-able sugars that are later on processed via fermentation, distillation, and dehydration, inan analogous way as the first-generation biofuels. Finally, the term “third-generation biofuels”refers to a technology where the full biomass is saccharified to its sugar components andlater sugars are converted—chemically or biochemically—to an array of molecules likehydroxymethylfurfural (HMF), valeric acid, levulinic acid, and their respective esters, togetherwith many other compounds [1–11].

In this field, it must be clarified that the generalist term “biomass” or “biorefinery” does notguarantee, as such, the setup of sustainable chemical processes. As a well-known example,ethanol currently produced from corn—within the so-called first generation of biofuels—has already been successful at commercial level for decades, yet quite often at the cost ofdiminishing natural food sources for societies, leading to an increase of food prices [10,11].The same consideration may stand for the production of biodiesel from edible crops or basedon massive deforestation [1–9]. Importantly, these strategies may be a sustainable option forlocal areas on a small-scale basis (e.g., internal consumption of sugar cane-based ethanol inBrazil since the 1970s). To overcome these issues and envisaging a worldwidemarket, secondand third generation of biofuels—using nonedible parts of lignocellulosic materials—arepresently assessed. Herein, an important aspect is the actual amount of biomass that canbe annually harvested in a particular area, without compromising the overall sustainability(soil degradation, use of nonmarginal lands for fuels, indirect land soil change, etc.) [12–16].For the energy needs, a broad portfolio of alternatives—biomass, electric automotive, photo-voltaic, wind, geothermal, etc.—may be combined to cope with the worldwide energydemand, biofuels just being a part of the solution [1]. In contrast, chemical supply will relyonly on biomass as a source of raw materials. Presently only �10-15% of the total extractedcrude petroleum is used for chemistry and material use [17], yet commodity prices are muchhigher than those of biofuels [1]. This aspect suggests that for compensating costs, biofuelswould be the coproducts of the commodities, and not vice versa. Current state of the art ischallenging, as biomass-derived products and processing routes have to compete with pet-rochemical processes, which have experienced already decades of intensive developmentand fine-tuning optimization. Although the use of biomass to obtain different chemicalsand materials is virtually as old as mankind (e.g., birch bark pitch use dates back in the late

44713.1 MOTIVATION FOR THE IMPLEMENTATION OF WHITE BIOTECHNOLOGY IN BIOREFINERIES

Paleolithic era), biomass technologies suffered a lack of research since the crude petroleumprices decreased in the 1940s. Recent research efforts aim to enhance the economics andefficiencies of novel approaches [1,12–16]. Especially, as this book is addressing fromdifferentviewpoints, the development of novel (bio)catalytic systems that may provide cleaner andmore efficient biomass treatment processes are of utmost importance for the provision of asustainable bio-based future.

Apart from resource availability and local vs. global considerations, to reach a completesustainability for second and third generation, two additional aspects must be considered.One is the water consumption [11]. All forms of lignocellulose contain a substantial amountof water that sooner or later must be removed for the chemical processing. Alternatively, bio-mass can be dried as a pre-step process, albeit at the cost of investing a considerable amount ofenergy on that. Conversely, pretreatment steps can be conducted in aqueous solutions, andthen performing the downstream processing of the different chemicals in subsequent steps(e.g., extraction in a second phase of produced more hydrophobic compounds). Large-scalewater consumption worldwide—as biofuels and biorefineries are necessarily envisaged—mayaggravate current environmental and social problems, with existing severe water shortage andfresh water contamination in reservoirs worldwide (e.g., from mining activities). Consideringwater as “the primary food,” biorefineries must cope with its rational use, developing sustain-able solutions valid for an increasing world human population, addressing concepts likeresource- and heat-integration, wastewater treatment, andwater reusability [11]. Likewise, cat-alysts should show a proven robustness by displaying activities in impure and reused watereffluents, while exhibiting a catalyst life under those conditions that enables the necessary(economic) number of reuses. Moreover, in a widest extent, catalysts and solvents should alsobe bio-based, to assure their worldwide large-scale availability [18]. Finally, catalyst wastes—which will be surely present in these aqueous effluents—must be easily degraded and assim-ilated by microorganisms. Overall, this must lead to a holistic picture in which value chainsare optimized and integrated in a “cradle-to-cradle” approach [19].

The second aspect is the economic need of a full valorization of lignocellulose [12–16].Pretreatment steps may be applied to selectively fractionate lignocellulosic materials in(at least) their three main components, xylose (from hemicellulose fractions), cellulosepulp, and lignin. This enables the achievement of more than one raw material from biomass,providing better cost-benefit balances [1,13,20–22]. Pretreatments will obviously needto be efficiently integrated, requiring low energy (power) inputs and providing virtually“stoichiometric” yields of the raw materials. Based on these closed-loop premises, it can beenvisaged that some pretreatments steps will involve steam explosion, hydrothermal treat-ments, and/or organosolv strategies, because these strategies often provide largely improvedmass balances and typically enable the recovery of high-quality nondegraded valorizablelignins (representing lignin in some lignocellulosic materials 25-30% of the wood content)[1,13,20–22].

Taking all these considerations in mind, this chapter deals with a broad palette of biotech-nological solutions for biorefineries, all of them within the so-called White Biotechnology.Applications in the area have been triggered by the impressive developments in molecularbiology, which have enabled the understanding of cell machineries, and have provided inte-grated bio-concepts for fermentative options, paving theway for the production of an array of(non)natural useful chemicals undermild and typically sustainable conditions.Moreover, theuse of free, isolated enzymes—for example, cellulases to depolymerize cellulose—has been


the matter of intense research as well, leading to several genetically improved enzymes. As amatter of fact, nowadays it is possible to clone and overexpress specific genes of a certain pro-tein, to produce such biocatalyst on an on-demand basis in a sustainable and improved form[23–28]. Whole-cells and enzymes are entirely biodegradable materials, and thus, the waterintegration in biorefineries appears quite feasible (e.g., recirculation of sterilized exhaustedwater to soils for the next harvest of crops). White Biotechnology strategies encompass apromising portfolio of options, provided that bio-processes can be economically integratedin a whole biomass-processing pipeline, fitting the on-spec and challenging price range ofbiofuels and bio-commodities.

An overview of enzymes and whole cells related to biomass and biorefineries is depictedin Scheme 13-1. Some enzymes have been assessed for lignin degradation. In fact, lignin isenzymatically degraded in Nature, and it is tempting to assess whether the same biocatalyticpathways might also be applied at commercial level. However, the long reaction times forenzyme-degrading lignin, and their costs—together with the need of mediators—representa hurdle that has not been overcome yet. Conversely, a different consideration may be madefor cellulases and hemicellulases, from which several commercial processes have been setup [1]. Likewise, fermentative approaches, especially the so-called metabolic engineering,

Lignocellulose

– Wood– Grass– Algae– Wastes

Fractionation

Lignin

Cellulose

Hemicellulose

Xylose

LaccasesPeroxidasesPerhydrolasesb-Etherases

Cellulases (enzyme cocktails)SwolleninsCellobiose dehydrogenasePolysaccharide monooxygenases

Phenylic compoundsDegraded lignin

Glucoseoligomers

HemicellulasesOxidoreductasesXylanases

Surfactants, xylose, xylitol

Chemical step

Polysaccharides

– Cellulose– Hemicellulose– Starch– Algae polysaccharides– Etc.

Depolymerization

Fermentation and metabolic engineering

Biocatalysis

(Bio)catalytic

C6 sugars

(e.g., Glucose)

C5 sugars

(e.g., Xylose)Fermentation

Metabolic Engineering

FuelsPlatform chemicalsCommoditiesEtc.

SCHEME 13-1 Overview of a fractionating-based biorefinery, addressing the type of enzyme or whole-cell, andsubstrate(s) where they have been assessed.

44913.2 BIOCATALYSIS FOR LIGNOCELLULOSE PROCESSING: FREE, ISOLATED ENZYMES

are emerging areas fromwhichmany applications can already be foreseen. Basically, the con-cept implies that starting from glucose—and eventually xylose and other C5 sugars present inhemicellulose—as a carbon source, genetically designed microorganisms can biosynthesize abroad number of valuable platform chemicals. Herein, integration between upstream (designof a microorganism able to produce a chemical economically) and downstream (extractionand purification of such chemical from an aqueous fermentative broth) will be crucial fora technical and economic success.

13.2 BIOCATALYSIS FOR LIGNOCELLULOSE PROCESSING:FREE, ISOLATED ENZYMES

The term “cellulases” for lignocellulose hydrolysis is referred to a pool of different glyco-sidic enzymes (“cocktail”) that digest the cellulose fibers to afford glucose and solubleoligomers under mild aqueous conditions [21,29,30]. Compared to other chemical methods,the major advantage of using cellulases relies on the intrinsic selectivity of the catalyst forcellulose hydrolysis [1]. This performance provides pure glucose as final product, preventingits degradation to other compounds (e.g., furfurals) and allowing its direct use as feedstockfor subsequent processing operations, for example, in fermentations. Conversely, cellulosehydrolysis by nonenzymatic strategies (e.g., mineral acid catalysis) often leads to the forma-tion of HMF, levulinic acid, etc., that can severely inhibit the microbial growth and loweringthe overall production yields [28,31]. Moreover, an enzymatic hydrolysis at mild tempera-tures allows the heat integration with the fermentative unit, which often takes place underthe same process conditions. Actually, several simultaneous saccharification and fermenta-tion (SSF) procedures have been successfully reported (see also Section 13.3).

Commercially available cellulase cocktails are mainly derived from the microorganismTrichoderma reesei (formerly known also as Trichoderma viride), a fungus isolated by Americanscientists from samples of cotton-based materials that had been “liquefied” in the pacificscenario of WWII [11]. There are multiple reasons—mostly biochemical, technical, and eco-nomic—to explain the preference for this glycosidic cocktail [32–35]. First of all, T. reesei is ableto produce itself the full set of enzymes required for the lignocellulose saccharification.It must be noted that for an efficient cellulose depolymerization, the concerted action ofthe various types of glycosidases present in the cocktail is needed. Thus, enzyme manufac-turers can directly produce such enzymatic cocktail in a single fermentation, contributing toreduce the overall costs. Moreover, such simple production of the T. reesei enzymes and theirin situ formulation allow the production of the enzymatic mixture directly at the biorefinerysite, lowering transportation costs of carbon source and enzymatic mixtures.

In most of the cases, the catalytic activity of a given wild-type enzyme is not sufficientto reach economic targets in an industrial application [24,36]. Thus, several rounds of geneticimprovement must normally be conducted, focusing on aspects like higher activity, stability,better suitability for pH or temperature, adaptation to other “real” conditions, etc. The opti-mization of T. reesei cellulase cocktail has traditionally followed two different lines:

• Insertion of other heterologous glycosidic enzymes, creating a recombinant T. reeseiwith stronger capabilities in polysaccharide depolymerization. For instance, the highly


active cellobiose-hydrolase from Aspergillus niger was expressed in T. reesei, providing asynergy between the two cellobiose hydrolases for the crucial step cellobiose to glucose.

• The improvement of the overall quantity of enzyme produced by the fungus (expressionlevels), leading to a more active mixture of glycosidases. In the 1980-1990s, randommutation methods were applied to isolate the variant with the highest activity on cellulosedegradation. After almost three decades of intensive research, a single mutant strain ofT. reesei (T. reesei RUT-C30) was isolated and used for the industrial production of theenzymatic cocktail. This work shed light on the fundaments of improved enzymaticsecretion in the T. reesei [1,32–35]. Based on the knowledge gained, and further judiciouschoice and optimization of the signal peptides for the different enzymes of interest, thelevel of expression of the enzymes was further improved. Current T. reesei strains producemore than 20-fold hydrolytic enzymes (compared to wild type), with improved catalyticefficiencies. All the different enzymatic components have been engineered, optimizingcatalytic activities, thermal stability, pH stability, higher stabilities in real biorefinery-based effluents, etc. [11,21]. As a consequence of these efforts, cellulase cocktails arepresently much cheaper than 20 years ago, with expectations for future lower prices [1].It must be noted that linking cellulase-cocktail prices just to the production of ethanol—asa typical product of second-generation biofuels—is perhaps not the wisest assessment. Byconsidering a whole biorefinery and the number of products that can be derived, theproduction of more expensive commodities—leaving biofuels as their coproducts—mayprovide better economic terms. The full integration and valorization of the wholelignocellulosic material may be crucial for an economic impact [11–16,20–22].

In any commercial cellulase cocktail, there are various hydrolytic enzyme families,namely, exoglucanases, endoglucanases, and cellobiose hydrolases, together with xylanases(hemicellulases) [32–35]. These enzymes cooperatively lead to the full cellulose saccharifica-tion. Exoglucanases are responsible for depolymerizing the cellulose fibers from both fiberextremes—reducing and not reducing-ends ones—forming cellobiose units (two glucoseunits), which are actually soluble in aqueous solutions. These exoglucanases are mainlycomposed of two domains joined through a linker: an anchor—also known as carbohydratebinding motif (CBM)—that allows the binding of the enzyme to the cellulose fibers, and acatalytic domain that is actually responsible for the cellulose hydrolysis. The linker is impor-tant because it must provide enough flexibility for both parts of the enzyme, and at the sametime, it must be rigid enough to prevent enzyme unfolding and subsequent loss of activity.These enzymes hydrolyze cellulose fibers starting from reducing or nonreducing ends ofcellulose processively, and after release of a cellobiose unit, theymove forward along the fiberto release the next cellobiose unit [21]. Likewise, endoglucanases are responsible for hydro-lyzing the cellulose fibers randomly and cleaving them to shorter fragments. These enzymesare constituted by the two above-mentioned domains as well. Although the catalytic domainalone is able to hydrolyze cellulose fibers, the overall saccharification rate is significantly higherwhen the protein is used as a complete structure, comprising the catalytic domain-linker-CBM[21]. Endoglucanases can digest both, amorphous and crystalline cellulose, albeit with higheractivities on amorphous celluloses, suggesting that those structures are the preferred sub-strates. In contrast to exoglucanases, most of the products that are released by endoglucanasesare often aqueous-insoluble cellulose fibers with a shorter number of glucose units that can


be further processed by other glycosidases. In addition to exo- and endoglucanases, cellobiosehydrolases are responsible for the digestion of produced soluble cellobiose to afford twoglucose units. As these enzymes are active on soluble substrates (cellobiose), they are only con-stitutedwith a catalytic domain (without showing a CBM). These enzymes are important in theoverall cellulose hydrolysis because increasing cellobiose concentration inhibits the action ofendoglucanases and cellobiose hydrolases, due to a negative-feedback enzymatic inhibition.For this reason, an efficient cellobiose hydrolysis is always a prerequisite to an overall efficientenzymatic cellulose hydrolysis. To reinforce this, a cellobiose-hydrolase from Aspergillus ssp.has been expressed in T. reesei as well (see also Table 13-1) [32–35].

Apart from T. reesei, several other bacteria or fungi are known to produce cellulases. Someof these bacteria produce cellulases with interesting properties different from those of T. reseeienzymes (e.g., stability at more alkaline pHs, different inactivation temperatures, etc.). Few ofthem, mainly the enzymes produced from aerobic bacteria like Bacillus spp., Pseudomonasspp., and the anaerobic ones such as Clostridium thermocellum and Clostridium acetobutylicum

TABLE 13-1 Enzymes Present in Glycosidic Cocktail of T. reesei and Proportion of them on It [21,32–35,37–43]

Enzyme Type of activity % in the cocktail

CBHI Exoglucanase 50-60

CBHII Exoglucanase 15-18

EGI Endoglucanase 12-15

EGII Endoglucanase 9-11

EGIII Endoglucanase 0-3

EGIV Endoglucanase 0-3

EGV Endoglucanase 0-3

EGVI Endoglucanase


have been characterized [37,44,45]. Others have been isolated in salty lakes, or were active athigh salty concentrations (up to 4 M NaCl) [46–49]. These biocatalysts may be valuable fortheir use in saline solutions, like possible “real effluents” in biorefineries that would comefrom either (concentrated) seawater or IL-containing aqueous effluents (after cellulose precip-itation) [46–51]. Likewise, directed evolution approaches for the design of more robustbiocatalysts in these media have been recently conducted, showing enzyme variants with en-hanced stability and catalytic activity in these media [51]. Moreover, other reported cellulasesdisplay interesting and different features for other types of biorefineries. Cellulase fromSulfulobus solfataricus—archaea isolated in a geothermal area with surprisingly not many cel-lulose resources are expected—catalyzes hydrolysis under acidic conditions (pH

PMO1

Oxidation at C1 position Oxidation at C4 position

PMO2O2O2

SCHEME 13-2 Postulated mechanism of PMOs, oxidating polysaccharides to create more reducing andnonreducing ends [39].


of enzymes—so-called PMOs—were reported [39–43,57,58]. This ubiquitous group of en-zymes were already known, yet wrongly considered as glycosidases with very low activity.Actually, PMOs are metallo-enzymes catalyzing the oxygen-mediated oxidative cleavage ofglycosidic bonds on the surface of (crystalline) cellulose (Scheme 13-2) [39–43,57,58]. Withthat action, more reducing and nonreducing ends are formed, thus facilitating the activityof the other hydrolytic enzymes.

Conclusively, the further addition of PMOs to the enzymatic cocktail of T. reseei allows de-creasing the protein/biomass ratio required for an efficient cellulose depolymerization, pro-viding better economics for the novel glycosidicmixtures. Overall it can be concluded that thedepolymerization of cellulose under mild conditions is a rather complex process, in whichseveral different (non)hydrolytic enzymes are concomitantly acting. Table 13-1 summarizesthe most important groups of them.

When changing from cellulose to lignocellulose, the enzymatic saccharification is furtherinfluenced by several different parameters, such as type of biomass, type of pretreatment,particle size, the ratio amorphous/crystalline cellulose, as well as enzyme loading, pH,temperature, and many other classical parameters for any industrial enzymatic reaction(e.g., inhibitions by products or substrate, affinities, etc.). Moreover, none of the known cel-lulases are able to catalyze an efficient cellulose depolymerization directly on untreatedlignocellulose, with lignin preventing the accessibility of the enzymes to cellulose fibers[53]. Therefore, for this area, the setup of pretreatments for an efficient separation of ligninand the provision of accessible polysaccharide fractions for cellulases are of pivotal impor-tance [1,13,20–22]. Well-known strategies involving steam explosion, hydrothermalprocesses, and organosolv-type approaches seem to have the potential to providenondegraded wood components [1,13,20–22]. The combination of pretreatment approaches


with enzymatic cellulose depolymerizations, showing that both concepts can becompatibilized, is crucial. For instance, by using oxalic acid as catalyst in biphasic media(water-organic solvent) at mild temperatures (100-140 �C), the one-pot selective depolymer-ization of hemicellulose is reached, rendering almost quantitative amounts of xylose in theaqueous solution [69,70]. Moreover, the nondegraded lignin is in situ extracted in the organicphase (typically bio-based 2-methyl-tetrahydrofuran, 2-MeTHF) [71] and recoveredupon evaporation of the solvent (which is further reused). As a third raw material, awater-immiscible delignified cellulose pulp is obtained by filtration of the aqueous effluent.Such cellulosic pulp can be depolymerized by cellulases (Scheme 13-3), probing that thepolysaccharide fraction is extensively delignified. Overall, three components of the lignocel-lulose may be achieved for further processing and valorization.

As an example of further valorization, the aqueous effluent containing xylose was directlyconverted in furfural bymeans of FeCl3 as catalyst, with no need of purifying xylose (and restof C5 sugars coming from hemicellullose), as the catalyst was highly active under these “real”aqueous and raw conditions [72]. It must be noted that the aqueous effluent of a wood-basedpretreatment approach contains high amounts of C5 sugars (mostly xylose ca. 30 g L

�1 forwood loading of 100 g L�1), but many other compounds coming from lignocellulose, whichmay certainly poison and inactivate (or inhibit) the catalyst. Therefore, the assessment of anew catalyst (in biomass field) under real conditions is typically recommended. By integrat-ing such a catalytic process, costs related to downstream processing can be directly avoided.Moreover, in the pretreatment approach, extracted lignin can be subjected to further researchand processing as well. Many innovative practical applications for nondegraded lignins havebeen already pointed out in the literature [1,73]. It must be noted that presently the onlylignin-fraction available at bulk scale is obtained from the Kraft process for pulp and paper in-dustry, yet suffering such lignin a considerable level of degradation/modification during thepulping procedure. Remarkably, the provision of nondegraded lignins at a large scale mightcertainly contribute to their valorization by means of already well-known applications [1].

As stated in Section 13.1, another important aspect in large-scale biorefineries is the waterconsumption. Typical biomass loadings in organosolv-type processes are in the range of 100-200 g biomass L�1 aqueous media (higher loadings may obviously lead to diffusional andstirring problems) [1,13,20–22]. Thus, to produce thousands of liters of biofuels, for example,

Lignocellulose

Lignin

Xylose(aqueous effluent)

Cellulose Glucose

CellulasesOxalic-acid-based pretreatment

(Biphasic media)

SCHEME 13-3 Selective one-pot fractionation of lignocellulose, based on oxalic acid as catalyst, and further use ofcellulosic pulp as effective substrate for commercial cellulase cocktails (dotted square step) [69,70].


considerable amounts of water would be needed worldwide. An obvious considerationwould be the smart integration of water cycles, by producing aqueous effluents with astraightforward and high biodegradability, which eventually might allow the recirculationof exhausted water—still with valuable organic matter—to the soil for the next crop cultiva-tion (or for animal feeding) [11]. Likewise, water depuration in populated areas and citiesshould not be considered as a waste, but as a useful resource to be integrated withbiorefineries. Furthermore, for algae-based processing, aswell as for coastal regions, seawatermay be a promising option as long as produced waste(sea)water may be treated in an eco-nomic and efficient way. Several commercial glycosidase cocktails are highly active for thedepolymerization of crystalline cellulose AvicelW in pure seawater [50]. Furthermore, othercellulases have been genetically improved to display a higher activity in these aqueous media[51]. Notably, enzymes were also active in concentrated seawater (2� to 4�) (Scheme 13-4)[50,51], suggesting that concentrated effluents produced in desalination plants could alsobe integrated in biorefineries. Production of drinkable water (desalination) might be coupledwith biofuel and the biosynthesis of commodities. Section 13.3 will further discuss the com-bination of cellulases and fermentations in (concentrated) seawater effluents.

Apart from their important use in glycosidic cocktails, hemicellulases and xylanaseshave found interesting applications in pulp and paper industry [74], as well as in the produc-tion of biosurfactants [75]. A recent example is the formation of alkyl-b-D-xylosides andoligoxylosides catalyzed by different (commercial) xylanases, and using pentanol and octanolas substrates for the alkylation. Wheat bran was hydrothermally pretreated for 1 h at 135 �C,from where hemicellulosic fraction was selectively removed, rendering an aqueous mixture

Component Concentration in

seawater (g L–1)

Cellulose(Avicel, amorphous, etc.)

Cellulases (recombinant and wild type)

(Concentrated) Seawater

Glucose, cellobiose,oligomers

MgCl2 2.50

MgSO4 3.40

CaCl2 1.17

NaHCO3

NaCl 27.13

KCl 0.74

0.21

NaBr 0.08

SCHEME 13-4 Conceptual use of (concentrated) seawater using commercially available and geneticallyimproved cellulases. Average composition of seawater [50].

Xylans(aqueous solution)

Xylanases / H2O / 60 °C

or

n

nm

m: 1-6

Biosurfactants

SCHEME 13-5 Xylanase-catalyzed production of biodegradable biosurfactants. As substrate, a nonpurified aque-ous solution of xylans coming from a hydrothermally based processing of wheat bran is used [75].


of different soluble oligoxylosides (from pure xylose to 7-ring-oligomers). Upon filtration, thesame aqueous solutionwas employed in subsequent enzymatic procedures. The use of a ther-mically based pretreatment delivers nondegraded biological materials at bulk scale, thusconferring an added value to processes, fromwhich biorefineries maywell economically ben-efit in the future (while producing biofuels as coproducts of those commodities at the sametime). The subsequent addition of xylanases at 60 �C in that nonpurified water effluentafforded several biosurfactants, formed with xylose, as well as with other solubleoligoxylosides (Scheme 13-5).

Apart from glycosidases, research has been performed with other enzymes (e.g., laccases,peroxidases, etc.) to process lignin for further potential use in biorefienies for differentpurposes (see Scheme 13-1). Another approach is the peracid-mediated delignification (e.g.,peracetic acid) [76]. Peracetic acid oxidizes hydroxyl groups in lignin, cleaving b-aryl bondsand reducing lignin molecular weight. Likewise, peracetic acid hydroxylates phenolic ringsto form hydroquinones, leading to a further oxidation/degradation of lignin (Scheme 13-6).

Upon such delignification (hemi)celluloses are more accessible for enzymatic hydrolysis,yielding fermentable sugars (typically as a mixture of xylose and glucose, together with therest of sugars coming from lignocellulose) [77,78]. Actually, in the pulp and paper industry,the use of peracids to oxidize lignin and for bleaching purposes has been known for de-cades. Yet, for a large-scale use, peracids cannot be stored and transported in a concentratedform, due to safety reasons. Therefore, the use of perhydrolases (as well as some geneticallyimproved esterases) for in situ producing peracetic acids to delignify biomass was assessed[77,78]. Perhydrolases catalyze the formation of organic peracids using hydrogen peroxideand carboxylic acids in aqueous media, under mild reaction conditions that avoid the deg-radation of other lignocellulosic materials. The efficient in situ peracetic acid formation inaqueous media was reported by means of wild type and mutants of Pseudomonas fluorescensesterase, with ethyl acetate (EtOAc) as second phase and substrate for the enzyme, uponaddition of diluted hydrogen peroxide as reagent. Significant improvements in cellulaseperformances in aspen wood were observed, reaching up to 98% of total fermentable sugars,after several cycles of peracid formation and processing (Scheme 13-7) [77,78].

O

O

OOH

OOH

Enzyme

PAA CellulasesLignocellulosicpretreatment

Sugars (C6 and C5)

Hydrolase, H2O2

(Ethyl acetate)

Aqueous

SCHEME 13-7 Hydrolase-mediated delignification via in situ formation of peracetic acid [77,78].

Lignin structure

HO

HO

O O

O

O

OO

OO

OHO

OH

Hydroxylation

Demethoxylation

Oxidation

HO

HO

O

O

SCHEME 13-6 Suggested pathways for the peracid-based oxidation and degradation of lignin, comprisinghydroxylation, demethoxylation, and oxidation [76].


Furthermore, these peracid-based enzymatic treatments would eventually sterilize thedelignified saccharide fraction as well, preparing the raw material for a subsequent fermen-tative step. However, these biocatalytic strategies have just been developed at the levelof proof of concept, and therefore, economic figures and the operational window arestill far from a real implementation. The delivery of a mixture of C5 and C6 sugars (hemicel-lulose and cellulose) might provide advantages for microorganisms able to grow on bothsources [1], as a full benefit of the saccharide fraction of lignocellulose would be then reached.


13.3 FERMENTATION AND METABOLIC ENGINEERING FOR THEPRODUCTION OF BIO-BASED COMMODITIES

Approaches in biorefineries using White Biotechnology comprise also a broad number ofpromising fermentations—using sugars from lignocellulose—or metabolic engineering ap-proaches (tailored “on-demand” design of microorganisms) [23–27]. To this end, wholecells—living or resting microorganisms—instead of isolated enzymes are used. In this area,two important aspects must be optimized and integrated to provide a strong case for a prac-tical application. First of all, the upstream part, in which a certain microorganism is geneticallydesigned for an ad hoc use, for example, by incorporating a new enzyme enabling a new bio-chemical step, or by attenuating/overexpressing biochemical pathways to enhance the bio-synthesis of a desired product or precursor, or to avoid the rapid degradation of some ofthem. Second, the downstream part, in which the desired chemical is extracted from the fer-mentative aqueous broth and purified until on-spec conditions. Both steps—and their corre-spondent integration—are crucial for the provision of economic figures, as well as for theecological footprint (e.g., amount of waste produced in a precipitation-based downstreamprocessing, and ways to treat or valorize that waste) [1]. In this section, several examplesof biosynthetic production of chemicals will be discussed.

Traditional fermentative routes have involved the use of wild-type or “process-driven”evolved microorganisms for the production of a desired chemical or fuel. Probably, the mostwell-known example in biosynthesis and second generation of biofuels is the fermentation ofsugars to produce ethanol as biofuel (as well as for the production of alcoholic beverages, etc.)[1]. With the purpose of generating biofuels, traditionally this has been done by using sugarcane or starch as a source of glucose (C6 sugars), as these raw materials permit a rapid andcheap depolymerization, within the so-called first-generation approach. As stated before, lastdecades havewitnessed a considerable research effort for the provision of cheap glucose fromlignocellulose as well. Regardless, the origin of sugars the biochemical pathway for theethanol formation is obviously common for all biosynthetic generations (Scheme 13-8).

Glucose

O

COO–

Pyruvate decarboxylase Alcohol dehydrogenase

NADH + H+ NAD+

OH

Whole-cell

Ethanol(Downstream)

H+ CO2

O

H

Glucose

1st Generation 2nd Generation

SCHEME 13-8 General biochemical pathway for the formation of ethanol using glucose as rawmaterial [1,11,53].

45913.3 FERMENTATION AND METABOLIC ENGINEERING FOR PRODUCTION OF BIO-COMMODITIES

It must be noted that not all microorganisms are able to process C5 sugars (e.g., xylose), andfurthermore such compounds may be toxic for some of them, leading to inhibition in theirgrowth, and thus diminishing the overall yield of the fermentation [79–81]. Thus a wise sep-aration of both fractions of wood (C5 and C6) would be desirable for many microorganismsable to produce ethanol. A pretreatment method that could separate—and further valorize—the xylose fraction of lignocellulose, together with lignin, would be then preferred for manybiorefineries (see, for instance, Scheme 13-3) [69,70]. In addition, the use of xylose for otherpurposes apart from being fermentable sugars—for example, for furfural or xylitol forma-tion—may contribute to enhance the profitability of the biomass-processing plant [72], asthese chemicals offer usually higher market prices than ethanol or other biofuels (e.g., buta-nol) [1]. However, for other industries or business models—fully focused on biofuel produc-tion—the complete use of the sugar fraction of lignocelluloses for ethanol production may beobviously privileged [80,81]. For this purpose, considerable research has been undertaken inthe design of microorganisms also accepting xylose and arabinose (C5 sugars) as carbonsource for growth and production, together with glucose and other C6 sugar sources(Scheme 13-9) [1,11,53,80]. To this end, it was necessary to clone and express several biochem-ical pathways involving several enzymes, in microorganisms (a metabolic engineeringapproach, see below), to confer them such capabilities in metabolism. By means of this strat-egy, several yeasts efficiently producing ethanol or butanol from complex C5/C6 sugar mix-tures have been successfully tailored [80,81]. For these latter approaches, the lignocellulosefractionation in their main three components provides less incentive for the “whole-picture,”and therefore only a selective delignification process would be desirable, enabling cellulasesan efficient accessibility to polysaccharides. Once the upstream is envisaged, for the down-stream processing of these systems, ethanol is usually recovered by distillation from the fer-mentation broth. Conversely, butanol forms a second phase over the aqueous fermentative

L-arabinose transporterL- arabinose isomeraseL-ribulokinaseL-ribulose5-P-4-epimerase

D-xylose transporterD-xylose isomeraseD-xylulokinase

D-xyloseL-arabinose

Classic1st/2nd generations

Ethanol

Butanol

GlycolysisGlucose

D-xylulose-5-P

SCHEME 13-9 Genetically designed microorganisms able to accept C5 sugars as carbon source (L-arabinose andD-xylose) to produce biofuels by fermentation [80,81].


fraction that can be thus separated from the fermentative broth. Several strains (e.g., C.acetobutylicum) have been identified and improved for that process [1,11,53,80,82].

Another important approach is the SSF [1], aiming at saving costs through process integra-tion. This case being enzymatic, cellulose hydrolysis is conducted at mild conditions, and itmay easily be combined with fermentations in one-pot two steps. Furthermore, biocatalystsdo not lead, often, to whole-cell poisoning or to growth inhibition, problems that otherchemo-catalysts might certainly bring. Moreover, the continuous in situ consumption offormed glucose will reduce inhibitory effects that glucose might have on cellulases(Scheme 13-10). Some examples of ethanol, butanol, or chemical productions by means ofSSF strategies have been reported in the literature, also in combination with metabolic engi-neering strategies (see below).

In a previous section, it was shown that some cellulases efficiently catalyzed the cellulosedepolymerization in concentrated seawater [50,51], as an alternative of nonedible water res-ervoirs for algae-based research and for coastal cities and regions. The use of seawater as fer-mentative reaction media (complemented with carbon sources as, e.g., glucose) has beenreported for the production of several compounds [83,84], for example, succinic acid byActinobacillus succinogenes, and combined with a further acid-catalyzed esterification of it[85]. Likewise, several halophilic microorganisms have been studied for the production of dif-ferent chemicals [86]. Moreover, very recently we have shown that fungus Ustilago maydismay grow in seawater to produce itaconic acid in analogous levels as in pure buffer culturemedia systems [87]. As carbon source both crude xylose-based effluents coming from frac-tionation systems with oxalic acid (see Scheme 13-2) and glucose produced from cellulosein seawater by cellulases were useful carbon sources (Scheme 13-11). The genetic improve-ment and adaptation of this fungus to such “real media” may certainly improve the produc-tivities and trigger research in the area [83–89].

Furthermore, last decades have witnessed the development of holistic metabolic ap-proaches for modern fermentations, often gathered under the name “Metabolic Engineering”[81]. Metabolic engineering aims at the increase of the overall production yield for a desiredtarget chemical through fermentation in a certain microbial host, through a combined

Polysaccharide fractions

Chemicals and biofuels

Cellulases

Whole-cells

StarchHemicellulosesCellulosesAlgae-based saccharidesEtc.

SCHEME 13-10 Conceptual approach for the simultaneous saccharification and fermentation to produce differ-ent chemicals and biofuels by designed microorganisms [1].

Ustilago maydis(seawater)

HOOC

Xylose(from beechwood)

Itaconic acid

Glucose(seawater)

SCHEME 13-11 Fermentation of Ustilago maydis to produce itaconic acid, using either xylose from beechwoodcrude effluent, or glucose produced in seawater by the enzymatic depolymerization of cellulose [87].

46113.3 FERMENTATION AND METABOLIC ENGINEERING FOR PRODUCTION OF BIO-COMMODITIES

attenuation and overexpression of biochemical pathways, coupled with a proper and eco-nomically feasible downstream processing. In addition, the overexpression of other externaldesired enzymes—to trigger a new biosynthetic activity in the tailored microorganism—canalso be carried out. New sustainable entries can be reached in two ways:

• By improving the overall yield of a certain molecule that is already produced by themicroorganism (e.g., outstanding examples reporting an increase in the ethanol yield inSaccharomyces cerevisiae fermentations) [80,81].

• By introducing in a targeted microorganism a new (whole) metabolic pathway for theproduction of a desired molecule that is not originally produced by the microorganism.

In virtue of the developments in molecular biology, it can be envisaged that metabolicengineering will have a bright future in the bio-based economy. A cell of an engineered or-ganism can be seen as a “micro-reactor”where sugars—and other rawmaterials andwastes—can be converted to the desired products under mild conditions and with the production ofwastewaters of low environmental impact. To this end, a proper combination of the upstreamand downstream steps must be set up. The production of these chemicals at competitive costswill, however, still be a challenge in many of these cases in the years to come.

Recent literature provides outstanding examples of metabolic engineering-based technol-ogies for the production of chemicals. For instance, several microorganisms have beendesigned to produce bio-based diamines [90,91], useful starting materials in the productionof nylons. To this end, several amino acid decarboxylases (e.g., ornithine or lysine decarbox-ylase) were cloned and overexpressed in Escherichia coli as microorganism, conferring a novelcapability to the microorganism. Upon addition of glucose, bacteria grow and produce theintended chemical. Another recent disclosure reports on the fermentative production ofhighly useful isoprene, by means of a SSF approach, starting from cellulose to afford unsat-urated chemical monomers (Scheme 13-12) [92].

Other promising concepts are the complementary formation of adipic acid and 1,3-propanediol, to afford biopolyesters starting from glucose. In the production of diols, for in-stance, several enzymes were cloned and overexpressed in E. coli cells to afford a competitiveroute starting from glucose (Scheme 13-13) [93].

Furthermore, very recently novel concepts on the production of alkenes from glucose werereported [81,94,95]. For instance, the formation of jet-fuel precursors (long-chain alkenes) canbe afforded by engineered cells in which different acyl-ACP reductases are cloned [96,97]. Inthis specific case, the downstream processing is straightforward, since such long-chain

CelluloseSSF

Glucose

Engineered whole-cell

Isoprene

SCHEME 13-12 Metabolic engineering for the production of isoprene from cellulose [92].

–

–

–

–

Glucose

Engineered whole cells

1,3-Propanediol

Glycerol-3-phosphate

dehydrogenase

Dehydratase

GlycerolPropanediol

Glucose

Oxidoreductase

Glycerol-3-phosphate

phosphatase

SCHEME 13-13 Metabolic process for the formation of 1,3-propanediol from glucose and enzymes involvedon it [93].


alkenes are not soluble in water, forming a second phase and reducing costs and waste pro-duction. Analogously, the production of short-chain alkenes (e.g., isobutylene, propylene,etc.) by means of engineered whole cells was reported as well [98]. Herein a direct connectionof “petrochemical-based” chemicals can be made, starting from glucose as the raw material(carbon source for microorganisms). The downstream processing appears again trivial, asthe intended chemicals are gases that may be stripped out from the fermentation broth. Inthis approach, to “train” microbial cells to produce alkenes from sugars, the biosynthesisof b-hydroxy-alkanoates was overexpressed, as these chemicals are the actual precursorsfor the formation of short-chain alkenes. Mevalonate diphosphate decarboxylase fromPicrophilus torridus was cloned and overexpressed, as this enzyme is able to decarboxylateand dehydrate such b-hydroxy-alkanoates to finally afford the alkenes, which are secretedby cells (Scheme 13-14) [98].

Glucose

Whole cell

Mevalonate

decarboxylasedecarboxylase

Mevalonate

SCHEME 13-14 Metabolic engineering approach for the synthesis of short-chain alkenes, recently developed[94,98].

46313.4 CONCLUDING REMARKS

Herein, with the provision of cheap sugars (at this moment, from first generation), alkeneprices of €0.4-0.9 kg�1 may be provided, depending on geographical areas. Of pivotal impor-tance will be the achievement of pure and cheap fermentable glucose—and eventually othersugars frombiomass—whichcouldbeachievedthrougha fullvalorizationof lignocellulose [94].


This chapter has discussed the options that White Biotechnology may bring in the use oflignocellulose as the source of biofuels and chemicals emerging arena.

First of all, free, isolated enzymes (or cocktails of) for their application in several specificcases were described, e.g., with depolymerization of cellulose as a core example. Key to thissuccess has been the development ofMolecular Biology techniques, which have permitted thegenetic optimization (and understanding) of enzymes, providing tailored variants withhigher stability, activity, and performance. Though enzymatic costs have significantly de-creased over the last years, due to these technical breakthroughs, further improvementsare still needed, in order to reach a complete economic scale in the production of biofuels.The recent discovery of novel oxidative enzymes may be a promising research avenue inthe future. In addition, an alternative would be the consideration of (part of the) glucoseas a rawmaterial for other chemicals different than biofuels, whichmay have a higher marketprice.

In a second package, whole cells (normally living cells in biomass field) may provide othersmart options for biorefineries. Starting from “classic” ethanol or butanol formation, otherapproaches like the formation of alkenes or diols have been reported. For these fermentativestrategies, the judicious combination of the upstream part (designing and tailoring the micro-organism) with the downstream part (purification until on-spec conditions) is critical for aneconomic success. Thus, in cases in which the downstream processing may be straightfor-ward (e.g., alkenes), the overall economic figures appear promising.

Furthermore, in this chapter it has been noted that the cost of fermentable sugars is a crucialparameter to be considered, not only for White Biotechnology, but virtually for any other


approach in biorefineries. The development of technical solutions that may provide suchsugars from biomass in a cheap and clean manner will be definitely crucial in the comingyears. Pretreatment approaches that enable the separation of the three main componentsof lignocellulose (xylose, cellulose, and lignin) may provide a full biomass valorization—leading to an array of different commodities and platform chemicals—while compensatingcosts at the same time. Aligned to this point, this chapter has also emphasized the pivotalimportance that water consumption will have in biorefineries. As alternatives, the uses ofdepurated water effluents, as well as seawater and desalination plant wastes, have been putforward. For all these options, the use of living organisms may be a clear asset.

In a nutshell, biorefineries will need to be built on such holistic concepts, with key wordslike integration, full valorization, waste-reuse, water-reuse, and closed-loops, if they areconsidered to be a sustainable solution for the future. For all such purposes, White Biotech-nologymay havemany options to offer, workingwithin an integrated and synergistic mannerwith other catalytic approaches.

Acknowledgments

This work was performed as part of the Cluster of Excellence “Tailor-Made Fuels from Bio-mass,” which is funded by the Excellence Initiative of the German Research Foundation topromote science and research at German universities. We thank the contribution of an anon-ymous referee who has provided very fruitful and stimulating suggestions for the improve-ment of this chapter.

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