Trends in bioconversion of lignocellulose: Biofuels, platform chemicals & biorefinery concept

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Progress in Energy and Combustion Science 38 (2012) 522e550

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Review

Trends in bioconversion of lignocellulose: Biofuels, platform chemicals& biorefinery concept

Vishnu Menon, Mala Rao*

Division of Biochemical Sciences, National Chemical Laboratory, Pune 411-008, India

a r t i c l e i n f o

Article history:Received 3 August 2011Accepted 14 February 2012Available online 17 March 2012

Keywords:LignocellulosePre-treatmentSaccharifying enzymesConsolidated biomass processingLifecycle assessmentValue-added products

* Corresponding author. Tel.: þ91 20 25902228; faE-mail address: [email protected] (M. Rao).

0360-1285/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.pecs.2012.02.002

a b s t r a c t

Bioconversion of renewable lignocellulosic biomass to biofuel and value added products are globallygaining significant prominence. Market forces demonstrate a drive towards products benign to naturalenvironment increasing the importance of renewable materials. The development of second generationbioethanol from lignocellulosic biomass serves many advantages from both energy and environmentalpoint of views. Biomass an inexpensive feedstock considered sustainable and renewable, is an optionwith the potential to replace a wide diversity of fossil based products within the energy sector; heat,power, fuels, materials and chemicals. Lignocellulose is a major structural component of woody and non-woody plants and consists of cellulose, hemicellulose and lignin. The effective utilization of all the threecomponents would play a significant role in the economic viability of cellulosic ethanol. Biomassconversion process involves five major steps, choice of suitable biomass, effective pretreatment,production of saccharolytic enzymes-cellulases and hemicellulases, fermentation of hexoses andpentoses and downstream processing. Within the context of production of fuels from biomass,pretreatment has come to denote processes by which cellulosic biomass is made amenable to the actionof hydrolytic enzymes. The limited effectiveness of current enzymatic process on lignocellulose isthought to be due to the relative difficulties in pretreating the feedstocks. The present review isa comprehensive state of the art describing the advancement in recent pretreaments, metabolic engi-neering approaches with special emphasis on the latest developments in consolidated biomass pro-cessing, current global scenario of bioethanol pilot plants and biorefinery concept for the production ofbiofuels and bioproducts.

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5232. Structure of lignocellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5243. Selected pretreatment categories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525

3.1. Physical pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5253.2. Physico-chemical pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526

3.2.1. Steam explosion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5263.2.2. Ammonia fiber explosion (AFEX) and ammonia recycle percolation (ARP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5263.2.3. Microwave-chemical pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5273.2.4. Liquid-hot water pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527

3.3. Chemical pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5273.3.1. Acid pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5273.3.2. Alkaline pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5283.3.3. Green solvents (ionic liquids) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528

3.4. Biological pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5293.5. Techno-economics of pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529

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4. Biocatalytic valorization of lignocellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5294.1. Saccharification systems for cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5294.2. Saccharification systems for hemicelluloses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531

5. Fermentation strategies & consolidated biomass processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5325.1. Future economic performance of hydrolysis process concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535

6. Metabolic engineering approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5367. Alternative liquid fuels - biobutanol and 2,3-butanediol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5388. Biorefinery perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5399. Lifecycle assessment of lignocellulosic ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 540

10. Current global status of lignocellulosic ethanol industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54211. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543

1. Introduction

The development of second generation biofuels from lignocel-lulosic biomass has many advantages from energy and environ-mental concerns. First generation biofuel derived mainly from foodcrops creates many problems ranging from net energy losses togreenhouse gas emission to increased food prices. The recentimbalance in oil market and hike in fuel costs have initiated a globalchallenge for biofuel production from lignocelluloses. Efficientconversion of lignocellulosic materials to ethanol and value addedbiochemicals are still to day a challenging proposition. In contrast tofossil fuels, cellulosic ethanol produced through fermentation ofsugars is a renewable energy source. Biomass is a primary source offood, fodder and fibre and as a bioenergy source provides about10.2% (50.3 EJ/yr) of the annual global total primary energy supply(TPES) from awide variety of biomass sources [1]. More than 80% ofbiomass feedstocks used for energy are derived from wood andshrubs. The remaining bioenergy feedstocks are from the agricul-tural sector (energy crops, residues and by-products) and from

Fig. 1. Thermochemical and biochemical p

various commercial and post-consumer waste and by-productstreams. According to the IEA report for the assesemnet of avail-able residues in 2030, it is concluded that 10% of global residuescould then yield around 155 billion lge (5.2 EJ) lignocellulosic-ethanol or roughly 4.1% of the projected transport fuel demand in2030 and 25% of global residues converted to either ethanol, dieselor syngas could contribute 385e554 billion lge (13e23.3 EJ) globally[2,3]. It is well documented that cellulosic ethanol offers greaterenvironmental benefits and sustainability; however the concern isthe economic viability of the process. The conversion process reliesheavily on major technological innovations centered on effectiveand low cost enzymes, feedstocks and efficient process design.

Globally large amount of agricultural residues are produced,most of which is burnt as waste disposal and small amount is usedfor mulching, for fuel or as fodder. Three types of energy can beproduced from lignocellulosic residues by thermochemical orbiochemical processing, liquid fuels such as ethanol or pyrolysis oil,gaseous fuels such as biogas (methane) and electricity (Fig. 1).Biomass is used with varying degrees of energy efficiency in various

rocessing of lignocellulosic biomass.

Table 1Composition of representative lignocellulosic feedstocks.

Feedstocks Carbohydrate composition (% dry wt) References

Cellulose Hemicellulose Lignin

Barley hull 34 36 19 [12]Barley straw 36e43 24e33 6.3e9.8 [13,14]Bamboo 49e50 18e20 23 [15,16]Banana waste 13 15 14 [17]Corn cob 32.3e45.6 39.8 6.7e13.9 [18,19]Corn stover 35.1e39.5 20.7e24.6 11.0e19.1 [20]Cotton 85e95 5e15 0 [21]Cotton stalk 31 11 30 [22]Coffee pulp 33.7e36.9 44.2e47.5 15.6e19.1 [23]Douglas fir 35e48 20e22 15e21 [24]Eucalyptus 45e51 11e18 29 [16,25]Hardwood stems 40e55 24e40 18e25 [26,27]Rice straw 29.2e34.7 23e25.9 17e19 [28,29]Rice husk 28.7e35.6 11.96e29.3 15.4e20 [30,31]Wheat straw 35e39 22e30 12e16 [29,32]Wheat bran 10.5e14.8 35.5e39.2 8.3e12.5 [33]Grasses 25e40 25e50 10e30 [34,35]Newspaper 40e55 24e39 18e30 [26]Sugarcane bagasse 25e45 28e32 15e25 [16,36]Sugarcane tops 35 32 14 [37]Pine 42e49 13e25 23e29 [25]Poplar wood 45e51 25e28 10e21 [38]Olive tree biomass 25.2 15.8 19.1 [39]Jute fibres 45e53 18e21 21e26 [40]Switchgrass 35e40 25e30 15e20 [26]Grasses 25e40 25e50 10e30 [26,27]Winter rye 29e30 22e26 16.1 [41]Oilseed rape 27.3 20.5 14.2 [41]Softwood stem 45e50 24e40 18e25 [26,27]Oat straw 31e35 20e26 10e15 [14]Nut shells 25e30 22e28 30e40 [42]Sorghum straw 32e35 24e27 15e21 [43,44]Tamarind kernel

powder10e15 55e65 e [45]

Water hyacinth 18.2e22.1 48.7e50.1 3.5e5.4 [46,47]

V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550524

sectors. Low-efficiency traditional biomass such as wood, straws,dung and other manures are used for cooking, lighting and spaceheating, generally by the poorer populations in developing coun-tries. High-efficiency modern bioenergy uses more convenientsolids, liquids and gases as secondary energy carriers to generateheat, electricity, combined heat and power and transport fuels forvarious sectors. High energy efficiency biomass conversion is foundtypically in the industry sector associated with the pulp and paperindustry, forest products, food and chemicals [4]. The biomassproduction costs can be combined with technological andeconomic data for related logistic systems and conversion tech-nologies to derive market potentials at the level of secondaryenergy carriers such as bioelectricity and biofuels for transport[5e7]. There are many favorable factors for the implementation oflignocellulose based biofuels. Geographical location of feedstocksources are more evenly distributed than the fossil sourcesenabling to a large extent the security of supply. The Intergovern-mental Panel on Climate Change in its Special Report on EmissionScenarios: A1, A2, B1 and B2 have developed different future land-use patterns. Acording to the report the largest contribution totechnical potential could come from energy crops on arable land,assuming that efficiency improvements in agriculture could out-pace food demand so as to avoid increased pressure on forests andnature areas. A range of 20e400 EJ/yr is presented for 2050, witha best estimate of 250 EJ/yr and 240 to 850 EJ/yr is presented for2100 [8]. The reforestation schemes can contribute significantly forbiomass production (8e110 EJ/yr). Although the low-yieldingbiomass production is more expensive, competition with foodproduction is almost absent and various co-benefits, such asregeneration of soils and carbon storage, improved water retentionand protection from further erosion may also offset part of theestablishment costs. The potential of low-productive land isnegligible and the rest land area is assumed to partly available,resulting in ranges of the geographical potential from 35 to 245 EJ/yr for the year 2050 and to 265 EJ/yr in 2100 [8]. Biofuel productionfrom lignocellulosic feedstocks may even help to a certain extent tocombat the unemployment status of rural areas. The greenhousegas impacts of lignocellulosic ethanol production based on lifecycleanalyses undertaken by the US environmental protection agency.These analysis show lignocellulosic ethanol generates 91% lessgreenhouse gases than fossil-based petrol or diesel in transportapplications, compared with just 22% for corn-based ethanol [9].However the assumed benefit is now under discussions, especiallydue to emissions from land-use changes (LUC) [10,11].

The compositions of various lignocellulosic feedstocks areillustrated in Table 1. Biomass conversion process involves fivemajor steps, choice of suitable biomass, effective pretreatment,production of saccharolytic enzymes such as cellulases and hemi-cellulases along with the accessory enzymes, fermentation ofhexoses and pentoses and the downstream processing. It is neces-sary to understand the complex structure of lignocellulose to designa suitable pretreatment. Within the context of production of fuelsfrombiomass, pretreatment has come to denote processes bywhichcellulosic biomass is made amenable to the action of hydrolyticenzymes. Research attention has been focused extensively for overtwo decades to enhance the digestibility of lignocellulosic biomassfor the efficient conversion of cellulose to ethanol, methane and inthe recent years to hydrogen. The present article is a comprehensivestate-of-the-art review discussing the pretreatment strategies withthe advantages, drawbacks and techno-economics, microbialsystems comprising the necessary enzymes (cellulases and hemi-cellulases) for valorization of lignocellulose, advancements infermentation technologies and metabolic engineering approacheswith special emphasis on consolidated biomass processing. Inaddition, the review addresses the current global scenario of

bioethanol industry, lifecycle assessment of lignocellulosic ethanol,biorefinery concept for the development of sustainable and bio-based sectors (biofuels and bioproducts) and recent advances inalternative biofuels such as biobutanol and 2,3-butanediol.

2. Structure of lignocellulose

Lignocellulosics are the most abundant source of unutilizedbiomass and their availability does not necessarily impact land use.Biomass in general consists of 40e50% cellulose, 25e30% hemi-cellulose and 15e20% lignin and other extractable components [48].The effective utilization of all the three components would playa significant role in economic viability of the cellulose to ethanolprocess. In nature except in cotton bolls, cellulose fibres areembedded in a matrix of other structural biopolymers, primarilyhemicellulose and lignin. Cellulose is a linear syndiotactic (alter-nating spatial arrangement of the side chains) polymer of glucoselinked together by b-(l / 4)-glycosidic bonds whereas hemi-cellulose is a branched heteropolymer of D-xylose, L-arabinose, D-mannose, D-glucose, D-galactose and D-glucuronic acid. Lignin iscomposed of three major phenolic components, namely p-cou-maryl alcohol, coniferyl alcohol and sinapyl alcohol. Lignin issynthesized by polymerization of these components and their ratiovaries between different plants, wood tissues and cell wall layers.Lignin is a complex hydrophobic, cross-linked aromatic polymerthat interferes with the hydrolysis process. A representative dia-grammatic framework of lignocellulosic biomass is illustrated inFig. 2. The cellulose chains are packed into microfibrils which arestabilized by hydrogen bonds. These fibrils are attached to eachother by hemicelluloses and amorphous polymers of different

Fig. 2. Diagrammatic illustration of the framework of lignocellulose.


sugars as well as other polymers such as pectin and covered bylignin. The cellulose microfibrils which are present in thehemicellulose-lignin matrix are often associated in the form ofbundles or macrofibrils. The molecules of individual microfibrils incrystalline cellulose are packed so tightly that not only enzymes buteven small molecules like water cannot enter the complex frame-work. Some parts of the microfibrils have a less ordered, non-crystalline structure referred to as amorphous region [49]. The highmolecular weight and ordered tertiary structure make naturalcellulose insoluble in water. The crystalline regions of cellulose aremore resistant to biodegradation than the amorphous parts.Cellulose with low degree of polymerization (DP) will be moresusceptible to cellulolytic enzymes. The isolation and derivatiza-tion/dissolution of cellulose are crucial steps in determining cellu-lose DP [50]. In general plant cell walls are subdivided as primary(PW) and secondary (SW) walls. The distribution of cellulose,hemicellulose and lignin varies considerably among these layers.The secondary wall is composed of SW1, SW2 and SW3where SW2is usually thicker than the others and contains the major portion ofcellulose. The middle lamella, which binds the adjacent cells, isalmost entirely composed of lignin [51].

The major impediments towards development of an economi-cally viable technology for biodegradation of cellulose are theassociation with lignin and hemicellulose, crystallinity, DP andsurface area. During the biocatalytic valorization of lignocellulosicsubstrate, a residual fraction survives the attack. This fractionabsorbs a significant amount of the original enzyme and restrictsthe use of these enzymes on added, fresh substrate [52]. Mostpotential cellulosic substrates for bioconversion are heavily ligni-fied. Thus, most of the cellulose in nature is unsuitable forbioconversion unless effective and economically viable procedures(pretreatments) are developed to remove or modify lignin.

3. Selected pretreatment categories

The crucial step in the production of biofuels from lignocellu-losic biomass is pretreatment. Pretreatment affords the solubili-zation or separation of the major components of biomass iecellulose, hemicellulose and lignin and thus render the digestibilityof lignocellulosic material. The choice of pretreatment shouldconsider the overall compatibility of feedstocks, enzymes and

organisms to be applied. Pretreatment is not only costly in its ownright but has a pervasive impact on the cost of virtually all otherbiological processing operations, including those precedingpretreatment, the handling of the liquid stream generated, theprocessing of the solids from pretreatment, waste treatment, andpotential production of co-products. To implement successfully thebioethanol production process, the first impediment to be resolvedis the efficient removal of lignin and hemicellulose through a costeffective pretreatment process.

During the past few decades, several approaches have been usedfor developing low cost pretreatments for generating sugar syrupsfrom cellulose and hemicellulose [53]. Different pretreatment studiespublished in literature are described in terms of the mechanismsinvolved, advantages and disadvantages, and economic assessment.Pretreatments for lignocellulosic biomass include biological,mechanical, chemical methods and various combinations thereof[54e56]. The choice of the optimum pretreatment process dependson the feedstocks and its economic assessment and environmentalimpact. There are a number of reports on pretreatment options forvarious biomass types. Table 2 illustrates some of themost promisingpretreatment categories that can be commercialized for the biofuelindustry. However, none of those can bedeclaredoutstanding as eachpretreatment has its intrinsic advantages and disadvantages. Aneffective pretreatment is characterized by several criteria: avoidingsize reduction, preservinghemicellulose fractions, limiting formationof inhibitors due to degradation products, minimizing energy input,andbeing cost-effective. Except for thesecriteria, severalother factorsare also needed to be considered, including recovery of high value-added co-products (e.g., lignin and protein), pretreatment catalyst,catalyst recycling, and waste treatment [57]. When comparingvariouspretreatmentoptions, all theabovementionedcriteria shouldbe comprehensively considered as a basis. At the moment efforts onthe production of ethanol from lignocellulose is growing rapidly andby analyzing the industrial scenario in this fieldmore knowledge canbe gained on the applied pretreatment methods.

3.1. Physical pretreatment

Most of the lignocellulosic biomass requires some of themechanical processing for size reduction. Several pretreatmentmethods such as milling, irradiation (using gamma rays, electron

Table 2Most promising pre-treatment technologies.

Method ofpre-treatment

Sugaryield

Inhibitorformation

Byproductgeneration

Reuse ofchemicals

Applicabilityto differentfeedstock’s

Equipmentcost

Success atpilot scale

Advantages Limitations &disadvantages

Mechanical L Nil No No Yes H Yes Reduce cellulose crystallinity High Power consumptionthan inherent biomassenergy

Mineral acids H H H Yes Yes H Yes Hydrolysis of cellulose andhemicellulose. alters ligninstructure

Hazardous, toxic andcorrosive

Alkali H L H Yes Yes Nil Yes Removal of lignin andhemicellulose, increasesaccessible surface area

Long residence time,irrecoverable salts formed

Liquid hotwater

H H L No e e Yes Removel of hemicellulosemaking enzymesaccessible to cellulose

Long residence time,less lignin removal

Organosolv H H H Yes Yes H Yes Hydrolyze lignin andhemicellulose

Solvents needs todrained, evaporated,condensed and reused

Wet oxidation H or L Nil L No e H e Removal of lignin, dissolveshemicellulose and causescellulose decrystallization

e

Ozonolysis H L H No e H No Reduces lignin content,no toxic residues

Large amount of ozonerequired

CO2 explosion H L L No e H e Hemicellulose removal,cellulose decrystallization,cost-effective

Does not modify lignin

Steamexplosion

H H L e Yes H Yes Hemicellulose removaland alteration in ligninstructure

Incomplete destructionof ligninecarbohydratematrix

AFXE H L e Yes e H e Removal of lignin andhemicellulose

Not efficient for biomasswith high lignin content

Ionic liquids H/L L e Yes Yes e e Dissolution of cellulose,increased amenabilityto cellulase

Still in initial stages

H:- High and L:- Low.


beam, microwave radiations etc) and extrusion are commonly usedto improve the enzymatic hydrolysis or biodegradability of ligno-cellulosic materials. Improved hydrolysis results due to the reduc-tion in crystallinity and improved mass transfer characteristicsfrom reduction in particle size. The energy requirements forphysical pretreatments are dependent on the final particle size andreduction in crystallinity of the lignocellulosic material. In mostcases where the only option available for pretreatment is physical,the required energy is higher than the theoretical energy contentavailable in the biomass. These methods are expensive and likelywill not be used in a full-scale process.

3.2. Physico-chemical pretreatment

Pretreatments that combine both chemical and physicalprocesses are referred to as physicoechemical processes. The mostimportant processes of this group includes: - steam explosion,catalyzed (SO2 or CO2) steam explosion, ammonia fiber explosion(AFEX), liquid hotwater,microwave-chemical pretreatment [20,58].

3.2.1. Steam explosionIn steam explosion the biomass is treated with high-pressure

saturated steam, and then the pressure is suddenly reduced, whichmakes the materials undergo an explosive decompression. Steamexplosion is typically initiated at a temperature of 160e260 �C (cor-responding pressure, 0.69e4.83 MPa) for several seconds to a fewminutes before the material is exposed to atmospheric pressure. Thebiomass/steam mixture is held for a period of time to promotehemicellulose hydrolysis, and the process is terminated by anexplosive decompression [59e61]. The process causes hemicellulosedegradation and lignin transformation due to high temperature, thusincreasing the potential of cellulose hydrolysis. A steam explosion

process using sugarcane bagasse as substrate was developed wherethe individual constituents were separated as pure cellulose, hemi-cellulose and lignin [62]. The steam-explosion pretreatment processhas been a proven technique for the pretreatment of differentbiomass feedstocks. It is able to generate complete sugar recoverywhile utilizing a low capital investment and low environmentalimpacts concerning the chemicals andconditionsbeing implementedand has a higher potential for optimization and efficiency [63].

The difference between ‘steam’ pretreatment and ‘steam explo-sion’ pretreatment is the quick depressurization and cooling downof the biomass at the end of the steam explosion pretreatment,which causes the water in the biomass to ‘explode’. During steampretreatment parts of the hemicellulose hydrolyze and form acids,which could catalyze the further hydrolysis of the hemicellulose.This process, in which the in situ formed acids catalyze the processitself, is called ‘auto-cleave’ steam pretreatment. The role of theacids, is probably however not to catalyze the solubilization of thehemicellulose, but to catalyze the hydrolysis of the soluble hemi-cellulose oligomers [64]. Steam explosion and thermal pretreat-ments are widely investigated for improving biogas productionfrom different dedicated materials such as forest residuals [65] andwastes of e.g. activated sludge [66,67], cattle manure [68] ormunicipal solidwastes. However, there are several investigations oncombining “thermal” pretreatment with addition of bases such asNaOH, which usually give a better result than individual thermal orchemical pretreatment [69,70]. The process of steam explosionwasdemonstrated on a commercial scale at the Masonite plants [71].

3.2.2. Ammonia fiber explosion (AFEX) and ammonia recyclepercolation (ARP)

Ammonia fiber explosion is a physicoechemical pretreatmentprocess in which lignocellulosic biomass is exposed to liquid


ammonia at high temperature and pressure for a period of time, andthen the pressure is suddenly reduced. The AFEX process is verysimilar to steam explosion. In a typical AFEX process, the dosage ofliquid ammonia is 1e2 kg of ammonia/kg of dry biomass, thetemperature is 90 �C, and the residence time is 30 min. AFEXpretreatment can significantly improve the fermentation rate ofvarious herbaceous crops and grasses [72]. The AFEX technology hasbeen used for the pretreatment of many lignocellulosic materialsincluding alfalfa, wheat straw, and wheat chaff. AFEX pretreatmentresults in the decrystallization of cellulose, partial depolymerizationof hemicellulose, removal of acetyl groups predominantly onhemicellulose, cleavage of ligninecarbohydrate complex (LCC)linkages, lignin CeOeC bond cleavage, increase in accessible surfacearea due to structural disruption, and increased wettability of thetreated biomass [73,74]. The AFEX process demonstrates attractiveeconomics compared to several leading pretreatment technologiesbased on a recent economic model [75] for bioethanol from cornstover. An advantage of AFEX is that the ammonia used during theprocess can be recovered and reused. Also, the downstream pro-cessing is less complex compared to other pretreatment processes.Over 90% hydrolysis of cellulose and hemicellulose was obtainedafter AFEX pretreatment of bermudagrass (approximately 5% lignin)and bagasse (15% lignin). Thus, AFEX is not a very efficient tech-nology for lignocellulosic biomasswith relatively high lignin contentsuch as woods and nut-shells [76]. Furthermore, ammonia must berecycled after the pretreatment to reduce the cost and protect theenvironment. However both the ammonia cost and the cost ofrecovery processes drive up the cost of the AFEX pretreatment [77].

Another type of process utilizing ammonia is the ammonia recy-cles percolation (ARP) method. In this process, aqueous ammonia(10e15 wt %) passes through biomass at elevated temperatures(150e170 �C)with afluid velocityof 1 cm/minanda residence timeof14 min, after which the ammonia is recovered [78]. In the ARPmethod, the ammonia is separated and recycled. Under theseconditions, aqueous ammonia reactsprimarilywith ligninand causesdepolymerization of lignin and cleavage of ligninecarbohydratelinkages. The ammonia pretreatment does not produce inhibitorsfor the downstream biological processes, so a water wash is notnecessary [79]. Generally, AFEX and ARP processes are not differen-tiated in the literature, although AFEX is carried out in liquidammonia and ARP is carried out in an aqueous ammonia solution(10e15%). The ammonia fiber explosion pretreatment simulta-neously reduces lignin content and removes some hemicellulosewhile decrystallizing cellulose. It can have a profound effect on therate of cellulose hydrolysis [80]. The cost of ammonia and especiallythe recovery process drives the AFEX pretreatment expensive.

3.2.3. Microwave-chemical pretreatmentThe microwave/chemical pretreatment resulted in a more effec-

tive pretreatment than the conventional heating chemical pretreat-ment by accelerating reactions during the pretreatment process[81,82]. Zhu et al. [82] examined three microwave/chemicalprocesses for pretreatment of rice straw e microwave/alkali, micro-wave/acid/alkali andmicrowave/acid/alkali/H2O2e for its enzymatichydrolysis and for xylose recovery fromthepretreatment liquid. Theyfound that xylose couldnot be recoveredduring themicrowave/alkalipretreatment process, but could be recovered as crystalline xyloseduring the microwave/acid/alkali and microwave/acid/alkali/H2O2

pretreatment. The enzymatic hydrolysis of pretreated rice strawshowed that the pretreatment by microwave/acid/alkali/H2O2 hadthe highest hydrolysis rate and glucose content in the hydrolyzate.

3.2.4. Liquid-hot water pretreatmentIn liquid hot water pretreatment (LHW), pressure is utilized to

maintain water in the liquid state at elevated temperatures

[83e86]. Biomass undergoes high temperature cooking in waterwith high pressure. LHW pretreatment has been reported to havethe potential to enhance cellulose digestibility, sugar extraction,and pentose recovery, with the advantage of producing prehy-drolyzates containing little or no inhibitor of sugar fermentation[87]. The sugar-enriched prehydrolyzates can be directly fermentedto ethanol. It has been shown to remove up to 80% of the hemi-cellulose and to enhance the enzymatic digestibility of pretreatedbiomass materials such as corn fiber and sugarcane bagasse [20].Perez et al. [88] used LHW to pretreat wheat straw and obtainedmaximum hemicellulose-derived sugar recovery of 53% and enzy-matic hydrolysis yield of 96%. Perez et al. [89] continued to optimizeprocess variables (temperature and residence time) in LHWpretreatment of wheat straw and achieved 80% and 91% xyloserecovery and enzymatic hydrolysis, respectively. LHW reduces theneed for neutralization of liquid streams and conditioning chem-icals since acid is not added [20,84,87]. Additionally, biomass sizereduction is not needed because the particles are broken apartduring pretreatment; therefore, LHW appears attractive for largescales [90]. A difference between the LHWand steam pretreatmentis the amount and concentration of solubilized products [64]. In anLHW pretreatment the amount of solubilized products is higherwhile the concentration of these products is lower compared tosteam pretreatment [91]. This is probably caused by the higherwater input in LHW pretreatment compared to steam pretreat-ment. The yield of solubilized (monomeric) xylan is generally alsohigher for LHW pretreatment; though this result diminishes whenthe solid concentration increases, because (monomeric) xylan isthen further degraded by hydrolytic reactions to, for example,xylose and furfural [92].

3.3. Chemical pretreatments

Chemical pretreatments were originally developed and havebeen extensively used in the paper industry for delignification ofcellulosic materials to produce high quality paper products. Thepossibility of developing effective and inexpensive pretreatmenttechniques bymodifying thepulpingprocesses has been considered.Chemical pretreatments that have been studied to date have had theprimary goal of improving the biodegradability of cellulose byremoving lignin and/or hemicellulose, and to a lesser degreedecreasing the degree of polymerization (DP) and crystallinity of thecellulose component. Chemical pretreatment is the most studiedpretreatment technique among pretreatment categories. Thevarious commonly used chemical pretreatments includes: acid,alkali, organic acids, pH-controlled liquidhotwater and ionic liquids.

3.3.1. Acid pretreatmentAcid pretreatment involves the use of concentrated and diluted

acids to break the rigid structure of the lignocellulosic material. Themost commonly used acid is dilute sulphuric acid (H2SO4), whichhas been commercially used for a wide variety of biomasstypesdswitchgrass [93,94], corn stover [95,96], spruce (softwood)[97], and poplar [98,99]. Dilute sulphuric acid has traditionally beenused to manufacture furfural [100] by hydrolyzing the hemi-cellulose to simple sugars, such as xylose, which continues toconvert into furfural. Other acids have also been studied, such ashydrochloric acid (HCl) [101], phosphoric acid (H3PO4) [102,103],and nitric acid (HNO3) [104]. Due to its ability to remove hemi-cellulose, acid pretreatments have been used as parts of overallprocesses in fractionating the components of lignocellulosicbiomass [102]. Acid pretreatment (removal of hemicellulose) fol-lowed by alkali pretreatment (removal of lignin) results in rela-tively pure cellulose. This chemical pretreatment usually consists ofthe addition of concentrated or diluted acids (usually between 0.2%


and 2.5% w/w) to the biomass, followed by constant mixing attemperatures between 130 �C and 210 �C. Depending on theconditions of the pretreatment, the hydrolysis of the sugars couldtake from a few minutes to hours [105e111]. Generally, thepretreatment should promote high product yields in a subsequentenzymatic hydrolysis and fermentation operations with minimumcost. Recently it has been demonstrated that the dilute acid pre-hydrolysis can achieve high reactions rates in short time andsignificantly improve cellulose hydrolysis [112]. However,pretreatment operating conditions must be tailored to the specificchemical and structural composition of the various sources ofbiomass. Despite continuing interest in the kinetic mechanism ofsolid-phase acid-catalyzed hydrolysis for several type of biomass,little attention has been given to incorporating those kineticmodels into the plant process modeling [113].

3.3.2. Alkaline pretreatmentAlkaline pretreatment involves the use of bases, such as sodium,

potassium, calcium, and ammonium hydroxide, for the pretreat-ment of lignocellulosic biomass. The use of an alkali causes thedegradation of ester and glycosidic side chains resulting in struc-tural alteration of lignin, cellulose swelling, partial decrystallizationof cellulose [114e116], and partial solvation of hemicellulose[116,117]. Sodium hydroxide has been extensively studied for manyyears, and it has been shown to disrupt the lignin structure of thebiomass, increasing the accessibility of enzymes to cellulose andhemicellulose [118e121]. Another alkali that has been used for thepretreatment of biomass is lime. Lignocellulosic feedstocks thathave been shown to benefit from this method of pretreatment arecorn stover, switchgrass, bagasse, wheat, and rice straw[80,122e124]. Sun and coworkers [125] studied the effectiveness ofdifferent alkaline solutions by analyzing the delignification anddissolution of hemicellulose in wheat straw. They found that theoptimal condition obtained was using 1.5% sodium hydroxide for144 h at 20 �C, which resulted in 60% release of lignin and 80%release of hemicellulose. The conditions for alkaline pretreatmentare usually less severe than other pretreatments. It can be per-formed at ambient conditions, but longer pretreatment times arerequired than at higher temperatures. The alkaline process involvessoaking the biomass in alkaline solutions and mixing it at a targettemperature for a certain amount of time. A neutralizing step toremove lignin and inhibitors (salts, phenolic acids, furfural, andaldehydes) is required before enzymatic hydrolysis. Recently, Zhaoand coworkers [120] showed the effectiveness of sodium hydroxidepretreatment for hardwoods, wheat straw, switchgrass, and soft-woods with less than 26% lignin content. A recent approach to limepretreatment eliminates the solideliquid separation step afterneutralization by neutralizing the lime with carbon dioxide beforehydrolysis resulting in 89% glucose recovery from leafstar rice straw[123]. Park and coworkers [123] also used this method to test SSFwhich resulted in an ethanol yield that was 74% of the theoreticalvalue using a mixture of Saccharomyces cerevisae and Pichia stip-itis. The advantage of lime pretreatment is that the cost of limerequired to pretreat a given quantity of biomass is lowest amongalkaline treatments. For example, in 2005, the estimated cost ofmaterials was $70/ton hydrated lime compared to $270/ton fertil-izer grade ammonia and $320/ton for 50 wt% NaOH and 45 wt%KOH [80]. Though lime pretreatment is energy intensive, CaCO3 canbe recovered by precipitation with CO2 after solideliquid separa-tion [126].

3.3.3. Green solvents (ionic liquids)Recently a new class of solvent has emerged referred as ionic

liquids/green solvents. These solvents are often fluid at roomtemperature, and consist entirely of ionic species. They have many

fascinating properties which make them of fundamental interest toall chemists, since both the thermodynamics and kinetics of reac-tions carried out in ionic liquids are different to those in conven-tional molecular solvents. In general, ionic liquids consist of a saltwhere one or both the ions are large, and the cation has a lowdegree of symmetry [127]. These factors tend to reduce the latticeenergy of the crystalline form of the salt, and hence lower themelting point [128]. Ionic liquids come in two main categories,namely simple salts (made of a single anion and cation) and binaryionic liquids (salts where equilibrium is involved). Ionic liquidshave been described as designer solvents [129], and which meansthat their properties can be adjusted to suit the requirements ofa particular process.

Very recently ionic liquids have been confirmed to be efficientfor dissolution of lignocellulosic materials, such as cellulose, wood,or wheat straw, switchgrass, corn stover [130e134]. Using 1-butyl-3-methylimidazolium chloride (BMIMCl) for pretreatment, Dadiet al. [135] found that the initial enzymatic hydrolysis rate and yieldof pretreated Avicel-PH-101 were increased by 50- and 2-fold incomparisonwith untreated Avicel. Kuo and Lee [136] also observedthat the 1, 3-N-methylmorpholine- N-oxide (NMMO) pretreatedsugarcane bagasse has 2-fold higher enzymatic hydrolysis yield ascompared to untreated bagasse. Nguyen et al. [137] reported thecombined use of ammonia and ionic liquid ([Emim]Ac) for therecovery of bio-digestible cellulose from rice straw. The treatmentexhibited a synergy effect for rice straw with 82% of the celluloserecovery and 97% of the enzymatic glucose conversion. The cellu-losic materials regenerated from ILs were found essentially amor-phous and porous and were much more prone to degradation bycellulases [133,135]. Furthermore, increased rate of cellulosehydrolysis via cellulase in ionic liquids could lead to increasedproduction of fermentable sugars that can be converted into fuels.In addition, ionic liquids involved processes are less energydemanding, easier to operate, and more environmentally friendlythan current dissolution processes [138e140].

However, more in-depth research involving environmentfriendly IL is needed to explore pretreatment green route forresolving the challenge of ionic liquid application [139e141]. Asa result, recent efforts have been focused on exploration of enzymeand environment-friendly ionic liquids [142], because greensolvent is promising for commercial application [143]. Therefore, itis a key point to evaluate the biocompatibility of ionic liquids andselect suitable IL for pretreatment process [144]. Application ofionic liquids has opened new ways for the efficient utilization oflignocellulosic materials in such areas as biomass pretreatment andfractionation. Binders and Raines [134] have reported a high-yielding chemical process for the hydrolysis of cellulose and cornstover using 1-ethyl-3-methylimidazolium chloride ([EMIM]Cl)and obtained 90% yield of glucose from cellulose and 70e80% yieldof sugars from untreated corn stover. Ion exchange chromatog-raphy allows the recovery of the ionic liquid and delivers sugarfeedstocks that support the vigorous growth of ethanologenicmicrobes. However, there are still many challenges in putting thesepotential applications into practical use, for example, the high costof ILs, regeneration requirement, lack of toxicological data andknowledge about basic physico-chemical characteristics, actionmode on hemicellulose and/or lignin contents of lignocellulosicmaterials and inhibitor generation issues. Consequently completesolvent recovery will be required to make biomass processing withIL economical. Further extensive research is required to addresssuch challenges. Recently Tadesse and Luque [145] have comparedthe environmental impact of different ionic liquids for theconversion of carbohydrates into useful biofuel intermediates withtheir inherent advantages for biomass valorisation processes interms of unique and tuneable physico-chemical properties.


3.4. Biological pretreatment

Biological pretreatment employs wood degrading microorgan-isms, including white-, brown-, soft-rot fungi, and bacteria tomodify the chemical composition and/or structure of the ligno-cellulosic biomass so that the modified biomass is more amenableto enzyme digestion. Although it has so far attracted little attentiondue to many inherent limitations, biological pretreatment hasmultiple benefits. Increasing understanding of termites and fungalsystems has provided insights for developing more effectivepretreatment technologies to realize these benefits. Fungi havedistinct degradation characteristics on lignocellulosic biomass. Ingeneral, brown and soft rots mainly attack cellulose whileimparting minor modifications to lignin, and white-rot fungi aremore actively degrade the lignin component [80]. Present researchis aimed towards finding those organisms which can degrade ligninmore effectively and more specifically. White-rot fungi wereconsidered the most promising basidiomycetes for bio-pretreatment of biomass and were the most studied biomassdegrading microorganisms [146]. The biological pretreatmentappears to be a promising technique and has very evident advan-tages, including no chemical requirement, low energy input, mildenvironmental conditions, and environmentally friendly workingmanner [147,148]. However, its disadvantages are as apparent as itsadvantages since biological pretreatment is very slow and requirescareful control of growth conditions and large amount of space toperform treatment. In addition, most lignolytic microorganismssolubilize/consume not only lignin but also hemicellulose andcellulose. Therefore, the biological pretreatment faces tech-noeconomic challenges and is less attractive commercially [149].

3.5. Techno-economics of pretreatments

Research is focused on converting biomass into its constituentsin amarket competitive and environmentally sustainable approach.Obstacles in the existing pretreatment processes include insuffi-cient separation of cellulose and lignin, formation of by-productsthat inhibit ethanol fermentation, high use of chemicals and/orenergy, and considerable waste production. Only few pretreatmentmethods have been reported for techno-economic analysis. Theseinclude steam explosion, liquid hot water, AFEX, lime and diluteacid pretreatments. Most of the techno-economic studies are basedon process simulations for bioethanol production from lignocellu-losic biomass to produce mainly sugar monomers (glucose, xylose,arabinose, and mannose) and acid-soluble lignin, but usingconversion fractions at fixed operating conditions. Some studies arebased on batch kinetics, where it has revealed that the main factorsaffecting the acid pretreatment are the type of biomass, the type ofacid, the feed acid concentration, the reaction time and the reactiontemperature. So that kinetic modeling and the operating conditionsof the pretreatment unit play an important role in the design,development, and operation of the complete process of bioethanolproduction. Processmodeling and simulation is critical and decisivefor the well design of the pretreatment process of lignocellulosicbiomass [113].

Efficient and economical pretreatment for one feedstock maynot translate to an efficient process for another biomass. Eggemanand Elander [149] carried out an economic analysis of differentpretreatment technologies (dilute acid, hot water, AFEX, ARP andlime) for the Biomass Refining Consortium for Applied Funda-mentals and Innovation (CAFI) as part of an initiative of the UnitedStates Department of Agriculture. Each pretreatment process wasembedded in a full bioethanol facility model and an economicanalysis done. The same feedstock (corn stover) for the differentpretreatment strategies was used in the analysis, and the resulting

solid and fluid streams after pretreatment were characterized, andgathered data were used for the material and energy balances.Their conclusion was that the low-cost pretreatment options areoften counterbalanced by the higher costs to recover catalysts/solvents and the higher costs of ethanol product recovery. Thus,there is little difference in the projected economic performance ofthe different pretreatment options. However, it was also clearlystated that further process improvements such as identification ofoptimum enzyme blends for each pretreatment approach andconditioning requirements of the hydrolyzates may lead to greaterdifferentiation of projected process economics. Sendich and Dale[150] used updated parameters and ammonia recovery configura-tions in the model of Eggeman and Elander and calculated the costof ethanol production using AFEX. They found that the minimumethanol selling price reduced from $1.41/gal to $0.81/gal.

A brief survey of minimum ethanol selling price (MESPs) fromrecent technoeconomic studies of biochemical cellulosic ethanolproduction revealed due to differences in feedstock cost, processassumptions, and co-product values, all of which vary considerablyacross the studies [151]. For example, the analysis by Laser et al. [92]assumes a fairly low feedstock cost and very high yields (indicatingaggressive process assumptions) as well as improved economies ofscale (if such a high feed rate can be sustained), while also receivingpositive revenue fromhigher-value co-products such as protein andhydrogen. Furthermore, this study assumes a consolidated bio-processing (CBP) approach, whichdalthough less developed thanseparate saccharification and co-fermentationdcould furtherimprove economics by reducing enzyme costs. Conversely, studieson the high end of the MESP range such as Kazi et al. [152] assumedhigher feedstock costs while achieving much lower ethanol yields.They have reported the comparison of techno-economic analysis ofseveral process technologies (dilute acid, 2-stage dilute acid, hotwater, AFEX) for the production of ethanol from lignocellulosicbiomass, based on a 5e8 year time frame for implementation.Economic analysis was performed for an nth plant to obtain totalinvestment and product value. It was observed that the productvalue was lowest ($1.36/liter of gasoline equivalent) for dilute acidpretreatment as compared to other pretreatments studied. Sensi-tivity analysis showed that the product value is most sensititve tofeedstock cost, enzyme cost and installed equipment cost. Theproduct value for the pioneer plant model with dilute-acidpretreatment is $2.30/liter of gasoline equivalent and the esti-mated total capital investment was more than double the nth plantcost. Compared to NREL’s 2002 design report (upon which many ofits cost inputs were based), the Klein-Marcuschamer study’s basecase assumes much longer batch times for saccharification andfermentation (thus higher associated capital costs), higher enzymecosts, and a lower carbohydrate fraction in the feedstock (contrib-uting to lower yields) [153].

4. Biocatalytic valorization of lignocellulose

4.1. Saccharification systems for cellulose

Over the past decades, a great amount of research interest andeffort has been generated in the area of enzyme systems for ligno-cellulose bioconversion. Cellulases play a significant role in theenzymatic process by catalyzing the hydrolysis of cellulose tosoluble fermentable sugars. Cellulases are synthesized by fungi,bacteria and plants. A list of microorganisms producing cellulasesare enlisted in Table 3. At least, three major type of cellulase activ-ities are believed to be involved in cellulose hydrolysis based ontheir structural properties: endoglucanases or 1,4-b-D-glucan- 4-glucanohydrolases (EC 3.2.1.4), exoglucanases, including 1,4-b-D-glucan glucanohydrolases (also known as cellodextrinases) (EC

Table 3Production of cellulases using microorganisms.

Microorganism Method Enzyme activities References

FPase CMCase b-glucosidase

Acinetobacter anitratus SmF ND 0.48 U/ml ND [154]Bacillus subtilis SSF 2.8 IU/gds 9.6 IU/gds ND [155]Bacillus pumilus SmF ND 1.9 U/ml ND [156]Cellulomonas biazotea SmF 7450 nkat/g 13,933 nkat/g 2850 nkat/g [157]Clostridium papyrosolvens SmF 35 IU/ml 45 IU/ml ND [158]Chaetomium globosum SmF 1.4 U/ml 30.4 U/ml 9.8 U/ml [159]Streptomyces drodowiczi SmF 4.4 U/gds 595 U/L ND [160]Thermomonospora sp SmF 0.11 IU/ml 23 IU/ml 0.02 IU/ml [161]Thermoascus auranticus SSF 4.4 U/gds 987 U/gds 48.8 U/gds [162]Neurospora crassa SmF 1.33 U/ml 19.7 U/ml 0.58 U/ml [163]Thermotoga maritima SmF ND ND 30 mU/ml [164]Trichoderma ressei SmF 2.49 IU/ml 7.15 IU/ml 2.17 IU/ml [165]T. ressei RUT C 30 SmF 6.2 U/ml 54.2 U/ml 0.39 U/ml [165]T. species A-001 SmF 18 U/ml 167 U/ml 49 U/ml [166]T. ressei ZU 02 SmF 0.25 IU/ml 5.48 IU/ml ND [167]T. viridae SmF 0.88 U/ml 33.8 U/ml 0.33 U/ml [168]Penicillium funiculosum SmF 1.4 IU/ml 4.55 IU/ml 9.29 IU/ml [165]Penicillum pinnophilum SmF 2 U/ml 65 U/ml 10 U/ml [36]P. janthinellum SmF 0.55 U/ml 21.5 U/ml 2.31 U/ml [168]P. decumbans SSF 20.4 IU/g ND ND [169]P. occitanis SmF-Fed 23 IU/ml 21 IU/ml ND [170]A. fumigatus IMI 246651 SmF 40 EU/ml 0.5 EU/ml 1.73 EU/ml [171]A. terreus SSF 243 U/g 581 U/g 128 U/g [172]Fusarium oxysporum SSF 304 U/g ND 0.140 U/g [173]


3.2.1.74) and 1,4-b-D-glucan cellobiohydrolases (cellobiohy-drolases) (EC 3.2.1.91), and b-glucosidases or b-glucoside glucohy-drolasess (EC 3.2.1.21) [174e176]. Cellulolytic systems can beassociated into multienzymatic complexes (called cellulosomes) orunassociated as individual enzymes. In both cases, enzymes haveamodular structure. The unassociated enzymes consist generally ofa catalytic domain responsible for the hydrolysis reaction and ofa cellulose-binding domain (CBD) mediating binding of theenzymes to the substrate. The two domains are joined by a linkerpeptide (Pro/Ser/Thr-rich), which must be sufficiently long andflexible to allow efficient orientation and operation of bothdomains. The cellulosomal enzymes are bound noncovalently to thecellulosome-integrating protein, which carries a CBD [177]. Themore inclusive term CBM (carbohydrate-binding module) hasevolved to reflect the diverse ligand specificity of these modules.Many CBMs have now been identified experimentally, and severalhundred putative CBMs can be further identified on the basis ofamino acid similarity. Similar to the catalytic modules of glycosidehydrolases, CBMs are divided into families based on amino acidsequence similarity. There are currently 64 defined families of CBMs[178] and display substantial difference in ligand specificity. In total,the three-dimensional structures of 36 different CBMs are nowknown, many of which were determined in the last few years (PDBdatabase). CBMs have three general roles with respect to the func-tion of their cognate catalytic modules: (i) a proximity effect, (ii)a targeting function and (iii) a disruptive function. The CBM hasbeen considered as the limiting factor in hydrolysis. CBMs playa pivotal role in degradative enzymes that mediate the recycling ofphotosynthetically fixed carbon in the biosphere. Understandingthe structural basis by which CBMs bind to their target ligandsprovides novel insights into the mechanisms ofcarbohydrateeprotein recognition. The harnessing of this infor-mation to inform strategies designed to manipulate carbohydrate-recognition through the use of ligands that act as agonists orantagonists will be of considerable biotechnological importance notonlywithin an industrial context, but also in the generation of novelpharmaceuticals that are designed tomodify cellecell signaling andhostepathogen recognition [179]. Despite the information availableon these enzyme systems and on the structure of plant cell walls,

application of this knowledge to cellulose degradation hasmetwithlimited success. Thismay be attributed to at least two factors: (i) theinherent complexity and heterogeneity of native cellulose, and (ii)our limited understanding of the basic hydrolysis processes [180].Therefore, an understanding of the molecular mechanisms under-lying cellulose degradation in combination with new and superiorenzymes may facilitate increased usage of this valuable renewableresource [181].

The most frequently reported source of cellulases is the fungusTrichoderma reesei, the most studied cellulolytic microorganismduring the last 60 years. Among the variousmicroorganisms capableof synthesizing cellulase enzymes, T. reesei produces an extracel-lular, stable, and efficient cellulase enzyme system [182,183].However, the low-glucosidase activity of the enzyme system fromT. reesei leads to incomplete hydrolysis of cellobiose in the reactionmixture and, as a result, to serious inhibition of the enzymes [77].Majority of reports on microbial production of cellulases utilizessubmerged fermentation technology (SmF) and thewidely studied Treesei is also grown in liquid media. Appropriate monitoring andhandling of cultures are still major factors in submerged fermenta-tion. The major technical limitation in fermentative production ofcellulases remains the increased fermentation time with lowproductivity. Solid state fermentation (SSF) for production ofcellulases is gaining as a cost effective technology, not only for theproduction of enzyme however for the bioconversion of lignocel-lulosic biomass employing cellulolytic microbes. Even though thereare reports on SSF for production of cellulases, commercial scaleproduction is still using the proven technology of SmF.

An eco-friendly approach towards the hydrolysis of poly-saccharides to monomeric sugars is by the use of enzymes, i.e.,cellulases and hemicellulases. The hydrolysis of cellulose by cellu-lolytic enzymes has been investigated intensively since the early1970s, with the objective of developing a process for the productionof ethanol. Even though soluble substrates have been developed formeasuring endoglucanase and b-glucosidase activities there arevery few substrates available for the estimation of exoglucanaseactivity. The hydrolysis data from soluble substrates cannot yielduseful information on the hydrolysis of insoluble substrates. Theenzymatic hydrolysis of the lignocellulosic biomass is preceeded by


a pretreatment process in which the lignin component is separatedfrom the cellulose and hemicellulose to make it amenable to theenzymatic hydrolysis. The lignin interferes with the hydrolysis byblocking the access of the cellulases to the cellulose and by irre-versibly binding to hydrolytic enzymes. Therefore, the removal ofthe lignin can dramatically increase the hydrolysis rate [54].Enzymatic hydrolysis methods have shown distinct advantagesover acid based hydrolysis methods; the very mild process condi-tions give potentially higher yields, the utility cost is low (nocorrosion problems), Therefore this is the method of choice forfuture wood-to-ethanol processes. The factors influencing enzy-matic hydrolysis can be divided into substrate related factors andenzyme related factors. The relationship between structuralfeatures of cellulose and rates of enzymatic hydrolysis has been thesubject of extensive study and several reviews have been published[176,184]. Most desired attributes of cellulases for lignocellulosebioconversion are the complete hydrolytic machinery, high specificactivity, high rate of turn over with native cellulose/biomass assubstrate, thermostability, decreased susceptibility to enzymeinhibition by cellobiose and glucose, selective adsorption oncellulose, synergism among the different enzymes and ability towithstand shear forces [185]. These parameters are fulfilledthrough protein engineering approaches, over expression tech-niques and developing optimal enzyme cocktails and conditions forhydrolysis. The hydrolytic efficiency of a multi-enzyme complex forlignocellulose saccharification depends both on properties ofindividual enzymes and their ratio in the multi-enzyme cocktail[186]. The ideal cellulase complex must be highly active on theintended biomass feedstock, able to completely hydrolyze thebiomass, operate well at mildly acidic pH, withstand process stress,and be cost-effective [48]. To improve the yield and rate of theenzymatic hydrolysis, research has been focused on optimizing thehydrolysis process, enhancing the cellulase activities, optimizingthe reaction conditions, enzyme and substrate cocktail composi-tion, enzyme recycling and recovery strategies. The yield and initialrate of enzymatic hydrolysis of the cellulose is affected mainly bythe substrate concentration. Realistic methods must be based onphysically and chemically relevant industrial substrates. Recentlythe enzymatic hydrolysis of lignocellulosic biomass has beenoptimized using enzymes from different sources and mixing in anappropriate proportion using statistical approach of factorial design[187]. A twofold reduction in the total protein required to reachglucan to glucose and xylan to xylose hydrolysis targets (99% and88% conversion, respectively), thereby validating this approachtowards enzyme improvement and process cost reduction forlignocellulose hydrolysis [188]. Research is also focused onenzymes that can tolerate both acid and heat whichmay contributetowards the improvement of lignocellulosic biomass processing.These enzymes are produced naturally by extremely thermophilicmicrobes/extremophiles [189,190]. Despite intensive research overthe few past decades, the enzyme hydrolysis step remains asa major techno-economic bottleneck in lignocellulose biomass-to-ethanol bioconversion process.

4.2. Saccharification systems for hemicelluloses

Hemicellulases facilitate cellulose hydrolysis by exposing thecellulose fibers, thus making them more accessible. Hemicellulasesare multi-domain proteins and generally contain structurallydiscrete catalytic and non-catalytic modules. The most importantnon-catalytic modules consist of carbohydrate binding domains(CBD) which facilitate the targeting of the enzyme to the poly-saccharide, interdomain linkers, and dockerin modules thatmediate the binding of the catalytic domain via cohesionedockerininteractions, either to the microbial cell surface or to enzymatic

complexes such as the cellulosome [191]. Based on the amino acidor nucleic acid sequence of their catalytic modules hemicellulasesare either glycoside hydrolases (GHs) which hydrolyse glycosidicbonds, or carbohydrate esterases (CEs), which hydrolyse esterlinkages of acetate or ferulic acid side groups and according to theirprimary sequence homology they have been grouped into variousfamilies. Most studies on hemicellulases have focused until now onenzymes that hydrolyse xylan [192]. Enzymes that hydrolysemannan have been largely neglected, even though it is an abundanthemicellulose, therefore the application of mannanases for cata-lysing the hydrolysis of b-1,4 mannans is as important as theapplication of xylanases.

The two main glycosyl hydrolases depolymerising the hemi-cellulose backbone are endo-1,4- b-D-xylanase and endo-1, 4-b-D-mannanase [193]. Endo-1,4-b-xylanase cleaves the glycosidicbonds in the xylan backbone, bringing about a reduction in thedegree of polymerization of the substrate. Xylan is not attackedrandomly, but the bonds selected for hydrolysis depend on thenature of the substrate molecule, i.e., on the chain length, thedegree of branching, and the presence of substituents [194].Initially, the main hydrolysis products are b-D-xylopyranosyl olig-omers, but at a later stage, small molecules such as mono-, di- andtrisaccharides of b-D-xylopyranosyl may be produced [195]. Theseenzymes are produced by fungi, bacteria, yeast, marine algae,protozoans, snails, crustaceans, insect, seeds, etc. [196]. Table 4 liststhe microorganisms producing different hemicellulases [27]. Fila-mentous fungi are particularly interesting producers of xylanasessince they excrete the enzymes into the medium and their enzymelevels are much higher than those of yeasts and bacteria [195].Aspergillus niger, Humicola insolens, Termomonospora fusca, T. reesei,Trichoderma longibrachiatum, T. koningii have been used as indus-trial sources of commercial xylanases. Nevertheless, commercialxylanases can also be obtained from bacteria, e.g., from Bacillus sp.Xylanases have many commercial uses, such as in papermanufacturing, animal feed, bread-making, juice and wine indus-tries, or xylitol production [196,197]. Several investigations so farhave indicated that xylanases are usually inducible enzymes [198],and different carbon sources have been analysed to find their role ineffecting the enzymatic levels. Xylanase biosynthesis is induced byxylan, xylose, xylobiose or several b-D-xylopyranosyl residuesadded to the culture medium during growth [193, 196 and 200].However, constitutive production of xylanase has also beenreported [199,200]. Catabolite repression by glucose is a commonphenomenon observed in xylanase biosynthesis [192]. Since xylanis a complex component of the hemicelluloses in wood, itscomplete hydrolysis requires the action of a complete enzymesystem, which is usually composed of b-xylanase, b-xylosidase, andenzymes such as a-L-arabinofuranosidase, a-glucuronidase, acetylxylan esterase, and hydroxycinnamic acid esterases that cleave sidechain residues from the xylan backbone. All these enzymes actcooperatively to convert xylan to its constituents [193]. Xylanasesattack randomly the backbone of xylan to produce both substitutedand non-substituted shorter chain oligomers, xylobiose and xylose.Xylosidases are essential for the complete breakdown of xylan asthey hydrolyse xylooligosaccharides to xylose [201]. The enzymesarabinosidase, a-glucuronidase and acetyl xylan esterase act insynergy with the xylanases and xylosidases by releasing thesubstituents on the xylan backbone to achieve a total hydrolysis ofxylan to monosaccharides [202].

The main sugar moiety of galactoglucomannans (GGM) is D-mannose, but for its complete breakdown into simple sugars, thesynergistic action of endo-1,4-b-mannanases (EC 3.2.1.78) andexoacting b-mannosidases (EC 3.2.1.25) is required to cleave thepolymer backbone. Additional enzymes, such as b-glucosidases (EC3.2.1.21), a-galactosidases (EC 3.2.1.22) and acetylmannan esterases

Table 4Microorganisms producing hemicellulases & accessory enzymes.

Enzyme Organism Substrate Specific activity (mmol min�1 mg�1)

Feruloyl esterase Clostridium stercorarium Ethyl ferulate 88b-1,4-xylosidase Thermoanaerobacter ethanolicus o-nitrophenyl-b-D-xylopyranoside 1073Exo-b-1,4-mannosidase Pyrococcus furiosus p-nitrophenyl-b-D-galactoside 31.1Endo-b-1,4-mannanase Bacillus subtilis Galactoglucomannan/glucomannans/mannan 514a-Larabinofuranosidase Clostridium stercoarium alkyl-a-arabinofuranoside/aryl-a-arabinofuranoside/

Larabinogalactan/L-arabinoxylan/methylumbelliferyl-a-Larabinofuranoside

883

a-Glucuronidase Thermoanaerobacteriumsaccharolyticum

4-O-methyl-glucuronosyl-xylotriose 9.6

a-Galactosidase Escherichia coli raffinose 27,350Endo-galactanase Bacillus subtilis arabinogalactan 1790b-Glucosidase Bacillus polymyxa 4-nitrophenyl-b-D-glucopyranoside 2417Acetyl xylan esterase Fibrobacter succinogenes Acetylxylan/a-naphthyl acetate 2933Feruloyl esterase Aspergillus niger Methyl sinapinate 156Endo-1,4-b-xylanase Trichoderma longibrachiatum 1,4-b-D-xylan 6630b-1,4-xylosidase Aspergillus nidulans p-nitrophenyl-b-D-xylopyranoside 107.1Exo-b-1,4-mannosidase A. niger b-D-Man-(1e4)-b-D-GlcNAc-(1e4)-b-DGlcNAc- AsneLys 188Endo-b-1,4-mannanase Sclerotium rolfsii Galactoglucomannan/mannans galactomannans/

glucomannans/380

Endo-a-1,5-arabinanase A. niger 1,5-a-L-arabinan 90.2a-L-arabinofuranosidase A. niger 1,5-a-L-arabinofuranohexaose/1,5-a- L-arabinotriose/

1,5-L-arabinan/a-Larabinofuranotriose396.6

a-Glucuronidase Phanerochaete chrysosporium 4-O-methyl-glucuronosyl-xylobiose 4.5a-Galactosidase Mortierella vinacea melibiose 2000Endo-galactanase A. niger 6593b-glucosidase Humicola insolvens (2-hydroxy methylphenyl)-b-Dglucopyranoside 266.9Acetyl xylan esterase Schizophyllum commune 4-methylumbelliferyl acetate/4-nitro phenyl acetate 227


are required to remove side chain sugars that are attached at variouspoints on mannans [203e205]. The property of mannanolysis iswidespread in the microbial world. A vast variety of bacteria, acti-nomycetes, yeasts and fungi are known to be mannan degraders[206,207]. Mannanases of microbial origin have been reported to beboth induced as well as constitutive enzymes and are usually beingsecreted extracellularly [203]. Although a number of mannanase-producing bacterial sources are available, only a few are commer-cially exploited as wild or recombinant strains, of these, theimportant ones are: Bacillus sp., Streptomyces sp., Caldibacillus cel-lulovorans, Caldicellulosiruptor Rt8B, Caldocellum saccharolyticum[208e210].

Many endoglucanases have been reported to hydrolyze xylo-glucan as a substrate analog [211], however few endo-b-1,4-glucanases have high activity toward xyloglucan, with little or noactivity towards cellulose or cellulose derivatives [212,213]. Theyhave been assigned a new EC number (EC 3.2.1.151) and designatedas xyloglucanase, xyloglucan hydrolase (XGH), or xyloglucan-specific endo-b-1,4-glucanases (XEGs) belonging to families 5, 12,44, and 74, according to a recent classification of glycoside hydro-lases (GHs) available at http://afmb.cnrs-mrs.fr/CAZY/ [214e216].They represent a new class of polysaccharide degrading enzymeswhich can attack the backbone at substituted glucose residues.Among these enzyme families, xyloglucanases placed in GH family74 are known to have high specific activity towards xyloglucan,with inversion of the anomeric configuration, and both endo-typeand exo-type hydrolases have been found in several microorgan-isms [217e227]. The exo-type enzymes recognize the reducing endof xyloglucan oligosaccharide (oligoxyloglucan reducing- end-specific cellobiohydrolase, EC 3.2.1.150, from Geotrichum sp. M128[217] and oligoxyloglucan reducing- end-specific xyloglucanobio-hydrolase from Aspergillus nidulans [224], whereas the endo-typeenzymes hydrolyze xyloglucan polymer randomly. In addition,XEG74 from Paenibacillus sp. KM21 and Cel74A from T. reesei havebeen reported to have endo-processive or dual-mode endo-like andexo-like activities [218,222]. Gloster et al. [228] have reported the

three dimensional structures in ligand free and xyloglucan-oligosaccharide complexed formed from two distinct xylogluca-nases from glycosyl hydrolyase families GH5 (Paenibacillus pabuliXG5) and GH12 (Bacillus licheniformis XG12). The structure ofClostridium thermocellum xyloglucanases, Xgh74A in both apo andligand-complexed forms reveals the structural basis for xyloglucanrecognition and degradation [229]. The complete hydrolysis ofgalactoxyloglucan requires an accessory enzyme, b-galactosidasewhich cleaves the galactose residue attached to the branchedxylose moiety in the b-D-glucopyranose backbone [230]. Thexyloglucan and the enzymes responsible for its modification anddegradation are finding increasing prominence, reflecting the drivefor diverse biotechnological applications in fruit juice clarification,textile processing, cellulose surface modification, pharmaceuticaldelivery and production of food thickening agents [228].

5. Fermentation strategies & consolidated biomassprocessing

The pretreated biomass can be processed using variety ofprocess configurations such as separate hydrolysis and fermenta-tion (SHF), simultaneous saccharification and fermentation (SSF),simultaneous saccharification and co-fermentation (SSCF) andconsolidated biomass processing (CBP) (Fig. 3). Process integrationreduces the capital cost [231]. SHF is a conventional two stepprocess where the lignocellulose is hydrolysed using the enzymesto form the reducing sugars in the first step and the sugars, thusformed, are fermented to ethanol in the second step using variousyeasts such as Saccharomyces, Kluveryomyces, Debaryomyces, Pichia,Zymomonas [232] as well as their recombinants. The advantage ofthis process is that each step can be carried out at its optimumconditions. Current technology using corn stover as feedstock,ammonia fibre expansion as the pretreatment technology andSaccharomyces cerevisiae 424A (LNH-ST) in SHF was able to achieve191.5 g of ethanol/Kg untreated biomass at ethanol concentration of

http://afmb.cnrs-mrs.fr/CAZY/

Multi-stage cellulosic ethanol production:

CBP:

BiomassModifiedbiomass

Biomassrecalcitrance

CBP microbescellulolytic

ethanologenEthanol

Solid/liquidseparation

NativePlant

Enzyme hydrolysis

Hexosefermentation

Ethanol

Pentosefermentation

Cellulose

Hemicelluloses

Pentose sugar

Enzyme hydrolysis

Enzyme production

Fig. 3. Fermentation strategies and consolidated biomass processing.


40 g/L. The process is carried out without washing of pretreatedbiomass, detoxification and nutrient supplementation [233].

The saccharification of the lignocellulosic biomass by theenzymes and the subsequent fermentation of the sugars to ethanolby yeast such as Saccharomyces or Zymomonas take place in thesame vessel in SSF. The compatibility of both saccharification andfermentation process with respect to various conditions such as pH,temperature, substrate concentration etc. is one of the mostimportant factors governing the success of the SSF process [234].The main advantage of using SSF for the ethanol bioconversion isenhanced rate of hydrolysis of lignocellulosic biomass (celluloseand hemicellulose) due to removal of end product inhibition.Another factor of great importance in the fermentative processes isthe cultivation conditions, which, if inadequate, can stimulate theinhibitory action of the toxic compounds [235]. Thermotolerantyeast is an added advantage for SSF and thermotolerant yeaststrains, e.g. Fabospora fragilis, Saccharomyces uvarum, Candidabrassicae, C. lusitaniae, and Kluyveromyces marxianus, have beenevaluated for future use in SSF, to allow fermentation at tempera-tures closer to the optimal temperature for the enzymes. However,in all these cases saccharification of pure cellulose (e.g. Sigmacell-50) or washed fibers, in defined fermentation medium, wereapplied. SSF of cellulose with mixed cultures of different thermo-tolerant yeast strains have also been carried out [236].

Running SSF in fed-batch mode does not necessarily give higherethanol yields than running in batch mode, but when the enzyme-adding method is suitable, similar or slightly higher ethanol yieldscould be obtained in fed batch SSF compared with batch mode. Theappropriate feeding strategy for the enzymes in fed-batch SSFappears to depend on the conditions during SSF [237]. Lu et al.[238] used a fed-batch separate enzymatic hydrolysis andfermentation approach to achieve high ethanol yields (49.5 g/L) ata 30% cumulative insoluble solids loading. This study also showedthat washing the pretreated material results in enhanced conver-sion of cellulose due to removal of inhibitory compounds. Astrategy for achieving high solids concentrations is to utilizealternative bioreactor designs for lignocellulose processing. At highsolids loadings (�15%), mixing via conventional shaking and stir-ring is ineffective [239]. Several novel mixing modes, including

gravitational tumbling in roller bottle reactors (RBRs) [240,241],horizontal rotating shaft with paddlers [242,243], and stirring withhelical impellers [242], have been designed and applied tosaccharification or SSF under high solids loadings. Recently, Zhanget al. [242] reported a bioreactor with a novel helical impeller forSSF at high solids loadings of 15e30% (w/w) of steam explosionpretreated corn stover. They found that the helical stirring systemhad better performances in terms of ethanol concentration andenergy consumption, compared to a Rushton impeller stirring. Atthe highest solids loading of 30%, the ethanol concentrationreached 40.0 and 64.6 g/L after 72 h SSF process, at enzyme dosagesof 7 and 30 FPU/g dry mass, respectively. Mixing energyconsumption was 58.6% of the total thermal energy of the ethanolproduced. A recombinant S. cerevisiae strain expressing the b-glucosidase gene from Humicola grisea was used for ethanolproduction from three different cellulosic sources by SSF. Initially,an enzymatic pre-hydrolysis step was donewith a solid: liquid ratioof 1:4, and an enzyme loading of 25 FPU.g�1 of cellulosic substrate.Using sugarcane bagasse pretreated cellulignin, crystalline cellu-lose and carboxymethyl cellulose, 51.7 g L�1, 41.7 g L�1 and13.8 g L�1 of ethanol was obtained, respectively, at the end of 55 hof fermentation. The highest ethanol productivity (0.94 g L�1 h�1)was achieved using sugarcane bagasse pretreated cellulignin [243].There is scarcity of literature from SSF experiments in whichhemicelluloses have been used together with thermotolerantstrains. Menon et al. [244] have reported for the first time the SSFexperiments using hemicellulosic substrates and thermotolerantDebaryomyces hansenii. Maximum ethanol concentrations of 9.1 g/Land 9.5 g/L were obtained in SSF with oat spelt xylan and wheatbran hemicellulose respectively. These concentrations wereattained in 36 h for OSX and 48 h for WBH from the onset of SSF.The increased ethanol yield in SSF systems is evidently due toremoval of xylose formed during hydrolysis which causes endproduct inhibition. The techno-economic study compares severalprocess technologies for the production of ethanol from lignocel-lulosic material, based on a 5e8 year time frame for implementa-tion [152]. Wringen et al. [245] have performed the techno-economic evaluation of producing ethanol from softwood by SHFand SSF and have discussed the bottleneck issues. Themain focus of


the techno-economic study conducted by Sassner et al. [246] wason the pretreatment and the SSF steps for three lignocellulosicsubstrates (spruce, salix and corn stover). Sensitivity analyses ofimportant process parameters showed their relative effects on theproduction cost and on the potential for cost reduction for each rawmaterial. The study clearly demonstrates the importance of a highethanol yield and the necessity of utilizing the pentose fraction forethanol production to obtain good process economy, especiallywhen using Salix or corn stover. Furthermore, a less energy-demanding process, here mainly achieved by increasing the drymatter content in SSF, reduces the capital cost and results in higherco-product credit, and therefore has a significant effect on theoverall process economy.

Simultaneous saccharification and co-fermentation (SSCF) hasbeen recognized as a feasible option for ethanol production fromxylose-rich lignocellulosic materials. Simultaneous saccharificationof cellulose and hemicellulose and co-fermentation of end productsglucose and xylose is reported to be carried out by geneticallyengineered microbes. A combination of enzyme and substratefeeding was shown to enhance xylose uptake by a recombinantxylose-fermenting strain of S. cerevisiae (TMB3400) and increaseoverall ethanol yield in SSCF of steam-pretreated wheat straw. Thisis conceptually important for the design of novel SSCF processesaiming at high-ethanol titers [247]. Zhang and Lynd [248] havereported the SSCF of waste paper sludge to ethanol using tworecombinant xylose-fermenting microbes: Zymomonas mobilis 8band S. cerevisiae RWB222. S. cerevisiae RWB222 produced over 40 g/L ethanol with a yield of 0.39 g ethanol/g carbohydrate on papersludge at 37 �C, while similar titers and yields were achieved byZ. mobilis 8b at 30 �C. Enzyme kinetics and yeast sugar uptake ratesfor a recombinant xylose utilizing strain of S. cerevisiae, TMB3400,were determined in a real hydrolyzate medium. The total xyloseuptake could be increased from 40% to as much as 80% bycontrolling the enzyme feed [249]. Synthetic biology approach forco-fermentation of hexose and pentose sugars has been success-fully demonstrated by incorporating two mutants of E. coli ina single system e one capable of utilizing only glucose, while theother capable of utilizing only xylose as a carbon source. In thisstudy, catabolite repression due to the presence of glucose wasmade irrelevant by the use of two strains, since one cannot utilizeglucose at all. Furthermore, the authors demonstrate by fed-batchexperiments that the system robustly adapts to fluctuations inthe feed stream, i.e., cultures actually grow in concert with thevarying feed composition [250]. Another promising alternative forsimultaneous utilization of pentoses and hexoses is the conversionof xylose into xylulose by xylose isomerase or oxidoreductase. Thefermentation of xylulose begins by its intracellular phosphorylationto xylulose-5-phosphate, which enters the pentose phosphatepathway and eventually forms ethanol. Yeasts like S. cerevisiae andSchizosaccharomyces pombe are capable of fermenting xylulose toethanol, along with glucose [251].

Lignocellulosic bioprospecting involving enzymes or microbialsystems commonly includes three major biologically mediatedprocesses: production of saccharolytic enzymes (cellulases andhemicellulases), hydrolysis of cellulose and hemicellulose tomonomeric sugars, fermentation of hexose and pentose sugars.These three biotransformations occurring in a single processconfiguration is called as consolidated biomass processing (CBP).CBP offers the potential for lower cost and higher efficiency thanprocesses featuring dedicated cellulase production. This result fromavoided costs for capital, substrate and other raw materials, andutilities associated with cellulase production. In addition, severalfactors support the possibility of realizing higher hydrolysis rates,and hence reduced reactor volume and capital investment, usingCBP. These include enzymeemicrobe synergy as well as the use of

thermophilic organisms and/or complexed cellulase systems. CBPrequires a microbial culture that combines properties related toboth substrate utilization and product formation. Desired substrateutilization properties include the production of a hydrolyticenzyme system allowing high rates of hydrolysis and utilization ofresulting hydrolysis products under anaerobic conditions witha practical growth medium. Desired product formation propertiesinclude high product selectivity and concentrations. A cellulolyticculture with this combination of properties has not been describedto date. Development of microorganisms for cellulose conversionvia CBP can be pursued according to two strategies. The nativecellulolytic strategy involves naturally occurring cellulolyticmicroorganisms to improve product-related properties such asyield and tolerance. The recombinant cellulolytic strategy involvesengineering noncellulolytic microorganisms that exhibit highproduct yields and tolerance so that they become able to utilizecellulose as a result of a heterologous cellulase system [176]. BothCBP organism development strategies involve very large challengesthat will probably require a substantial sustained effort to over-come. The feasibility of CBP will be fully established only whena microorganism or microbial consortium is developed thatsatisfies the requirements. Jin et al. [252] have evaluated theperformance of CBP organism Clostridium phytofermentans (ATCC700394) on AFEX-treated corn stover (AFEX-CS). At optimalconditions with 0.5% (w/w) glucan loading of AFEX-CS,C. phytofermentans hydrolyzed 76% of glucan and 88.6% of xylanin 10 days. These values reached 87% and 102% of those obtained bySSCF using commercial enzymes and S. cerevisiae 424A. Ethanoltiter for CBP was found to be 2.8 g/L which was 71.8% of that yieldedby SSCF (3.9 g/L). Fundamental and applied topics relevant to CBPare comprehensively reviewed in 177, 255 and 256.

Process integration plays a major role in efficient process design.When several operations can be performed in a same single unit,the possibilities for improving the performance of the process arehigher. Reactionereaction integration has been proposed for theintegration of different biological transformations taking placeduring ethanol production [255]. This type of integration can bedone between different steps of ethanol production processinvolving chemical transformations as well. In this sense, theintegration of detoxification step with fermentation can play animportant role in their intensification and in the reduction ofethanol production costs. Palmqvist and Hahn-Hägerdal [256]propose a way to implement this kind of integration consisting incarrying out the detoxification in the same vessel in whichfermentation is to be accomplished just before the cultivationprocess. Separation is the step where major costs are generated inprocess industry. Therefore, reactioneseparation integration couldhave the highest impact on the overall process in comparison withhomogeneous integration of processes (reactionereaction,separationeseparation) [257]. The development of technologiesfor separationeseparation integration has been linked to thedevelopment of the different involved unit operations and to newapproaches for process intensification. The examples ofseparationeseparation integration in the case of ethanol produc-tion mostly correspond to integration of the conjugated type, i.e.,when integrated processes are carried out in different equipmentsclosing the flowsheet by fluxes or refluxes [258]. In this context, theapplication of saline extractive distillation and membrane tech-nologies for the integration of several configurations for separationand dehydration of ethanol should be highlighted [259]. Similarly,the integration of different chemical and biological processes forthe complete utilization of the feedstocks should lead to thedevelopment of big “biorefineries” that allow the production oflarge amounts of fuel ethanol and many other valuable co-productsat smaller volumes, improving the overall economical effectiveness


of the conversion of a given rawmaterial. Integration opportunitiesmay provide the ways for a qualitative and quantitative improve-ment of the process tomake it techno-economical and eco-friendly.

5.1. Future economic performance of hydrolysis process concepts

Lignocellulosic ethanol is expected to be commercializedduring the next decade as renewable energy for transport. Thetechno-economic analysis for commercial stage evaluates thecompetitiveness with first generation bioethanol and gasoline.During few decades, researchers are analyzing the techno-economic status of second generation biofuels. The logistics ofproviding a competitive, all-year-round, supply of biomass feed-stock to a commercial-scale plant is challenging, as is improvingthe performance of the conversion process to reduce costs [259].The biochemical route, being less mature, probably has a greatercost reduction potential than the thermo-chemical route, but herea wider range of synthetic fuels can be produced to better suitheavy truck, aviation and marine applications. Continued invest-ment in research and demonstration by both public and privatesectors, coupled with appropriate policy support mechanisms, areessential if full commercialisation is to be achieved within the nextdecade. After that, the biofuel industry will grow only at a steadyrate and encompass both 1st- and 2nd-generation technologiesthat meet agreed environmental, sustainability and economicpolicy goals [260].

Although conversion of cellulosic biomass to ethanol has beenstudied for decades, the uncertainty of techno-economic feasibility,particularly at large scale production, prohibits commercializationof such processes. Besides the relatively high cost of some pro-cessing stages (i.e. pretreatment and enzymatic hydrolysis), thecost of feedstocks share a large portion of operating costs. Duringthe late 19th and early 20th century techno-economic evaluation oflignocellulosic ethanol involves two major studies by NREL inassociation with other US research institutes and universities[261,262]. The NREL 2002 report projects that for a production scaleof 2000 ton of feedstock per day, at $30/ton corn stover, accountsfor 31.3% of the overall operating costs [263]. At a larger scale of5000 tons of corn stover per day and a higher corn stover price of$40/ton, feedstock costs were estimated to account for 71.8% of theoperating costs with advanced bioconversion processes [254]. Onthe other hand, using seasonally harvested feedstocks, such asagricultural wastes and energy crops, also raises questions ofobtaining year-long supply or feedstock storage for large scaleproduction. Therefore, lower feedstock costs along with achievinghigh yields of ethanol can result in significant improvements in theeconomics of cellulosic ethanol.

The techno-economic evaluation strategies have been changedfrom 2002, with the launch of the Biomass Program of the USDepartment of Energy (DOE). Since 2007, the design of this programhas acquired a clear strategic goal with the aim of the publicauthorities to reduce the use of gasoline by20% by2017 andproduce35�109 L of renewable and alternative fuels in 2017 [259]. DOE haslaunched a multi-year program plan (MYPP) and is updated everytwo years, including so far 2004 [263], 2005 [264], 2007 [265], 2009[266], 2011 [267]. The estimation of the ethanol program cost target(EPCT) in 2012 for the biochemical ethanol is based on the referencescenario by the Energy Information Administration (EIA, 2009)which forecasts the wholesale price of gasoline in 2012 at US$ 2.62/gal gasoline (US$ of 2007). Assuming a conversion factor of0.67 gallon gasoline per gallon of ethanol, the EPCT is set at US$ 1.76/gal ethanol (US$ of 2007). The contribution made by feedstockproduction to the ethanol production cost increases from oneMYPPto the other due to progress in understanding and estimating thefeedstock production and logistics [259].

Sassners et al. [246] compare the techno-economic performanceof conversion of lignocellulosic-to-ethanol based on three differentfeedstocks, a softwood (spruce), a hardwood (salix) and an agri-cultural residue (corn stover). The process consists of SO2-catalysedsteam explosion pre-treatment and SSF. The process capacity of theethanol plants is supposed to be 200,000 dry tons of biomass peryear. The process parameters are adjusted to experimental data andadapted to each feedstock. Enzymes are assumed to be purchasedwhile the yeast is produced on-site. The authors find the followingenergy efficiencies for ethanol output: 25% (salix), 25% (corn stover)and 31% (spruce) for the base case and the maximum energy effi-ciencies in the range of 52e53% for salix, 55% for corn stover and56% for spruce. The economic evaluation consists in estimatingannual production cost including annulaised capital cost using 7%interest rate and 15 years depreciation period, and annual opera-tion costs. For the base case the annual production cost significantlyvaries i.e. US$0.69/l ethanol (spruce), 0.86 (corn stover) and 0.87(salix). For alternative cases the cost estimates were 0.66 (spruce),0.67 (corn stover) and 0.72 (salix). Wingren et al. [268] performa techno-economic evaluation of SSF-based softwood-to-ethanol,with the objective to compare the impact of various downstreamconfigurations. The base case consists in conversion of wood chipsof spruce to ethanol. The conversion process and the economicevaluation use the same approach as in Sassners et al. [246]. Theproduction cost (US$/l) varies between 0.546 for the mechanicalvapour recompression option to 0.591 for the base case. The case ofanaerobic digestion results in 0.549 (US$/l) production cost. Atechno-economic evalution of the spruce-to-ethanol process, basedon SO2 catalysed steam pretreatment followed by SSF, has beenperformed using the commercial flow-sheeting program AspenPlusTM [269]. Various process configurations of anaerobic diges-tion of the stillage, with different combinations of co-products,have been evaluated in terms of energy efficiency and ethanolproduction cost. Anaerobic digestion of stillage showed a signifi-cantly higher overall energy efficiency (87e92%) based on thelower heating values, the production cost varied between 4.00 and5.27 Swedish kronor per liter (0.38e0.50 euro/l).

A comprehensive techno-economic analysis was performed forconversion of cellulosic feedstock (Tall Fescue) to ethanol usingsome of the common pretreatment technologies: dilute acid, dilutealkali, hot water and steam explosion [270]. Detailed processmodels incorporating feedstock handling, pretreatment, simulta-neous saccharification and co-fermentation, ethanol recovery anddownstream processing were developed using SuperPro Designer.Projected ethanol yields were 252.62, 255.80, 255.27 and 230.23 L/dry metric ton biomass for conversion process using dilute acid,dilute alkali, hot water and steam explosion pretreatment tech-nologies respectively. Price of feedstock and cellulose enzymeswere assumed as $50/metric ton and 0.517/kg broth (10% protein inbroth, 600 FPU/g protein) respectively. Capital cost of ethanolplants processing 250,000 metric tons of feedstock/year was $1.92,$1.73, $1.72 and $1.70/L ethanol for process using dilute acid, dilutealkali, hot water and steam explosion pretreatment respectively.Ethanol production cost of $0.83, $0.88, $0.81 and $0.85/L ethanolwas estimated for production process using dilute acid, dilutealkali, hot water and steam explosion pretreatment respectively.The results demonstrated the importance of addressing the trade-offs in capital costs, pretreatment and downstream processingtechnologies.

Lynd et al. [271] indicates that there is no capital or operationexpenditures required for the enzyme production within the CBPprocess. Similarly, part of the substrate is not deviated to enzymeproduction. According to the projections, the reduction ofproduction costs due to an advanced configuration involving CBP isthree times greater than the reduction related to the scale of


economy of the process and ten times greater than the reductionassociated with a lower cost of the feedstock. This diminish isaccomplished, thanks to the reduction of more than eight times inthe cost of biological conversion [271]. The comparative cost ofethanol production by CBP and SSCF (featuring a dedicated cellu-lase production) processes are simulated assuming aggressiveperformance parameters intended to be representative of maturetechnology [253]. Their results indicated that the total biologicalprocessing cost for ethanol production reaches 18.9 ¢/L, taking intoaccount the 9.90 ¢/gal ethanol for dedicated cellulase productionand 9 ¢/gal for SSCF which is more than fourfold larger than the4.2 ¢/gal projected for CBP. Hamelinck et al. [272] have analysed thetechno-economic performance of promising conversion conceptsfor the short-, middle- and long-term systems. The current avail-able technology, which is based on dilute acid hydrolysis, has about35% efficiency (HHV) from biomass to ethanol. The overall effi-ciency, with electricity co-produced from the not fermentablelignin, is about 60%. Improvements in pre-treatment and advancesin biotechnology, especially through process combinations canbring the ethanol efficiency to 48% and the overall process effi-ciency to 68%. They estimated the current investment costs at2.1 kh/kWHHV (at 400 MWHHV input, i.e. a nominal 2000 tonnedry/day input). A future technology in a 5 times larger plant(2GWHHV) could have investments of 900 kh/kWHHV. A combinedeffect of higher hydrolysis-fermentation efficiency, lower specificcapital investments, increase of scale and cheaper biomass feed-stock costs (from 3 to 2 h/GJHHV), could bring the ethanol produc-tion costs from 22 h/GJHHV in the next 5 years, to 13 h/GJ over the10e15 year time scale, and down to 8.7 h/GJ in 20 or more years. InDe Vries et al. [273](based on the analyses of Hoogwijk et al. [8]), itis indicated that the biofuel production potential around 2050could lay between about 70 and 300 EJ fuel production capacitydepending strongly on the development scenario. Around thattime, biofuel production costs would largely fall in the range up to15 U$/GJ, competitive with equivalent oil prices around 50e60 U$/barrel. The process efficiency and potential technical advances andchallenges along with cost information (USD2005/GJ) for severalbioenergy processes for biofuel production are summarized inTable 5. The relatively higher production cost of ethanol is the mainobstacle to be resolved. Process engineering plays a central role forthe generation, design, analysis and implementation of technolo-gies improving the indexes of global process. Undoubtedly, processintensification through integration of different phenomena andunit operations as well as the implementation of consolidatedbioprocessing of different feedstocks into ethanol (that requires thedevelopment of tailored recombinant microorganisms), will offer

Table 5Projected cost for developing biochemical technologies for 2030 (adapted from [4]).

Process Feedstock Efficiency and process economicsEff. ¼ energy product/biomassenergy component costs in USD20

SHF Barley straw Steam explosion, enzyme hydrolyfermentation, High solids 15%

SSF Bagasse Standalone plant 370 L/t dry (ethþ 0.56 kWh/L ethanol (electricity

Corn stover Dilute acid hydrolysis, 260 million

Lignocellulosic variousEff. 35% ethanol þ 4% power

Eff. Kg/L ethanol (poplar, Miscantswitchgrass, corn stover, wheat: 32.6, 2.4). Plant size 1500 to 1000

SSCF Lignocellulosic Eff.w39% ethanol þ 10% power

CBP Eff.w49% for wood and 42% for st(ethanol) þ 5% power

the most significant outcomes during the search of the efficiency infuel ethanol production. Additionally, the intensification of bio-logical processes implies a better utilization of the feedstocks andthe reduction of process effluents improving the environmentalperformance of the proposed configurations [255].

6. Metabolic engineering approaches

One of the strategies for improving the economics and yields oflignocellulosic bioethanol process is optimization of metabolicpathways through genetic modifications for effective manipulationof metabolic capabilities of microbes. The exploitation of thediverse metabolic pathways leading to energy-rich, fuel-likehydrocarbons opens up a path to develop renewable fuels whichare beyond the restrictions of bioethanol and plant-derived bio-diesel [274]. The increasing number of sequenced genomescollected globally, including potential energy crops and cellulolyticmicrobes will be significant for cellulosic biofuel production [275].Considerable progress has been made to understand the inductionmechanism of cellulases by lactose in Trichoderma ressei, whichinvolves an alternative D-galactose metabolism pathway. With therecent availability of the complete genome sequence, T ressei hasentered the post-genomic era and the knowledge in advancementof metabolic engineering approaches will enhance the probabilityof obtaining new hyper active mutants strains. Trichoderma sp.dominantly occupies ecological niches and a better understandingof signal transduction pathways initiating and/or modulating, mayhelp to develop new strategies for improving cellulase geneexpression [276].

Metabolic redirection is an alternative strategy, i.e., redirec-tion of the central metabolism of the ethanologenic organism bygene knockout to block undesirable metabolic pathways, inorder to divert the carbon flow of pyruvate from organic acids toethanol production [277,278]. The yeast S. cerevisiae, rarelypossess native pathways to efficiently ferment both hexoses andpentoses [279]. Escherichia coli is the most convenient startingpoint for engineering microbial catalysts for biofuel production,owing to the wealth of genetic and metabolic knowledge avail-able [280]. E. coli can actively metabolize a wide range ofsubstrates, including hexoses and pentoses [281], but its hexosemetabolism is inferior to that of Z. mobilis, an obligate ethano-logenic bacterium [282]. As a metabolic enhancement strategy,E. coli has been genetically engineered for cofermentation of allconstitutive sugars from lignocellulose by importing the highlyefficient fermentation pathway for ethanol production fromZ. mobilis [283].

,

05/GJ

Potential technical advances andchallenges

Production cost by2030 (USD2005/GJ)

sis, ethanol System integration, high solids,decrease toxicity for fermentation

30 (Finland)from pilot data

anol))

Mechanical harvest improvementssugarcane residues (occurring)

6e15

L/yr Pretreatment process integration,enzyme cost

15.5 (US)nth plant, future

hus,.7, 3.2, 2.6,t/day

Process integration, project a 25%reduction in operation cost by2025 and 40% by 2035

18e22 (2020)

Efficient 5 carbon conversiontechnology, advanced enzymes,thermotolerant microbes

25e2728e35

raw Lignin engineering facilitatescellulose access, developefficient CBP microbes

15.5 future


Plant genetic engineering technology offers great potential toreduce the cost of lignocellulosic biofuel. All necessary cell walldegrading enzymes such as cellulases and hemicellulases could beproduced within the crop biomass. However, research is needed todetermine the stability of the biological activity of extracted plant-produced hydrolysis enzymes. Studies also need to be focused onincreasing the titre of enzyme production and biological activity ofthe heterologus enzymes. The plant genetic engineeringapproaches could be applied to modify lignin amount and/orconfiguration in order to reduce the needs for expensive pretreat-ment technologies. Recent advances in the understanding of lignincomposition, polymerization and regulation have revealed newopportunities for the rational manipulation of lignin in futurebioenergy crops, augmenting the previous successful approach ofmanipulating lignin monomer biosynthesis. Furthermore, recentstudies on lignin degradation in nature may provide novelresources for the delignification of dedicated crops and othersources of lignocellulosic biomass [278]. Recently Fu et al. [284]have showed that genetic modification of switchgrass canproduce phenotypically normal plants that have reduced thermal-chemical (�180 �C), enzymatic, and microbial recalcitrance. Down-regulation of the switchgrass caffeic acid O-methyltransferase genedecreases lignin content modestly, reduces the syringyl:guaiacyllignin monomer ratio, improves forage quality, and, most impor-tantly, increases the ethanol yield by up to 38% using conventionalbiomass fermentation processes. The down-regulated lines requireless severe pretreatment and 300e400% lower cellulase dosages forequivalent product yields using simultaneous saccharification andfermentation with yeast. Furthermore, fermentation of dilutedacid-pretreated transgenic switchgrass using C. thermocellum withno added enzymes showed better product yields than obtainedwith unmodified switchgrass. Therefore, this apparent reduction inthe recalcitrance of transgenic switchgrass has the potential tolower processing costs for biomass fermentation-derived fuels andchemicals significantly. Finally, future research on the upregulationof cellulose and hemicellulose biosynthesis pathway enzymes forincreased polysaccharides will also have the potential to increasecellulosic biofuel production [285]. The genes necessary for thebiosynthesis of cellulose and hemicellulose are identified by func-tional genomics and mutant studies. However the completebiosynthetic pathway is not yet well studied. Although research hasbeen tremendously focused on plant genetic engineering for bio-fuel production, this science is still premature. The major challengeis to develop efficient genotype-nonspecific genetic engineeringsystems in feedstock crops.

Advancements in the fields of metabolic engineering, syntheticbiology, and systems biology have further increased the ability tosuccessfully implement and analyze different strategies to engineermicrobes and plants for the production of a broad range of novelbiofuels through the exploitation of various metabolic pathways.The continued development of synthetic biology tools that bothreduce the time required to make genetic constructs as well asincreasing their predictability and reliability should greatlyimprove metabolic engineering techniques for the effectiveproduction of a wide variety of fuels [286] and chemicals [287]. Forthe biomass-derived fuel production, the pathway design isunderway to expedite the hydrolytic conversion from cellulose tomonosaccharides and the subsequent fermentation to obtain C3/C4alcohols or fatty acids. For successful construction of syntheticorganisms, a hybrid approach has been commonly practiced: basedon the alternate routes and enzymes found in multiple naturalorganisms, one may develop efficient transport systems, improvethe hosts’ tolerance against toxic intermediates, optimize carbonflux to the desired fuels, and establish robust strains that fit massproduction settings [288]. When exposed to a mixture of carbon

sources, wild-type E. coli exhibits a specific hierarchy for theirutilization. As long as they are present in sufficient amounts in thegrowth medium, sugars such as glucose, sucrose, and fructoserepress the synthesis of enzymes necessary for the transport andmetabolism of less favorable carbon sources such as xylose, arabi-nose, maltose, and lactose. The development of recombinant E. colistrains capable of efficiently utilizing sugar mixtures is essential forthe large scale production of biofuels from lignocellulosic materials[289]. While the efforts to coutilize hexose and pentose sugars inE. coli through the inactivation of the phosphotransferase system(PTS) were successful, the existence of other, less understoodregulatory systems that prioritize the sequential metabolism ofsugar mixtures provides another avenue to engineer E. coli strainsfor the simultaneous consumption of hexose and pentose sugars.Fermentative pathways have been utilized to engineer E. coli for theproduction of a variety of alcohols, while several non-fermentativepathways have been engineered to enable the production of bio-fuels such as higher chain alcohols from the amino acid biosyn-thetic pathways and novel fuels derived from the fatty acid andisoprenoid biosynthetic pathways. In contrast to the native E. colipathway, the pathway for ethanol production in Zymomonasmobilis is a two-step process in which pyruvate decarboxylase(PDC) converts pyruvate directly to acetaldehyde and CO2 andalcohol dehydrogense (ADH) then converts acetaldehyde intoethanol. Expression of Z. mobilis pdc and adhB genes (encoding forpyruvate decarboxylase and alcohol dehydrogenase, respectively)in E. coli, via a plasmid bearing an artificial pet (production ofethanol) operon, resulted in much higher ethanol yields, withethanol accounting for 95% of the fermentation products [290].Another recent study developed a minimal E. coli cell for efficientethanol production from hexoses and pentoses using elementarymode analysis to dissect the metabolic network into its basicbuilding blocks [291]. Analysis and construction of a minimal E. colicell expressing the Z. mobilis pdc and adhB genes, resulted in theconversion of glucose or xylose to ethanol at theoretical yields.However, recent studies have found that succinate and acetatewere also produced during glycerol fermentation and thereforerepresented competing by-products whose synthesis decreases theyield of ethanol [292,293]. Therefore, metabolic engineeringstrategies were implemented in order to minimize by-productformation through the disruption the genes encoding fumaratereductase (frdA) and phosphotransacetylase (pta), key enzymes inthe synthesis of succinate and acetate, respectively [294]. Arabinoseis another pentose sugar obtained upon deconstruction of biomass.Recently, a mutant yeast strain which anaerobically convertsarabinose to ethanol in batch fermentationwas reported [295]. Thisstrain was obtained by introducing the bacterial pathway forarabinose utilization from Lactobacillus plantarum, overexpressingS. cerevisiae genes encoding the nonoxidative pentose phosphatepathway enzymes, and subsequent evolutionary engineering. Anethanol yield of 0.43 g/g carbohydrate consumed and a specificethanol production rate of 0.29 g/g/h from arabinose as the solecarbon source were achieved. Peralta-Yahya and Keasling [296]have reported the heterologus expression of the clostridial C3eC4biosynthetic pathway for the production of isopropanol and 1-butanol in E. coli and S. cerevisiae. The highest reported produc-tion of isopropanol by the engineered E.coli is 4.9 g/L compared toClostridium (1.8 g/L). The butanol yield obtained from the engi-neered S. cerevisiae (2.5 mg/L) was two orders-of-magnitude lowerthan that obtained by E. coli (550 mg/L). It should be noted that allthese engineering processes will shift toward more and more denovo design and synthesis, and the pace will heavily depend on thestandardization and quantitative characterization of the biologicalparts using miniaturized technologies, such as single-moleculegene sequencing, gene synthesis on a DNA microchip, and the


multifunctional technology found in microenvironments. Finally,new tools for better de novo design of synthetic pathways need tobe developed. Several databases, such as BNICE (BiochemicalNetwork Integrated Computational Explorer) [297] and ReBiT(Retro-Biosynthesis Tool) [298], have already been established tofacilitate identification of enzymes to construct a completesynthetic pathway for producing target compounds. It is importantto establish guidelines, such as redox balance, energy production,and thermodynamic feasibility, to screen among these enormouspathways for the optimal routes [299].

7. Alternative liquid fuels - biobutanol and 2,3-butanediol

Biobutanol or Biobased butanol fuel is second generation alco-holic fuel with a higher energy density and lower volatility ascompared to ethanol. Butanol is a 4-carbon alcohol (butyl alcohol),an advanced biofuel, offers a number of advantages and can helpaccelerate biofuel adoption in countries around the world. It can beproduced through processing of domestically grown crops, such ascorn and sugar beets, and other biomass, such as fast-growinggrasses and agricultural waste products. Biobutanol’s primary useis as an industrial solvent in products such as lacquers and enamelsand is also compatible with ethanol blending and can improve theblending of ethanol with gasoline [300]. Butanol’s application asa replacement for gasoline will outpace ethanol, biodiesel andhydrogen when its safety and simplicity of use are seen. Butanol’sapplication for the Department of Defense as a clean-safereplacement for batteries when used in conjunction with fuel celltechnology is seen as an application for the future. Disposablecanisters made of PLA that carry butanol to be reformed and used togenerate electricity for computers, night vision and stealth equip-ment can be easily disposed of [301]. Using fermentation to replacethe chemical process for the production of butanol depends largelyon the availability of inexpensive and abundant raw materials andefficient conversion of these materials to solvents. SolventogenicAcetone Butanol Ethanol (ABE)-producing Clostridia have an addedadvantage over many other cultures as they can utilize both hexoseand pentose sugars [302], which are released from wood andagricultural residues upon hydrolysis, to produce ABE. Parekh et al.[303] produced ABE from hydrolysates of pine, aspen, and cornstover using Clostridium acetobutylicum P262. Similarly Marchalet al. [304] used wheat straw hydrolysate and C. acetobutylicum,while Soni et al. [305] used bagasse and rice straw hydrolysates andClostridium saccharoperbutylacetonicum to convert these agricul-tural wastes into ABE. Qureshi et al. [306] have studied theproduction of butanol from corn fibre hydrolysate using Clos-tridium beijerinckii BA101. Sun and Liu [307] have reported theproduction of butanol from sugar maple hemicellulose hydrolysateusing C. acetobutylicum ATCC824. As compared to the existingstrains of C. beijerinckii BA101, C. acetobutylicum PJC4BK and C.acetobutylicum P260, which produces a total ABE on the order of25e33 g/L [306,308e310], development of more robust and effi-cient second generation cultures are essential for the economicviability of biobutanol production. Improvements in yields anda better understanding of the butanol toxicity are among thepotential benefits of engineering the butanol production pathwayinto a well known host such as E.coli or S. cerevisiae [311]. Thehighest butanol productivity observed in E. coli was 1.2 g L�1 overa 60-h period, with a carbon yield that was 15% of the theoreticalmaximum of 0.41 g butanol g�1 glucose [312]. Although the lattervalue is significantly lower than in native Clostridia, it representsa promising starting point for further engineering.

Research on the production of butanol from other agriculturalresidues including corn stover, barley straw and switchgrass hassteadily progressed. Use of several product-recovery technologies

such as liquideliquid extraction, gas stripping, perstraction, andpervaporation has been successfully applied in laboratory-scalebioreactors [313]. It is expected that these recovery technologieswill play a major role in commercialization of this fermentation[306]. The production of biobutanol from biomass has led to the re-examination of ABE fermentation, including strategies for reducingor eliminating butanol toxicity to the culture and for manipulatingthe culture to achieve better product specificity and yield. Advancesin integrated fermentation and in situ product removal processeshave resulted in a dramatic reduction of process streams, reducedbutanol toxicity to the fermentating microbes, improved substrateutilization and overall improved bioreactor [310]. Recently butanolis a potential gasoline replacement that can also be blended insignificant quantities with conventional diesel fuel. These effortshave transitioned to research focused on the development of viablemethods for the production of an array of oxygenated and fullysaturated jet and diesel fuels from butanol [301].

2,3-Butanediol, also known as 2,3-butylene glycol (2,3-BD) isa valuable chemical feedstock because of its application asa solvent, liquid fuel, and as a precursor of many synthetic polymersand resins [314]. Butanediol is produced during oxygen-limitedgrowth, by a fermentative pathway known as the mixed acid-butanediol pathway [315]. The 2,3-BD pathway and the relativeproportions of acetoin and butanediol serve to maintain theintracellular NAD/NADH balance under changing culture condi-tions. All of the sugars commonly found in hemicellulose andcellulose hydrolysates can be converted to butanediol, includingglucose, xylose, arabinose, mannose, galactose, and cellobiose. Thetheoretical maximum yield of butanediol from sugar is 0.50 kg perkg. With a heating value of 27,200 J/g, 2,3-BD compares favorablywith ethanol (29,100 J/g) and methanol (22,100 J/g) for use asa liquid fuel and fuel additive [316]. Hexose and pentose can beconverted to 2,3-BD by several microorganisms including Aero-monas [317], Bacillus [318], Paenibacillus [319], Serratia, Aerobacter[320], Enterobacter [321] and also Klebsiella [322e327]. Amongthese, especially Klebsiella is often used for 2,3-BDO productionbecause of its broad substrate spectrum and cultural adaptability.At first, 2,3-BD was produced worldwide from glucose or xylose byfermentation. Starch [317,328], molasses [329], water hyacinth[330] and Jerusalem artichoke tubers [331] were also tested fortheir suitability as substrates for 2,3-BD fermentation. Some studieshave been conducted utilizing the hemicellulose portion of forestresidues like wood for 2,3-BD production [332]. An industrialmedium containing urea as a sole nitrogen source, low levels ofcorn steep liquor and mineral salts as nutrition factors to retainhigh 2,3-butanediol production through co-fermentation ofglucose and xylose (2:1, wt/wt) by Klebsiella oxytocawas developedto yield 0.428 g/g of 2,3-BD, which was 85.6% of theoretical value[326]. Cheng et al. [327] have reported production of 2,3-butanediol from corn cob hydrolysate by fed batch fermentationusing K. oxytoca. A maximal 2,3-butanediol concentration of 35.7 g/l was obtained after 60 h of fed-batch fermentation, giving a yield of0.5 g/g reducing sugar and a productivity of 0.59 g/h/l.

There are numerous reports on ABE fermentation processesfrom the last 30 years, which together accomplishes advancementsin butanol production. In recent years several economic studieshave been performed on the production of butanol from corn (drycorn and wet corn milling) whey permeate, and molasses[309,333]. In these studies it was determined that the distillativerecovery of biobutanol from the fermentation broth is noteconomical when compared with butanol derived from the currentpetrochemical route. Nevertheless, studies employing C. beijerinckiiBA101, C. acetobutylicum P260, hydrolyzed fiber-rich distillersdried grains and solubles (DDGS) and wheat straw suggest thatcommercial production of biobutanol from agricultural byproducts/


wastes is drawing closer. Increased butanol concentrations,productivity, and yields were achieved by the establishment of highcell density continuous cultures by cell immobilization or by cellrecycling together with cell bleeding, fed-batch culture integratedwith in-situ butanol recovery system, and butanol production frombutyric acid in living cells [334]. The enzymes responsible forbutanol production should be characterized in order to deepen ourunderstanding of the thermodynamics and kinetics of the meta-bolic pathway. Industrial biobutanol production currently lagsbehind production of bioethanol and biodiesel however significantamount of progress has been made on the development of newprocess technologies, including studies with high-productivityreactors and energy-efficient product-recovery systems. Recently,DuPont (US) and BP (UK) announced their plans to invest in bio-butanol production research. It is anticipated that the first plantswould operate on sugar or corn starch; however, it is likely thatagricultural waste would become a potential substrate in the nearfuture. The use of lignocellulosic substrates in combination withdeveloped process technologies is expected tomake the productionof biobutanol economically viable [310,335].

8. Biorefinery perspectives

The development and implementation of biorefinery processesis of upmost importance to meet the vision towards a sustainableeconomy based on bio-resources. Biorefinery concept is to utilizeinedible lignocellulosic biomass to produce biofuels, cellulose,hemicellulose, lignin and byproducts. Replacement of petroleum-derived chemicals with those from biomass will play a key role insustaining the growth of the chemical industry. Contrary to petro-resources which are limited in nature and composition, the bio-resource are composed of heterogenous compounds such ascellulose, hemicellulose, oils, lignin, starch and proteins. Eachconstituent in the biomass (plant) can be functionalized in order toproduce non-food and food fractions, intermediate agro-industrialproducts and synthons [336]. Thus, a complete set of specifictechnologies must be developed in order to convert each fraction asefficiently into value added products. These fractions can be useddirectly as desired biochemicals or can be converted by chemical,enzymatic, and/or microbial approaches. Conversion of these by-products to high-value co-products will offset the cost of biofuel,improve the economy of lignocellulose biorefinery, minimize thewaste discharge, and reduce the dependence of petroleum-basedproducts. The biorefinery will offer new economic opportunitiesfor agriculture and chemical industries by the production ofa tremendous variety of chemicals, transportation fuels, and energy[337]. To develop technologically sustainable bio-refinery routes,the entire chain of biomass production, i.e., from breeding, culti-vation, and harvest, its (pre)treatment, and conversion to productsshould be considered. The relatively low energy content, season-ality and discrete geographic availability of biomass feedstockshave been noted as barriers to the large volume demands forenergy and fuel [338]. In addition, it will be shown that small scale(pre)processing of the biomass may be advantageous over large-scale processing [339]. Conceptually, a biorefinery would applyhybrid technologies from different fields including polymerchemistry, bioengineering and agriculture [340].

The U.S. Department of Energy (DOE) identified high-volumecommodity chemicals that could be produced from biomass andcan serve as starting materials for many chemical products viabiological processes [341]. Ethanol is perhaps the most widelyknown example of a bio-based chemical produced worldwide;however there is a range of bioproducts produced and used ina variety of industrial applications. These fall into several genericcategories such as naturally occurring carbohydrate polymers, fats

and oils from plant origin, terpene based materials, chemicalproducts of carbohydrate containing materials, fermentationproducts of carbohydrate containing sources. Cellulosic plantmaterials are used as fuel, lumber, mechanical pulps and textiles.Purified cellulose is currently used to make wood-free paper,cellophane, photographic film, membranes, explosives, textilefibres, water-soluble gums, and organic-solvent-soluble polymersused in lacquers and varnishes. The principal cellulose derivative iscellulose acetate a biodegradable, which is used to make photo-graphic film, acetate rayon, various thermoplastic products, andlacquers [342]. Lyocell is the first new textile fibre, commerciallyproduced from a solvent spinning process. Specialty chemicals canbe made using fermentation and enzymatic processes. Severalcommodity chemicals are already produced by fermentation,including glutamic acid (w1.7 billion kg/year worldwide), citricacid (w1.6 billion kg/year), and lysine (w850 million kg/year)(346). In addition to these commodity chemicals, commoditypolymers may also be produced using biologically producedmonomers combined with classical chemical methods, as well asthrough biological polymerization methods, either with isolatedenzymes or in actively growing cells. Lactic acid produced byfermentation [344] can be converted chemically to methyl lactate,lactide, and polylactic acid. The latter polymer commerciallyavailable under different trade names is a fully biodegradablereplacement for polyethylene terephthalates [345]. Efficientprocesses for converting biologically produced lactic and hydrox-ypropionic acids to methacrylic and acrylic acids are being devel-oped. Genencor and DuPont, who have developed a cost-effectivefermentative route to 1,3-propanediol, the key building block inpoly- (propyleneterephthalate), which is not readily available frompetrochemical feedstocks [346]. Fermentation by various microor-ganisms has been used to produce succinic acid [347,348], whichmay potentially replace maleic anhydride, now produced frombutane. Recently, chemical companies such as Dow ChemicalCompany, Huntsman Corporation, Cargill and Archer DanielsMidland Corporation, have begun to use glycerol as a low-costbuilding block material for conversion to higher value propyleneglycol. Furthermore, Dow Chemical Company and Solvay areexploring the use of glycerol in the production of epichlorohydrin,which can be used in the manufacture of epoxy resins andepichlorohydrin elastomers [343]. Due to its versatility, it is antic-ipated that glycerol could become a substitute for many commonlyused petrochemicals [349]. A promising sugar-lignin platformpotential for the generation of value-added bioproducts under thelignocellulosic biorefinery concept is illustrated in Table 6. Thecurrent situation indicates that the demand for food, energy,mobility, chemicals andmaterials will increase tremendously in thenear future. 80e90% of the fossil resources are used for energysupply and mobility. Being aware that a coupling between worldmarkets for energy and fuels, raw materials for chemicals andmaterials, feed and food and biomass exists one has to solve theproblems of raw material supply for food production as well as formaterials, fuels and energy production [336].

The effective use of biomass feedstocks, particularly lignocel-lulosic materials in large-scale applications will evolve from inno-vative research aimed at the development and implementation ofbiorefineries e multi-step, multi-product facilities established forspecific bio-sourced feedstocks (340). In the realization of a bio-based chemical industry two distinct approaches can be identi-fied. In the first approach, the value chain approach, value addedcompounds in biomass are identified and isolated in differentprocessing and (bio)conversion steps. The remaining biomass isthen transformed into a universal substrate from which chemicalproducts can be synthesized. In this approach it is thought that istechnologically and economically beneficial to extract valuable

Table 6Value added biochemicals potentially derived from cellulose, hemicellulose andlignin.

Lignocellulosicbiomass

Cellulose PolymersLevulinic acid Succinic acid, THF,

MTHF, 1,4 butanediol,NMP, Lactones

EthanolLactic acid Acrylic acid,

Acetaldehyde2,3-pentanedione,Pyruvic acid

3-hydroxy-propanioc acidItaconic acid 3-methyl THF,

3-methyl pyrrolidone2,methyl-1,4- butanediamineItaconic diamide

Glutamic acidGlucuronic acidSuccinic acid 2-pyrrolidones,

1,4- butanediol,Tetrahydrofurane

Hemicellulose XylitolEthanol, butanol,hydrogen2,3-butanediolFerulic acid Vanillin, Vanillic acid,

Protocatechuic acidLactic acidFurfuralChitosanXylo-oligosaccharides

Lignin SyngasSyngas products Methanol/Dimethy

ether, Ethanol, Mixedliquid fuels

Hydrocarbons Cyclohexanes, higheralkylates

Phenols Cresols, Eugenol,Coniferols, Syringols

Oxidized products Vanillin, vanillic acid,DMSO, aldehydes,Quinones, aromaticand aliphatic acids

Macromolecules Carbon fibres,Activated carbon,polymer alloys,polyelectrolites,substituted lignins,thermosets,composites, woodpreservatives,Neutraceuticals/drugs,adhesives and resins


chemicals and polymers from biomass rather then building thesecompounds from universal building blocks. It can be concluded thatthe main technological challenges to aid the economic feasibility ofthis approach lay in the area of biomass refining, separation tech-nology and bioconversion technology. Moreover, a far reachingintegration of food, feed and chemical industries is required as wellas a major investment in infrastructure. The second approach, theintegrated process chain approach, follows the analog of thepetrochemical industry. In this scheme a ”universal” substrate isfirst transformed into universal building blocks, based on whichchemical products are produced. In this approach it is thought thatit is economically and technologically beneficial to build chemicalsin highly integrated production facilities. The main technologicalchallenges for this approach lay in the high-efficient trans-formation of biomass into commonly known building blocks for thepetrochemical industry [337]. The current situation indicates that

the demand for food, energy, mobility, chemicals and materials willincrease tremendously in the near future. 80e90% of the fossilresources are used for energy supply andmobility. Being aware thata coupling between world markets for energy and fuels, rawmaterials for chemicals and materials, feed and food and biomassexists one has to solve the problems of rawmaterial supply for foodproduction as well as for materials, fuels and energy production[336]. Biomass feedstocks available decentrally will be morecommodious for localized biorefinery approach than the exhaustivelarge scale and centralized plants driven by cost intensive tech-nology. Depending on regional and technological boundary condi-tions the most economic solution for a biorefinery may be providedby large centralized facilities as well as by smaller decentralizedplants [350].

Energy and GHG emission savings from bio-based polymers inspecific terms were found to be 20e50 GJ/t polymer and 1.0e4.0 tCO2eq/t polymer respectively. Bio-based polymers are attractive interms of specific energy and emissions savings and cannot offsetthe additional environmental burden due to the growth of petro-chemical polymers [351]. Within the next two decades, bio-basedpolymers will not be able to compensate the environmentalimpacts of the economy as a whole. The potential environmentaland socio-economic effects studied concluded that while environ-mental effects in specific terms are high, effects in absolute termsrelative to those of total industry or society are low. There is low jobcreation potential. It must be emphasized that these relatively lowcontributions have their reason in the comparatively low produc-tion volumes of bio-based polymers until 2020 [352,353].

9. Lifecycle assessment of lignocellulosic ethanol

A life-cycle assessment (LCA) takes into account all resource andenergy inputs required to make a product, the wastes, and thehealth and ecological burdens associatedwith the product. In effect,performing a life-cycle assessment of the entire ethanol productionsystem (preproduction through consumption) would quantify thetotal benefits as well as any drawbacks that the process mightcontain and identify opportunities for process improvement [353].LCA techniques allowdetailed analysis ofmaterial and energyfluxeson regional and global scales. At the same time if not used properly,LCA can lead to incorrect and inappropriate actions on the part ofindustry and/or policymakers [354]. Earlier technoeconomic or life-cycle analyses of ethanol from lignocellulosic biomass have focusedon energy, greenhouse gases, criteria pollutants, and economics.Lynd et al. [355] identified costs of production as the key barrier,while acknowledging environmental and energy benefits usinga limited life-cycle analysis that excluded pre-manufacturingstages. Wang et al. [356] showed that corn ethanol used inconventional internal combustion engines reduces fossil energyconsumption between 28% and 50%, but only ethanol from ligno-cellulose can achieve reductions over 90%. Similarly, Sheehan et al.[357] conducted a thorough life-cycle assessment of ethanolproduction from corn stover in Iowa and discovered that E85 (85%ethanol blend with gasoline) reduces petroleum consumption 95%per kilometer traveled compared to conventional gasoline, reducesozone precursor emissions, reduces total fossil energy consumptionand greenhouse gas emissions by 102% and 113%, respectively, butincreases emissions of CO, NOx, and SOx. However, none of thesestudies investigated the effects of process improvements on life-cycle impacts. Kemppainen and Shonnard [353] have studied theLCA approach to evaluate to evaluate the environmental impacts ofa lignocellulosic biomass-to-ethanol process using two differentregional biomass options: timber and recycled newsprint. Readersare referred to various monographs on LCA for potential use oflignocellulosic feedstocks in bioethanol production [354,358e360]


which elaborates various factors (type of biomass, ethanol conver-sion technologies, utilization of produced ethanol, systemboundary, byproduct allocation and reference system) affecting theoutcome of the analysis. Mu et al. [361] have explored the potentialsof the biochemical and thermochemical conversion pathways,different technological scenarios were modeled, including current,2012 and 2020 technology targets, as well as different production/co-production configurations. The modeling results suggest thatbiochemical conversion has slightly better performance on green-house gas emission and fossil fuel consumption, but that thermo-chemical conversion has significantly less direct, indirect, andlifecycle water consumption. Also, if the thermochemical plantoperates as a biorefinery with mixed alcohol coproducts separatedfor chemicals, it has the potential to achieve better performancethan biochemical pathway across all environmental impact cate-gories considered due to higher co-product credits associated withchemicals being displaced. The results from this work serve asa starting point for developing full life cycle assessment model thatfacilitates effective decision-making regarding lignocellulosicethanol production.

Biofuels production as a strategy to mitigate global warming iscurrently under debate due to the influence of including direct andindirect land-use changes in the GHG balance of biofuels. Increaseddemand for biofuels in order to partially substitute fossil fuels indifferent parts of the world will require the use of a significantamount of biomass. Energy crops production on current land anduse of biomass in a given region can induce displacement ofactivities and land-use changes elsewhere. This effect is known asindirect land-use change (ILUC). Due to changes in the carbon stockof the soil and the biomass, ILUC has consequences on the GHGbalance of a biofuel that are not presently considered in the eval-uation of the environmental merits of biofuels. Significant changesin land-use are expected to occur in biofuel producing countriesand their consequences may affect global markets. Greenhouse gas(GHG) emissions reduction is one of the main drivers of biofuelsdevelopment. However, this assumed benefit is now underdiscussion, especially due to emissions from land-use change (LUC).Therefore some countries and regions worldwide being experi-enced biofuels blending policies (e.g. the EU, the UK, Switzerland,the Netherlands, State of California in the US) have defined a carbonconservation criterion (i.e. an emissions reduction target) that mustbe fulfilled in order to trade and supply biofuels. Thus biofuelimporting countries should select production regions that satisfythis criterion. Recent studies have highlighted both positive andnegative environmental and socioeconomic effects of bioenergyand the associated agriculture and forestry LUC [362,363]. Likeconventional agriculture and forestry systems, bioenergy canexacerbate soil and vegetation degradation associated with over-exploitation of forests, too intensive crop and forest residueremoval, and water overuse [364,365]. Diversion of crops or landinto bioenergy production can influence food commodity pricesand food security [366]. With proper operational management, thepositive effects can include enhanced biodiversity [367,368], soilcarbon increases and improved soil productivity [369,370], reducedshallow landslides and local flash floods, reduced wind and watererosion and reduced sediment volume and nutrients transportedinto river systems [371]. For forests, bioenergy can improve growthand productivity, improve site conditions for replanting and reducewildfire risk [372]. However, forest residue harvesting can havenegative impacts such as the loss of coarse woody debris thatprovides essential habitat for forest species.

It is overly simplistic and inaccurate to view land use changeworldwide as being driven primarily by increased agriculturalproduction, as has been assumed [373]. There is a rich academicliterature on the subject of land use change [374,375]. According to

these studies, land use change is driven by three primary forces:timber harvest, infrastructure development (e.g., road building),and agricultural expansion. Any one of these variables taken aloneexplains less than 20% of documented land use changes worldwide.Taken together, they explain over 90% of observed cases of land usechange. Agricultural expansion alone is therefore seldom thereason for land use change. Thus it is arbitrary and unreasonable toassume that all land use change worldwide is driven primarily byagricultural expansion. Whatever the final result of the ongoingdebate about the validity and limits of the indirect land use changeanalysis, both direct and indirect LUC analyses depend on a numberof variables and assumptions [376].

Studies of environmental effects, including those focused onenergy balances and GHG emission balances, usually employmethodologies in line with the principles, framework, require-ments and guidelines in the ISO 14040:2006 and 14044:2006standards for Life Cycle Assessment (LCA) [4]. LCA studies ofprospective bioenergy options involve projections of technologyperformance and have relatively greater uncertainties. The waythat uncertainties and parameter sensitivities are handled acrossthe supply chain to fuel production significantly impacts theresults. Studies combining several LCA models and/or Monte Carloanalysis provide bioenergy system uncertainties and levels ofconfidence for some bioenergy options [377e379]. The chosencombination of production options to satisfy a given demand ofbiofuels is the result of a multi-criteria decision based oneconomical, political, technological, agro-ecological, social andenvironmental factors, which determined the local framework forbiofuels production. This decision will have an impact on LUC, andtherefore on the GHG emissions balance of the biofuel. Conse-quently, reducing GHG emissions from direct (DLUC) and indirectLUC (ILUC) is a key element to optimize the environmentalperformance of a given biofuel pathway. It can be concluded thatLUC can affect GHG balances in several ways, with beneficial ordetrimental outcomes for bioenergy’s contribution to climatechange mitigation, depending on conditions and context. Whenland high in carbon (notably forests and especially peat soil forests)is converted to bioenergy, upfront emissions may cause a time lagof decades to centuries before net emission savings are achieved.But the establishment of bioenergy plantations can also lead toassimilation of CO2 into soils and aboveground biomass in the shortterm. Increased utilization of forest biomass can reduce forestcarbon stocks. The use of post-consumer organic waste and by-products from the agricultural and forest industries does notcause LUC if these biomass sources were not utilized for alternativepurposes. Bioenergy feedstocks can be produced in combinationwith food and fibre, avoiding land use displacement and improvingthe productive use of land. Lignocellulosic feedstocks for bioenergycan decrease the pressure on prime cropping land. Stimulation ofincreased productivity in all forms of land use reduces the LUCpressure.

Air pollutant emissions from bioenergy production depend ontechnology, fuel properties, process conditions and installedemission reduction technologies. Compared to coal and oilstationary applications, sulphur dioxide (SO2) and nitrous oxide(NOx) emissions from bioenergy applications are mostly lower.When biofuel replaces gasoline and diesel in the transport sector,SO2 emissions are reduced, but changes in NOx emissions dependon the substitution pattern and technology [4]. Bioenergyproduction can have both positive and negative effects on waterresources. Bioenergy production generally consumes more waterthan gasoline production [380,381]. However, this relationship andthe water impacts of bioenergy production are highly dependenton location, the specific feedstock, production methods and thesupply chain element. Feedstock cultivation can lead to leaching


and emission of nutrients that increase eutrophication of aquaticecosystems [363,382]. The subsequent processing of the feedstockinto biofuels and electricity can increase chemical and thermalpollution loads from effluents and generate waste to aquaticsystems [383,384]. These environmental impacts can be reduced ifsuitable equipment is installed [385,386]. Increased biomassoutput for bioenergy can directly impact wild biodiversity throughconversion of natural ecosystems into bioenergy plantations orthrough changed forest management. Habitat and biodiversity lossmay also occur indirectly, such as when productive land use dis-placed by energy crops is re-established by converting naturalecosystems into croplands or pastures elsewhere. Because biomassfeedstocks can generally be produced most efficiently in tropicalregions, there are strong economic incentives to replace tropicalnatural ecosystemsdmany of which host high biodiversity values[387]. However, forest clearing is mostly influenced by local social,economic, technological, biophysical, political and demographicforces [388]. The considerable soil impacts of increased biofuelproduction include soil carbon oxidation, changed rates of soilerosion, and nutrient leaching. However, these effects are heavilydependent on agronomic techniques and the feedstock underconsideration [389]. The overall performance of bioenergyproduction systems is therefore interlinked with management ofland use and water resources.

10. Current global status of lignocellulosic ethanol industry

The commercialization of cellulose to ethanol technology in thepresent-day scenario is not economically viable. Currently there arespecial programs in a number of countries targeting production ofbiofuel such as biogas, bioethanol, biodiesel and fuel cells fromrenewable resources [390]. Global production of bioethanolincreased from 17.25 billion liters in 2000 to over 46 billion liters in2007, which represented about 4% of the 1300 billion liters ofgasoline consumed globally [391]. With all of the new governmentprograms in America, Asia, and Europe in place, total global fuelbioethanol demand could grow to exceed 125 billion liters by 2020[392]. Bioenergy ranks second (to hydropower) and accounts for 3%in renewable primary energy production in U.S [393]. The UnitedStates is the world’s largest producer of bioethanol fuel, accountingfor nearly 47% of global bioethanol production in 2005 and 2006[394]. The “Biofuels Initiative” in theU.S. (USDepartmentof Energy),strives to make cellulosic ethanol cost-competitive by 2012 andsupposedly to correspond and account for one third of the U.S. fuelconsumption by 2030. In 2007, the U.S. president signed the EnergyIndependence and Security Act of 2007 (EISA, 2007),which requires34 billion liters of bio-fuels (mainly bioethanol) in 2008, increasingsteadily to 57.5 billion liters in 2012 and to 136 billion liters in 2022.Companies such as POET (earlier called Broin) and Abengoa arebuilding refineries that canprocess biomass and turn it into ethanol,while companies such as Genencor, Diversa, Novozymes and Dyadicare producing enzymes which could enable a cellulosic ethanolfuture. Brazil is the world’s largest exporter of bioethanol andsecond largest producer after the United States. Ethanol fromsugarcane provides 40% of automobile fuel in Brazil and approxi-mately 20% is exported to the U.S., EU, and other markets.

Commercial scale plants are expected to be operational by 2012.In US, plants built with substantial federal funding totaling 12million gallon per year are operational with an additional 21.3million gallon of capacity- 26 new plants- under construction.There are more than 10 ethanol biofuel facilities either in operationor under construction in Canada [395,396]. In eastern Canada andthe U.S., corn is used as the feedstock while in western Canadawheat is used. Many Asian countries such as China, India, Japan,and Indonesia are also developing ethanol production capacity

[397,398]. Iogen Corp, Canada, is a developer of cellulosic ethanolprocess technology. Iogen has developed a proprietary process andoperates a demonstration-scale plant in Ontario. The facility hasbeen designed and engineered to process 40 tons of wheat strawper day into ethanol using enzymes from in-house enzymemanufacturing facility. Lignol is a Canadian company which isundertaking to construct biorefineries for the production of fuel-grade ethanol and biochemicals from Canadian forests and vastsupplies of biomass feedstocks. Lignol has acquired and sincemodified, a solvent based pre-treatment technology Lignol hasrecently produced ethanol and other biochemicals from bothhardwood and softwood species representative of Canadian forests.

Ethtec is building a pilot plant in Harwood, New south Wales,Australia which uses wood residues as a feedstock. Another planthas been built by Queensland University of Technology near Bris-bane. SEKAB, Sweden has developed an industrial process forproduction of ethanol from biomass feedstocks, including woodchips and sugarcane bagasse. The technology will be graduallyscaled up to commercial production in a newbreed of bio-refineriesfrom 2013 to 2015. A $ 400 million investment programme to coverthe construction of a World scale ethanol plant and a high tech-nology demonstration plant to advance development work on thenext generation biofuels has been announced by BP, AssociatedBritish Foods (ABF) and DuPont. The bioethanol plant with anannual production capacity of 420 million liters of biofuel fromwheat feedstock will be built on BP’s existing chemicals site atSaltend, Hull, UK. In India, Praj Industries have recently starteda demonstration plant that can process 2 tonnes per day of ligno-cellulosic feedstocks such as corn stover, corn cob, bagasse, agrowaste and wood chips for biofuel production [57]. A few companieshave operated pilot plants (Table 7), however no commercialindustrial scale plants for biofuel production is in operation. ChinaResources Alcohol Corporation (CRAC) has announced their inten-tion to construct sufficient cellulosic ethanol facilities to generate330 million gallons of ethanol by 2012. SunOpta provided itspatented systems and technology to CRAC in September 2006 andthe plant began production of ethanol from local corn stover inOctober 2006. This facility is reported to be the first cellulosicethanol production facility operational in the People’s Republic ofChina. The SunOpta system is currently operating on a continuousbasis and steps are currently being taken to scale the SunOptaprocess up to full commercial levels for use in future plants in China.China has committed $5 billion to cellulosic ethanol production andrecently announced that they would allow no further increase inethanol production from starch (corn), due to the needs for starch asfood. Nippon Oil Corporation and other Japanese manufacturersincluding ToyotaMotors Corporation plans to set up a research bodyto develop cellulose-derived biofuels. The consortium plans toproduce 1.6 million barrels per year of bioethanol by March 2014and produce bioethanol at $0.437 per litre by 2015.

Recently research is directed towards identifying, evaluating,developing and demonstrating different pretreatment methodsthat result in efficient enzymatic hydrolysis. Several physical,physico-chemical, chemical and biological pretreatments or theircombinations are under evaluation. Due to the high cost of enzymethe current fuel grade ethanol produced from lignocellulosicmaterial is still not able to compete with gasoline. In a contempo-rary process of lignocellulosic ethanol which is being worked outfor more than 2e3 decades is still not materialized into a viabletechnology. The permissible cost of enzymes is 15e30 cents/gallonof ethanol which is still not a reality. The lignocellulosic biomassfeedstock including pretreatment costs around 50e80 cents/gallonof ethanol [399]. Today cellulosic ethanol still lies around approxUS$ 4 per gallon of ethanol based on best estimates [400]. Thecellulase cost has been reduced dramatically from US$5.40 per

Table 7Demonstration and pilot plant facilities developed worldwide for production of bioethanol.

Company Location Products Status Raw material Pretreatment/technology Fate of lignin

Abengoa Spain 4.000 t/a EtOH Demo facility,start-up 2009

Wheat straw Acid catalysed steamexplosion, Enzymatichydrolysis

As co-product, Recovered afterdistillation

Inbicon Denmark 4.000 t/a EtOHC5-molassesSolid biofuel

Demofacility,start-up 2009

Wheat straw Liquid hot water(hydro-thermal, autocatalysed)

Solid biofuel for power-plant,Recovered after distillation

Iogen Canada 70.000 t/a EtOH Commercial facility,start-up 2011

Straw (wheat, barley, oat) Modified steam explosion,Enzymatic hydrolysis

For steam and electricitygeneration,Recovered after enzymatichydrolysis

KL Energy USA 4.500 t/a EtOH Demo facility,operationalsince 2007

Wood waste, cardboardand paper

Thermo mechanical For steam or electricitygeneration, or as wood pelletco-product,Recovered after distillation

SEKAB Sweden 4.500 t/a EtOH Demo facility,start-up 2011d

Wood chips or sugarcanebagasse

Acid pre-treatment For energy production or otheruses Recovered after(enzymatic) hydrolysis

Vereniumprocess

USA 4.200 t/a EtOH Demo facility,operationalsince 2009

Sugarcane bagasse,energy crops, woodproducts and switchgrass

Mild acid hydrolysisand steam explosion

Lignin-rich residue burned forsteam generation Recoveredafter distillation

Range fuels Georgia 20 million gallon/annum EtOH

Commercial facility,start up at 2010

Woody biomass andgrasses

Thermo-chemicalprocess

For energy production

POET LLC EmmetsburgIA

55 milliongallon/annumEtOH

Pilot plant facility Corn cob Biogas production

Sources: Biotechnology Industry Organization (BIO), companies.


gallon of ethanol to approximately 20 cents per gallon of ethanol;further efforts are carried out to reduce the costs below 5 cents pergallon ethanol. The hydrolysis and subsequent fermentation oflignocellulose is complicated and the economic viability of theprocess is yet to be achieved.

11. Future prospects

Although bioethanol production has been greatly improved bynew technologies there are still challenges that need further inves-tigations. Tremendous focus is essential for developing a detailedunderstanding of lignocellulose, the main structural material inplants, from cellulose synthesis and fibril formation to a matureplant cell wall, forming a foundation for significant advancement insustainable energy and materials. Characterization, understandingand overcoming the barriers for enzymatic hydrolysis of differentraw material is essential for the development of economicallycompetitive processes based on enzymatic treatments.

Availability of feedstocks for biofuel production, their variabilityand sustainability are major criteria’s to be addressed. Each rawmaterial requires a different processing and pretreatment strategywhich has to be tailored taking in to consideration their composi-tion and susceptibility to such treatments. Physical/chemical/physico-chemical or combinations thereof needs to be optimizedfor pretreatment of each feedstocks. The pretreatment must beadvanced and appropriately integrated with the rest of the processto achieve the complete potential of lignocellulosic ethanol.Another major challenging area of research is to develop low costeffective enzymes for lignocellulose saccharification of the pre-treated biomass. Extensive research in developing new technolo-gies for high solid handling is also a major objective. Improvementsin fermentation technology and media optimization approacheshave to be performed along with genetic engineering techniques toimprove the yield and efficiency of cellulases. Although enzymecosts have decreased in the last few years, this is still at its pre-mature state. The high cost of enzyme production and therequirement of higher enzyme dosage for hydrolysis of biomass areconsidered to be main hurdles for the economic viability of ligno-cellulosic bioethanol. Synthetic biology and metabolic engineering

approaches has to be utilized for developing efficient and robustmicrobes for SSF, SSCF and CBP processes. Integration of processesfor reducing the number of process steps, energy demands and re-use of process streams to make the conversion process economical.

Advances in the cost-effective conversion of lignocellulosicbiomass are often difficult to assess accurately because of the lack ofintegrated process configurations. Despite current expectations,significant uncertainty remains on the different environmental andsustainability factors on the performance of second generationbiofuel in a commercial scale. Tremendous R&D studies areimproving the conversion process but the issue of feedstock avail-ability and revenue stability remain uncertain and subject topolitical risks. Keeping a realistic perspective one can conclude thatseveral pieces still remain to be properly assembled and optimizedbefore an efficient industrial configuration is acquired. Even thoughdeveloping the technology for cost-effective motor fuel productionby 2030 is challenging, the advances in scientific understandingnecessary to achieve this goal appear realizable.

Acknowledgements

The authors acknowledge the financial support and the seniorresearch fellowship from CSIR Emeritus Scheme, Govt. of Indiarespectively.

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Trends in bioconversion of lignocellulose: Biofuels, platform chemicals & biorefinery concept

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