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Value added products from hemicellulose: Biotechnological perspective Vishnu Menon, Gyan Prakash and Mala Rao*

Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India

*Corresponding Author

Dr. Mala Rao, mb.rao@ncl.res.in , Phone : +91-20-25902228

Fax No: +91-20-25902648

Division of Biochemical Sciences

National Chemical Laboratory

Pune - 411 008,

India.

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Abstract Lignocellulosic biomass represents a renewable, widespread and low cost source of

sugars which ca be used as substrate for the production of specialilty chemicals. Various

agricultural residues contain about 20-30% hemicellulose, the second most abundant

biopolymer found in nature. They are heterogeneous polymer of pentoses (xylose and

arabinose) hexoses (mannose, glucose and galactose) and sugar acids. Xylans is the major

component of hemicellulose and are heteropolysaccharides with homopolymeric

backbone chains of 1, 4 linked β-D-xylopyranose units. The conversion of hemicellulose

to bioethanol and other value added products is challenging problem.and is foreseen to be

essential for the economic success of lignocellulosic ethanol. The xylose from the

hemicellulose hydrolysate can be fermented to ethanol/ xylitol/lactic acid and other

valuable products by pentose fermenting microbes. Xylitol a non caloric sugar has

important applications in pharmaceuticals and food industries due to high sweetening

power, non- and anticariogenicity property. . The acid or enzymatic hydrolysis of

hemicellulose to fermentable sugars will be outlined. The enzymatic and fermentation

routes for the conversion of hemicellulose into useful products will be described.

Keywords

Hemicellulose, Hemicellulases, Hydrolysis, Fermentation route, Biochemicals,

Recombinant approaches

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1 Introduction

In collation to first generation bioethanol from starch and molasses, the

development of second generation bioethanol from lignocellulosic biomass serves many

advantages from both energy and environmental concerns. Efficient conversion of

lignocellulosic materials is a global challenge for eco-friendly value addition. Cellulose is

the primary constituent of lignocellulosic biomass and is the most abundant organic

compound available. The estimated global annual production of biomass is 1×1011 tons

[1] and is considered to be one of the significant renewable resources for the production

of biofuel and value added products. Unlike fossil fuels, cellulosic ethanol produced

through fermentation of sugars is a renewable energy source. Biomass in general consists

of 40-50% cellulose, 25 to 30% hemicellulose and 15 to 20% lignin. The effective

utilization of all the three components would play a significant role in economic viability

of the cellulose to ethanol process [2].

In Indian scenario, bagasse and starchy biomass are expected to be primary

feedstocks in preference to other materials such as lipids. The setting up of biorefinary

requires that technologies be developed for biomass pretreatment, hydrolysis of cellulose

and hemicellulose and creation of microbes by recombinant DNA techniques for the

production of desired biochemicals and further their strain improvement. The biorefinary

concept is being built on two different platforms by National Renewable Energy Lab

(NREL) to promote different product areas. The sugar platform based on biochemical

conversion process focuses on the fermentation of sugar extracted from biomass

feedstocks and the other platform is based on thermo chemical conversion processes and

focuses on the gasification of biomass feedstocks and byproducts from the conversion

process (www.nrel.gov/biomass/biorefinery.html). Biorefineries using fibrous

lignocellulosic biomass as feed stock will produce C6 and C5 sugars (predominantly

xylose and arabinose) and lignin.

Various agricultural residues contain about 20-30% hemicellulose, the second

most abundant biopolymer found in nature. They are heterogeneous polymer of pentoses,

hexoses and sugar acids. In recent years, bioconversion of hemicellulose has received

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much attention because of its practical applications in various agro-industrial processes,

for the production of fuels and chemicals, delignification of paper pulp, digestibility

enhancement of animal feedstock, clarification of juices, and improvement in the

consistency of beer [3]. The present review is a comprehensive state-of-the-art describing

the acid and enzymatic hydrolysis of hemicellulose to fermentable sugars. The article

also covers the recent developments in the biotechnological and recombinant aspects for

the fermentation of hemicellulosic hydrolysates to value added products.

2 Structures of Hemicelluloses Hemicelluloses are heterogeneous polysaccharides, which are located between the

lignin and cellulose fibres and, depending on wood species, constitute about 20-30% of

the naturally occurring lignocellulosic plant biomass [4]. They are composed of both

linear and branched heteropolymers of D-xylose, L-arabinose, D-mannose, D-glucose, D-

galactose, L-rhamnose, L-fucose and D-glucuronic acid, which may be acetylated or

methylated. Most hemicelluloses contain two to six of these sugars [5]. They are usually

classified according to the main sugar residues in the backbone. Homopolymers of

xylose, so-called homoxylans only occur in seaweeds (red and green algae). The two

main hemicelluloses in wood are xylans and glucomannans both of which are present in

softwood, whereas in hardwood, xylan is the major component. Xylan is the second most

abundant polysaccharide, comprising up to 30% of the cell wall material of annual plants,

15-30% of hardwoods and 7-10% of softwoods [6]. Xylan occurs as a

heteropolysaccharide with a homopolymeric backbone chain of 1,4-linked β-D-

xylopyranose units, which consists of O-acetyl, α-L-arabinofuranosyl, α-1, 2-linked

glucuronic or 4-O-methylglucuronic acid substituents. In hardwoods, xylan exists as O-

acetyl- 4-O-methylglucuronoxylan with a higher degree of polymerization (150-200) than

softwoods (70-130), which occurs, as arabino-4-O-methylglucuronoxylan.

Approximately one in ten of the β-D-xylopyranose backbone units of hardwood xylan are

substituted at C-2 position with 1,2-linked 4-O-methyl-α-D-glucuronic acid residue,

whereas 70% are acetylated at C-2 or C-3 positions or both [4]. Softwood xylans are not

acetylated but the 4-O-methylglucuronic acid and the β-arabinofuranose residues are

attached to the C-2 and C-3 positions, respectively of the relevant xylopyranose backbone

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units [7]. Birch wood (Roth) xylan contains 89.3% xylose, 1% arabinose, 1.4% glucose,

and 8.3% anhydrouronic acid [8]. Rice bran neutral xylan contains 46% xylose, 44.9%

arabinose, 6.1% galactose, 1.9% glucose, and 1.1% anhydrouronic acid [9]. Wheat

arabinoxylan contains 65.8% xylose, 33.5% arabinose, 0.1% mannose, 0.1% galactose,

and 0.3% glucose [10]. Corn fiber xylan is one of the complex heteroxylans containing β-

(1,4)-linked xylose residues [11]. It contains 48–54% xylose, 33–35% arabinose, 5–11%

galactose and 3-6% glucuronic acid [12]. The xylan of grasses occurs as arabino-4-O-

methylglucuronoxylan with a degree of polymerisation of 70 and contains β-

arabinofuranosyl side chains linked to C-2 or C-3 positions, or both, of the β-D-

xylopyranose main chain residues. The 1,2-linked 4-O-methyl-α-D-glucuronic acid is

found less frequently in grasses than in hardwood xylan. Hardwood xylan contains 2-5%

by weight of O-acetyl groups linked to C-2 or C-3 positions of the xylopyranose units,

and 6% of the arabinosyl side chains are themselves substituted at position 5 with

feruloyl groups, whereas 3% are substituted with p-coumaroyl residues [13]. The relative

proportions of the various components of grass arabinoxylan vary from species to species

and from tissue to tissue within a single species. The main mannan group in the cell walls

of higher plants is galacto-glucomannan with the backbone composed of 1, 4-β-linked D-

glucose and D-mannose units that are distributed randomly within the molecule.

Softwood glucomannan has a backbone composed of β-1, 4- linked D-glucopyranose and

D-mannopyranose units, which are partially substituted by α-galactose units and acetyl

groups. Hardwoods contain a small amount of glucomannan (2-5%) that has no galactose

or acetic acid side groups present [14]. Table 1 describes the composition of

representative hemicellulosic feedstocks. Xylans are usually available in large amounts as

by-products of forest, agriculture, agro-industries, wood and pulp and paper industries.

2.1 Arabinoxylans

Arabinoxylans represent the major hemicellulose structures of the cereal grain cell

walls and are similar to hardwoods xylan but the amount of L-arabinose is higher. The

linear β-(1,4)-D-xylopyranose backbone is substituted by α-L-arabinofuranosyl units in

the positions 2-O and/or 3-O and by α-D-glucopyranosyl uronic unit or its 4-O-methyl

derivative in the position 2-O [40, 41]. O-acetyl substituents may also occur [42,43].

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Arabinofuranosyl residues of arabinoxylan may also be esterified with hydroxycinnamic

acids residues, e.g., ferulic and p-coumaric acids [43]. Dimerization of esterified phenolic

compounds may also lead to inter- and intra-molecular cross-links of xylan. The physical

and/or covalent interactions with other cellwall constituents, restricts the extractability of

xylan. In lignified tissues for example, xylan is ester linked through its uronic acid side

chains to lignin [44].

2.2 Glucuronoxylans

Glucuronoxylans (O-acetyl-4-O-methylglucuronoxylan) are the main

hemicellulose of hardwoods, which may also contain small amounts of glucomannans

(GM). In hardwoods, it represent 15– 30% of their dry mass and consist of a linear

backbone of β-D-xylopyranosyl units (xylp) linked by β-(1,4) glycosidic bonds. Some

xylose units are acetylated at C2 and C3 and one in ten molecules has an uronic acid

group (4-O-methylglucuronic acid) attached by α-(1,2) linkages. The percentage of acetyl

groups ranges between 8% and 17% of total xylan, corresponding, in average, to 3.5–7

acetyl groups per 10 xylose units [45]. The 4- O-methylglucuronic side groups are more

resistant to acids than the Xylp and acetyl groups. The average degree of polymerization

(DP) of glucuronoxylan is in the range of 100–200 [46]. Besides these main structural

units, it may also contain small amounts of L-rhamnose and galacturonic acid. The later

increases the polymer resistance to alkaline agents.

2.3 Arabinoglucuronoxylans

Arabinoglucuronoxylans (arabino-4-O-metylglucuronoxylans) are the major

components of non-woody materials (e.g., agricultural crops) and a minor component for

softwoods (5–10% of dry mass). They also consist of a linear β-(1,4)-D-xylopyranose

backbone containing 4-O-metil-α-D-glucopiranosyl uronic acid and α- L-

arabinofuranosyl linked by α-(1,2) and α-(1,3) glycosidic bonds [47,48]. The typical ratio

arabinose: glucuronic acid:xylose is 1:2:8 [45]. Conversely to hardwood xylan,

arabinoglucuronoxylan might be less acetylated, but may contain low amounts of

galacturonic acid and rhamnose. The average DP of AGX ranges between 50 and 185

[46].

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2.4 Xyloglucans

Xyloglucan is a predominant hemicellulose found in the primary cell wall of

dicots and several monocots ([49], consists of a β-(1→ 4) - linked β-D-Glcp (β-D-

glucopyranose) backbone having up to 75% of the β-D-Glcp residues covalently linked to

α-D-Xylp (α-D-xylopyranose) at the O-6 position. While the extent of xylose substitution

varies considerably, the glucose backbone structure is typically substituted by 50% or

75% xylose [50]. Xylose can be further substituted by galactose or arabinose and the

galactose may be fucosylated. The glucose, xylose and galactose residues are present in

the ratio of 2.8: 2.25:1.0. In addition, xyloglucans can contain O-linked acetyl groups

[51, 52]. Xyloglucans interact with cellulose microfibrils by the formation of hydrogen

bonds, thus contributing to the structural integrity of the cellulose network [49, 53]. XG

is also a storage polymer in the seed endosperm of many plants. Tamarind kernel powder

(TKP) a rich source of hemicellulose, galactoxyloglucan (GXG) is a crude extract

derived from the seeds of Tamarindus indica Linn., an abundantly grown tree of

Southeast Asia, India, Mexico, and Costa Rica [54].

2.5 Galactoglucomannans

Galactoglucomannans (O-acetyl-galactoglucomannans) are the main

hemicelluloses of softwoods, accounting up to 20–25% of their dry mass [46]. It consist

of a linear backbone of β-D-glucopyranosyl and β-D-mannopyranosyl units, linked by β-

(1,4) glycosidic bonds, partially acetylated at C2 or C3 and substituted by a-D-

galactopyranosyl units attached to glucose and mannose by α-(1,6) bonds. Some are

water soluble, presenting in that case higher galactose content than the insolubles [55].

Acetyl group’s content of galactoglucomannan is around 6%, corresponding, to 1 acetyl

group per 3–4 hexoses units. Glucomannans occur in minor amounts in the secondary

wall of hardwoods (<5% of the dry wood mass) [45]. They have linear backbone of β-D-

glucopyranosyl (Glcp) and β-D-mannopyranosyl (Manp) units but the ratio Glcp:Manp is

lower. The extent of galactosylation governs the association of galactoglucomannan and

glucomannans to the cellulose microfibrils and hence, their extractability from the cell

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wall matrix [44]. The average DP of galactoglucomannans ranges between 40 and 100

[46].

2.6 Complex heteroxylans

Complex heteroxylans are present in cereals, seeds, gum exudates and lucilages

and they are structurally more complex [56]. The β-(1,4)-D-xylopyranose backbone is

substituted with single uronic acid and arabinosyl residues and also with various mono-

and oligoglycosyl side chains.

3 Separation and saccharification of hemicellulose Without a profitable use of the hemicellulose fraction, bioethanol is too expensive

to compete in commercial markets [57]. Therefore to foster the commercial production of

lignocellulosic ethanol, bioconversion of the hemicelluloses into fermentable sugars is

essential. The initial step for the production of bioethanol is pre-treatment. The

hemicelluloses are commonly removed during the initial stage of biomass processing

aiming to reduce the structural complexity for further enzymatic cellulose hydrolysis.

Various pretreatment processes are available to fractionate, solubilize, hydrolyse and

separate cellulose, hemicellulose and lignin. These include concentrated acid [58], dilute

acid [11], alkaline [59], SO2 [60], hydrogen peroxide [61], steam explosion

(autohydrolysis) [62], ammonia fiber explosion (AFEX) [63], wet-oxidation [64], lime

[65], liquid hot water [66], CO2 explosion [67], organic solvent treatments [68], ionic

liquids [69, 70] and supercritical fluids [71]. The main processes for the selective

separation of hemicelluloses from biomass include the use of acids, water (liquid or

steam), organic solvents and alkaline agents. The later two are not selective towards

hemicellulose as they also remove lignin, which in turn can hinder the valorisation

process, e.g., fermentation or bioconversion, as the lignin-derived compounds are usually

microbial growth inhibitors. Therefore, acid/water/steam pretreatments are the most

commonly applied technologies yielding a selectively solubilisation of hemicelluloses

and producing hemicellulose-rich liquids totally or partially hydrolysed to oligomeric and

monomeric sugars and cellulose-enriched solids for further bioprocessing [72].

Depending on the operational conditions degradation products are also formed, both from

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sugars (furan and its derivates and weak acids) and, to a less extent, from lignin

(phenolics) [73]. These compounds may also inhibit the fermentation processes, leading

to lower ethanol yields and productivities; however a prior detoxification step is essential.

The following section describes few selected pretreatments wherein most of the

hemicellulose is obtained in the form of oligosaccharides and monomeric sugars.

3.1 Acid hydrolysis

3.1.1 Dilute acid hydrolysis

The dilute acid hydrolysis of the biomass is, by far, the oldest technology for

converting the biomass to ethanol. The first attempt at commercializing a process for the

ethanol from the wood was carried out in Germany in 1898. It involved the use of dilute

acid to hydrolyze the cellulose to glucose, and was able to produce 7.6 liters of ethanol

per 100 kg of wood waste (18 gal per ton). The hydrolysis occurs in two stages to

accommodate the differences between the hemicellulose and the cellulose [74] and to

maximize the sugar yields from the hemicellulose and cellulose fractions of the biomass.

The first stage is operated under milder conditions to hydrolyze the hemicellulose, while

the second stage is optimized to hydrolyze the more resistant cellulose fraction. The

liquid hydrolyzates are recovered from each stage, neutralized, and fermented to ethanol.

National Renewable Energy Laboratory (NREL) a facility of US Department of

energy (DOE) operated by Midwest Reseach institute, Bettelle, NREL outlined a process

where the hydrolysis is carried out in two stages to accommodate the differences between

hemicellulose and cellulose. NREL has reported the results for a dilute acid hydrolysis of

softwoods in which the conditions of the reactors were as follows: Stage 1: 0.7% sulfuric

acid, 190°C, and a 3-minute residence time; Stage 2: 0.4% sulfuric acid, 215°C, and a 3-

minute residence time. The liquid hydrolyzates are recovered from each stage and

fermented to alcohol. Residual cellulose and lignin left over in the solids from the

hydrolysis reactors serve as boiler fuel for electricity or steam production. These bench-

scale tests confirmed the potential to achieve yields of 89% for mannose, 82% for

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galactose and 50% for glucose. The fermentation with Saccharomyces cerevisiae

achieved ethanol conversion of 90% of the theoretical yield [75].

Typical sulphuric acid concentrations for hemicellulose hydrolysis are in the

range 0.5–1.5% and temperatures above 121–160°C. From hemicelluloses, dilute-acid

processes yield sugar recoveries from 70% up to >95% [76-79]. The advantages of dilute-

acid hydrolysis are the relatively low acid consumption, limited problem associated with

equipment corrosion and less energy demanding for acid recovery. Under controlled

conditions, the levels of the degradation compounds generated can also be reduced [72].

3.1.2 Concentrated Acid Hydrolysis

This process is based on concentrated acid decrystallization of the cellulose

followed by the dilute acid hydrolysis to sugars at near theoretical yields. The separation

of acid from the sugars, acid recovery, and acid re-concentration are critical unit

operations. The fermentation converts sugars to ethanol. A process was developed in

Japan in which the concentrated sulfuric acid was used for the hydrolysis and the process

was commercialized in 1948. The remarkable feature of their process was the use of

membranes to separate the sugar and acid in the product stream. The membrane

separation, a technology that was way ahead of its time, achieved 80% recovery of acid

[80]. The concentrated sulfuric acid process has also been commercialized in the former

Soviet Union. However, these processes were only successful during the times of the

national crisis, when economic competitiveness of the ethanol production could be

ignored. Concentrated hydrochloric acid has also been utilized and in this case, the

prehydrolysis and hydrolysis are carried out in one step. Generally, acid hydrolysis

procedures give rise to a broad range of compounds in the resulting hydrolysate, some of

which might negatively influence the subsequent steps in the process. A weak acid

hydrolysis process is often combined with a weak acid prehydrolysis.

In 1937, the Germans built and operated commercial concentrated acid hydrolysis

plants based on the use and recovery of hydrochloric acid. Several such facilities were

successfully operated. During World War II, researchers at USDA's Northern Regional

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Research Laboratory in Peoria, Illinois further refined the concentrated sulfuric acid

process for corncobs. They conducted process development studies on a continuous

process that produced a 15%-20% xylose sugar stream and a 10%-12% glucose sugar

stream, with the lignin residue remaining as a byproduct. The glucose was readily

fermented to ethanol at 85%-90% of theoretical yield. The Japanese developed a

concentrated sulfuric acid process that was commercialized in 1948. The remarkable

feature of their process was the use of membranes to separate the sugar and acid in the

product stream. The membrane separation, a technology that was way ahead of its time,

achieved 80% recovery of acid [80]. R&D based on the concentrated sulfuric acid

process studied by USDA (and which came to be known as the "Peoria Process") picked

up again in the United States in the 1980s, particularly at Purdue University and at

Tennessee Valley Authority (TVA) [81]. Among the improvements added by these

researchers were 1) recycling of dilute acid from the hydrolysis step for pretreatment, and

2) improved recycling of sulfuric acid. Minimizing the use of sulfuric acid and recycling

the acid cost-effectively are critical factors in the economic feasibility.

(http://www1.eere.energy.gov/biomass/printable_versions/concentrated_acid.html). The

conventional wisdom in the literature suggests that the Peoria and TVA processes cannot

be economical because of the high volumes of acid required [82]. The improvements in

the acid sugar separation and recovery have opened the door for the commercial

application. Two companies namely Arkenol and Masada in the United States are

currently working with DOE and NREL to commercialize this technology by taking

advantage of niche opportunities involving the use of biomass as a means of mitigating

waste disposal or other environmental problems

(http://www1.eere.energy.gov/biomass/concentrated_acid.html). Minimizing the use of

the sulfuric acid and recycling the acid cost-effectively are the critical factors in the

economic feasibility of the process. U.S.Pat. No. 5,366,558 [83] uses two "stages" to

hydrolyze the hemicellulose sugars and the cellulosic sugars in a countercurrent process

using a batch reactor, and results in poor yields of glucose and xylose using a mineral

acid. Further, the process scheme is complicated and the economic potential on a large-

scale to produce inexpensive sugars for fermentation is low. U.S. Pat. No. 5,188,673 [84]

employs concentrated acid hydrolysis which has benefits of high conversions of biomass,

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but suffers from low product yields due to degradation and the requirement of acid

recovery and recycles. Sulfuric acid concentrations used are 30-70 weight percent at

temperatures less than 100°C. Although 90% hydrolysis of the cellulose and

hemicellulose is achieved by this process, the concentrated acids are toxic, corrosive and

hazardous and require reactors that are resistant to corrosion. In addition, the

concentrated acid must be recovered after the hydrolysis to make the process

economically feasible [85]. A multi-function process for hydrolysis and fractionation of

lignocellulosic biomass to separate hemicellulosic sugars using mineral acid like sulfuric

acid, phosphoric acid or nitric acid has been described [86]. A process for treatment of

hemicellulose and cellulose in two different configurations is described [87].

Hemicellulose is treated with dilute acid in a conventional process. The cellulose is

separated out from the "prehydrolyzate" and then subjected to pyrolysis at high

temperatures. Further, the process step between the hemicellulose and cellulose reactions

require a drying step with a subsequent pyrolysis high temperature step at 400-600°C for

conversion of the cellulose to fermentable products.

3.2 Steam explosion

Autohydrolysis and steam explosion processes selectively hydrolyze

hemicellulose from lignocellulosic biomass. The process is economic and ecofriendly as

it doesn’t involves chemical catalyst. A relatively high hemicellulose recovery in the

range of 55–84%, together with low levels of inhibitory by-products, has been obtained

through autohydrolysis [88-91]. Cellulose and lignin are not significantly affected,

yielding a cellulose- and lignin-rich solid phase together with a liquid fraction with a

relative low concentration of potential fermentation inhibitors. The main drawback of this

process is that the solubilised hemicellulose appears mainly in oligomeric form [16, 22,

90, 92, 93,]. In principle steam explosion is one of the attractive pretreatment methods

that can cause disintegration of the material, thereby creating a large surface area on

which cellulase enzyme complex can act upon. Simultaneously hemicellulose is

separated during the steam explosion process thereby improving the accessibility to the

enzymes and enhancement of the over all lignocellulose degradation [94]. Most steam

treatments yield high hemicellulose solubility (producing mainly oligosaccharides) along

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with slight lignin solubilisation. Studies without added catalyst report sugars recoveries

between 45% and 69% [95-98]. Cara et al., [99] have reported a 30% increase in

maximum pentose yield for olive tree prunings when steam explosion was followed by

acid hydrolysis.

3.3 Enzymatic hydrolysis The most promising method for hydrolysis of polysaccharides to monomer sugars

is by use of enzymes, i.e., cellulases and hemicellulases. Moreover, hemicellulases

facilitate cellulose hydrolysis by exposing the cellulose fibres, thus making them more

accessible [100]. The enzymatic hydrolysis of the lignocellulosic biomass is preceeded by

a pretreatment process in which the lignin component is separated from the cellulose and

hemicellulose to make it amenable to the enzymatic hydrolysis. The lignin interferes with

the hydrolysis by blocking the access of the cellulases to the cellulose and by irreversibly

the binding hydrolytic enzymes. Therefore, the removal of the lignin can dramatically

increase the hydrolysis rate [101]. Recently the enzymatic hydrolysis of lignocellulosic

biomass has been optimized using enzymes from different sources and mixing in an

appropriate proportion using statistical approach of factorial design. A twofold reduction

in the total protein required to reach glucan to glucose and xylan to xylose hydrolysis

targets (99% and 88% conversion, respectively), thereby validating this approach towards

enzyme improvement and process cost reduction for lignocellulose hydrolysis [102].

3.3.1 Hemicellulose degrading enzymes

3.3.1.1 Xylanases and Mannanases Hemicellulases are multi-domain proteins and generally contain structurally

discrete catalytic and non-catalytic modules. The most important non-catalytic modules

consist of carbohydrate binding domains (CBD) which facilitate the targeting of the

enzyme to the polysaccharide, interdomain linkers, and dockerin modules that mediate

the binding of the catalytic domain via cohesion-dockerin interactions, either to the

microbial cell surface or to enzymatic complexes such as the cellulosome [100]. Based on

the amino acid or nucleic acid sequence of their catalytic modules hemicellulases are

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either glycoside hydrolases (GHs) which hydrolyse glycosidic bonds, or carbohydrate

esterases (CEs), which hydrolyse ester linkages of acetate or ferulic acid side groups and

according to their primary sequence homology they have been grouped into various

families. Most studies on hemicellulases have focused until now on enzymes that

hydrolyse xylan [7]. Enzymes that hydrolyse mannan have been largely neglected, even

though it is an abundant hemicellulose, therefore the application of mannanases for

catalysing the hydrolysis of β-1,4 mannans is as important as the application of

xylanases.

Due to the complex structure of hemicelluloses, several different enzymes are

needed for their enzymatic degradation or modification. The two main glycosyl

hydrolases depolymerising the hemicellulose backbone are endo-1, 4- β-D-xylanase and

endo-1, 4-β-D-mannanase [5]. Endo-1,4-β-xylanase cleaves the glycosidic bonds in the

xylan backbone, bringing about a reduction in the degree of polymerization of the

substrate. Xylan is not attacked randomly, but the bonds selected for hydrolysis depend

on the nature of the substrate molecule, i.e., on the chain length, the degree of branching,

and the presence of substituents [103]. Initially, the main hydrolysis products are β-D-

xylopyranosyl oligomers, but at a later stage, small molecules such as mono-, di- and

trisaccharides of β-D-xylopyranosyl may be produced [104]. These enzymes are

produced by fungi, bacteria, yeast, marine algae, protozoans, snails, crustaceans, insect,

seeds, etc. [105]. Filamentous fungi are particularly interesting producers of xylanases

since they excrete the enzymes into the medium and their enzyme levels are much higher

than those of yeasts and bacteria [7]. Aspergillus niger, Humicola insolens,

Termomonospora fusca, Trichoderma reesei, T. longibrachiatum, T. koningii have been

used as industrial sources of commercial xylanases. Nevertheless, commercial xylanases

can also be obtained from bacteria, e.g., from Bacillus sp. Xylanases have many

commercial uses, such as in paper manufacturing, animal feed, bread-making, juice and

wine industries, or xylitol production [104, 106]. Several investigations so far have

indicated that xylanases are usually inducible enzymes [107], and different carbon

sources have been analysed to find their role in effecting the enzymatic levels. Xylanase

biosynthesis is induced by xylan, xylose, xylobiose or several β-D-xylopyranosyl

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residues added to the culture medium during growth [7, 104, 108]. However, constitutive

production of xylanase has also been reported [109]. Catabolite repression by glucose is a

common phenomenon observed in xylanase biosynthesis [7].

The main sugar moiety of galactoglucomannans (GGM) is D-mannose, but for its

complete breakdown into simple sugars, the synergistic action of endo-1,4-β-mannanases

(EC 3.2.1.78) and exoacting β-mannosidases (EC 3.2.1.25) is required to cleave the

polymer backbone. Additional enzymes, such as β-glucosidases (EC 3.2.1.21), α-

galactosidases (EC 3.2.1.22) and acetyl mannan esterases are required to remove side

chain sugars that are attached at various points on mannans [110, 112]. The property of

mannanolysis is widespread in the microbial world. A vast variety of bacteria,

actinomycetes, yeasts and fungi are known to be mannan degraders [113, 114].

Mannanases of microbial origin have been reported to be both induced as well as

constitutive enzymes and are usually being secreted extracellularly [110]. Although a

number of mannanase-producing bacterial sources are available, only a few are

commercially exploited as wild or recombinant strains, of these, the important ones are:

Bacillus sp., Streptomyces sp., Caldibacillus cellulovorans, Caldicellulosiruptor Rt8B,

Caldocellum saccharolyticum [115-117].

3.3.1.2 Accessory enzymes for hemicellulose hydrolysis

Since xylan is a complex component of the hemicelluloses in wood, its complete

hydrolysis requires the action of a complete enzyme system, which is usually composed

of β-xylanase, β-xylosidase, and enzymes such as α-L-arabinofuranosidase, α-

glucuronidase, acetylxylan esterase, and hydroxycinnamic acid esterases that cleave side

chain residues from the xylan backbone. Figure 1 describes the mechanism of xylanolytic

enzymes involved in the degradation of hemicellulose. All these enzymes act

cooperatively to convert xylan to its constituents [5]. Xylanases attack randomly the

backbone of xylan to produce both substituted and non-substituted shorter chain

oligomers, xylobiose and xylose. Xylosidases are essential for the complete breakdown

of xylan as they hydrolyse xylooligosaccharides to xylose [118]. The enzymes

arabinosidase, α-glucuronidase and acetylxylan esterase act in synergy with the xylanases

16

and xylosidases by releasing the substituents on the xylan backbone to achieve a total

hydrolysis of xylan to monosaccharides [4]. Table 2 lists the microorganisms producing

different hemicellulases [119].

3.3.1.3 Xyloglucanases

Many endoglucanases have been reported to hydrolyze xyloglucan as a substrate

analog [120], however few endo-β-1,4-glucanases have high activity toward xyloglucan,

with little or no activity towards cellulose or cellulose derivatives [121, 122]. They have

been assigned a new EC number (EC 3.2.1.151) and designated as xyloglucanase,

xyloglucan hydrolase (XGH), or xyloglucan-specific endo-β-1,4-glucanases (XEGs)

belonging to families 5, 12, 44, and 74, according to a recent classification of glycoside

hydrolases (GHs) available at http://afmb.cnrs-mrs.fr/CAZY/ [123–126]. They represent

a new class of polysaccharide degrading enzymes which can attack the backbone at

substituted glucose residues. Among these enzyme families, xyloglucanases placed in

GH family 74 are known to have high specific activity towards xyloglucan, with

inversion of the anomeric configuration, and both endo-type and exo-type hydrolases

have been found in several microorganisms [127– 134]. The exo-type enzymes recognize

the reducing end of xyloglucan oligosaccharide (oligoxyloglucan reducing- end-specific

cellobiohydrolase, EC 3.2.1.150, from Geotrichum sp. M128 [129] and oligoxyloglucan

reducing- end-specific xyloglucanobiohydrolase from Aspergillus nidulans [134]),

whereas the endo-type enzymes hydrolyze xyloglucan polymer randomly. In addition,

XEG74 from Paenibacillus sp. KM21 and Cel74A from Trichoderma reesei have been

reported to have endo-processive or dual-mode endo-like and exo-like activities [127,

132]. The complete hydrolysis of galactoxyloglucan requires an accessory enzyme, β-

galactosidase which cleaves the galactose residue attached to the branched xylose moiety

in the β-D-glucopyranose backbone [135]. The xyloglucan and the enzymes responsible

for its modification and degradation are finding increasing prominence, reflecting the

drive for diverse biotechnological applications in fruit juice clarification, textile

processing, cellulose surface modification, pharmaceutical delivery and production of

food thickening agents [136].

17

3.3.2 Role of thermostable hemicellulases in hydrolysis

Thermostable enzymes are gaining wide industrial and biotechnical interest due to

the fact that they are more stable and thus generally better suited for harsh process

conditions. Thermostability is expressed as a measure of the half-life of enzyme activity

at elevated temperatures [137]. Thermostable enzymes are produced both by thermophilic

and mesophilic organisms. Although thermophilic microorganisms are a potential source

for thermostable enzymes, the majority of industrial thermostable enzymes originate from

mesophilic organisms. Thermophilic bacteria have, however, received considerable

attention as sources of highly active and thermostable enzymes. Thermostable enzymes in

the hydrolysis of lignocellulosic materials have several potential advantages: higher

specific activity (decreasing the amount of enzyme needed), higher stability (allowing

elongated hydrolysis times) and increased flexibility for the process configurations [138].

The two first characteristics would expectedly improve the overall performance of the

enzymatic hydrolysis even at the range of conventional enzymes active at around 50°C.

Thus, carrying out the hydrolysis at higher temperature would ultimately lead to

improved performance, i.e. decreased enzyme dosage and reduced hydrolysis time and,

thus, potentially decreased hydrolysis costs. Thermostable enzymes would expectedly

also allow hydrolysis at higher consistency due to lower viscosity at elevated

temperatures and allow more flexibility in the process configurations. In addition to

improved performance in the hydrolysis of lignocellulosic substrates, thermophilic

enzymes allow the design of more flexible process configurations [138]. Menon et al,

[139] have analyzed the comparative data on hydrolysis of hemicellulosic substrates, oat

spelt xylan and wheat bran hemicellulose at different temperatures with thermostable

xylanase from alkalothermophilic Thermomonospora sp. At higher temperature, rapid

hydrolysis of hemicellulosic substrates were achieved, favouring a reduction in process

time and enzyme dosage.

18

4 Production of biochemicals from hemicellulosic hydrolysates

4.1 Fermentation of hemicellulosic hydrolysates to fuel ethanol

D-xylose, the predominant sugar of hemicellulose can be produced from

xylan/hemicellulose by the action of acid/enzymes. D-xylose is not readily utilized as D-

glucose for the production of ethanol by microorganisms such as Saccharomyces

cerevisiase. Bacteria, yeast and mould differ in their mode of conversion of D-xylose to

xylulose, the intial step in xylose fermentation. Few yeasts uses the enzymes xylose

reductase to reduce xylose to xylitol which is subsequently oxidized to D-xylulose by

xylitol dehydrogenase. Yeasts such as Pichia, Klyveromyces, Pachysolen are reported to

ferment xylose to ethanol under micro-aerophilic condition. The general requirements of

an organism to be used in ethanol production is that it should give a high ethanol yield, a

high productivity and be able to withstand high ethanol concentrations in order to keep

distillation costs low. In addition to these general requirements, inhibitor tolerance,

temperature tolerance and the ability to utilize multiple sugars are also essential. Figure 2

describes the catabolism of hemicellulose derived monosaccharide.

Essentially, there are three different types of processes namely: Separate

Hydrolysis and Fermentation (SHF), Direct Microbial Conversion (DMC) and

Simultaneous Saccharification and Fermentation (SSF). SSF has been shown to be the

most promising approach to biochemically convert cellulose to ethanol in an effective

way [140].

4.1.1 Separate hydrolysis and fementation (SHF)

SHF is a conventional two step process where the hemicellulose is hydrolysed

using the enzymes to form the reducing sugars in the first step and the sugars, thus

formed, are fermented to the ethanol in the second step using Pichia or Zymomonas. The

advantage of this process is that each step can be carried out at its optimum conditions.

Table 3 summarizes the ethanol production from hemicellulosic hydrolysates. A recent

study by Wilkins et al, [149] reveals an ethanol production by Kluyveromyces sp from D-

xylose at 40°C and 45°C with a yield of 0.15g/g and 0.08g/g respectively under anaerobic

19

condition. H. polymorpha could ferment both glucose and xylose up to 45°C [150].

Debaryomyces sp used both pentoses and hexoses to similar extents in sugar mixtures

and a preference for one carbohydrate did not inhibit the consumption of other. This

could be important, since the usual substrates of hemicelluloses, which often consists of

sugar mixtures [151].

4.1.2 Direct microbial conversion (DMC)

This process involved three major steps, namely enzyme production, hydrolysis of

the lignocellulosic biomass and the fermentation of the sugars all occurring in one step

[152]. The relative lower tolerance of the ethanol is the main disadvantage of this

process. A lower tolerance limit of about 3.5% has been reported as compared to 10% of

ethanologenic yeasts. Acetic acid and lactic acid are also formed as the by-products in

this process in which a significant amount of carbon get utilized [153]. Neurospora

crassa is known to produce ethanol directly from the cellulose/hemicellulose, since it

produces both the cellulase and the xylanase and also has the capacity to ferment the

sugars to ethanol anaerobically [154].

4.1.3 Simultaneous saccharification and fermentation (SSF)

The saccharification of the lignocellulosic biomass by the enzymes and the

subsequent fermentation of the sugars to ethanol by the yeast such as Saccharomyces or

Zymomonas takes place in the same vessel in this process [155]. The compatibility of

both saccharification and fermentation process with respect to various conditions such as

pH, temperature, substrate concentration etc. is one of the most important factors

governing the success of the SSF process. The main advantage of using SSF for the

ethanol bioconversion is enhanced rate of hydrolysis of lignocellulosic biomass (cellulose

and hemicellulose) due to removal of end product inhibition. Several inhibitory

compounds are formed during hydrolysis of the raw material, the hydrolytic process has

to be optimized so that inhibitor formation can be minimized. When low concentrations

of inhibitory compounds are present in the hydrolyzate, detoxification is easier and

fermentation is cheaper. The choice of a detoxification method has to be based on the

degree of microbial inhibition caused by the compounds. As each detoxification method

20

is specific to certain types of compounds, better results can be obtained by combining

two or more different methods. Another factor of great importance in the fermentative

processes is the cultivation conditions, which, if inadequate, can stimulate the inhibitory

action of the toxic compounds. Thermotolerant yeast is an added advantage for SSF and

thermotolerant yeast strains, e.g. Fabospora fragilis, Saccharomyces uvarum, Candida

brassicae, C. lusitaniae, and Kluyveromyces marxianus, have been evaluated for future

use in SSF, to allow fermentation at temperatures 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, were applied. SSF of

cellulose with mixed cultures of different thermotolerant yeast strains have also been

carried out [156]. However, there is scarcity of literature from SSF experiments in which

hemicelluloses have been used together with thermotolerant strains. Menon et al., [139]

have reported for the first time the SSF experiments using hemicellulosic substrates and

thermotolerant Debaryomyces hansenii. Maximum ethanol concentrations of 9.1 g/L and

9.5 g/L were obtained in SSF with oat spelt xylan and wheat bran hemicellulose

respectively. These concentrations were attained in 36h for OSX and 48h for WBH from

the onset of SSF. The increased ethanol yield in SSF systems is evidently due to removal

of xylose formed during hydrolysis which causes end product inhibition.

4.2 Production of xylitol from hemicellulosic hydrolysates

The conversion of xylose to value-added product xylitol will have significant role

in the economic viability of cellulose bioconversion process. The pretreatment (acid or

enzyme) of hemicellulose predominantly liberates xylose and arabinose and

hydrogenation products are xylitol and arabinitol. Xylitol is a naturally occurring five-

carbon sugar alcohol [157], has important applications in pharmaceuticals and food

industries due to high sweetening properties, non- and anticariogenicity property and

microbial growth inhibition capacity [158, 159]. Xylitol is used as sugar substitute for

diabetics, as it does not require insulin for its metabolism [160]. Recently it has been

demonstrated that xylitol can prevent acute otitis media (AOM) [161] in children. The

value of xylitol market is currently $340 million with applications in mouthwashes,

21

toothpastes and chewing gums as well as in foods for special dietary uses. Global xylitol

consumption was 43,000 t in 2005, the U.S. and Western Europe accounting for 30 and

37% of the total xylitol consumption [162]. Currently the major product of xylan which is

of considerable importance is xylitol derived mainly from agricultural and wood residues

respectively.

Xylitol is produced from xylan-rich biomass by both chemical and biological

methods [163]. The chemical process adapted so far is not ecofriendly and hydrogenation

of xylose demands huge production costs in terms of temperature and pressure input as

well as the formation of by-products that require expensive separation and purification

steps. In addition, xylose should be purified before it is hydrogenised, which further

increases the capital investment and costs for xylitol production [164]. Certain microbial

species have potential to utilize xylose as carbon source by converting it to xylitol. Yeasts

naturally produce xylitol as an intermediate during D-xylose metabolism. Xylose

reductase (XR) is typically an NADPH or NADH-dependent enzyme which oxidizes

xylose to xylitol [165], while enzyme xylitol dehydrogenase (XDH) requires NAD+

[166] for reduction of xylitol to xylulose. The cofactors imbalance results in the secretion

of xylitol as a xylose fermentation by-product [167]. Various bioconversion methods

have been exploited for the production of xylitol from hemicellulose using

microorganisms or their enzymes [168]. Xylitol production from hemicellulosic

hydrolysates using various microorganisms is illustrated in Table 4. Xylitol production by

fermentation is usually operated under mild conditions and could achieve higher yield

and will lead to an ecofriendly process. Hence the biological process for xylitol

production is receiving wider attention.

4.3 Production of other value added products from hemicellulosic hydrolysates

4.3.1 Butanediol

2,3-Butanediol, also known as 2,3-butylene glycol (2,3-BD) is a valuable

chemical feedstock because of its application as a solvent, liquid fuel, and as a precursor

22

of many synthetic polymers and resins [3]. Butanediol is produced during oxygen-limited

growth, by a fermentative pathway known as the mixed acid-butanediol pathway [175].

The 2,3-BD pathway and the relative proportions of acetoin and butanediol serve to

maintain the intracellular NAD/NADH balance under changing culture conditions. All of

the sugars commonly found in hemicellulose and cellulose hydrolysates can be converted

to butanediol, including glucose, xylose, arabinose, mannose, galactose, and cellobiose.

The theoretical maximum yield of butanediol from sugar is 0.50 kg per kg. With a

heating value of 27,200 J/g, 2,3-BD compares favorably with ethanol (29,100 J/g) and

methanol (22,100 J/g) for use as a liquid fuel and fuel additive [176].

Hexose and pentose can be converted to 2,3-BD by several microorganisms

including Aeromonas [177], Bacillus [178], Paenibacillus [179], Serratia, Aerobacter

[180], Enterobacter [11] and also Klebsiella [181–186]. Among these, especially

Klebsiella is often used for 2,3-BDO production because of its broad substrate spectrum

and cultural adaptability. At first, 2,3-BD was produced worldwide from glucose or

xylose by fermentation. Starch [177,187], molasses [188], water hyacinth [189] and

Jerusalem artichoke tubers [190] were also tested for their suitability as substrates for 2,3-

BD fermentation. Some studies have been conducted utilizing the hemicellulose portion

of forest residues like wood for 2,3-BD production [191]. Cheng et al, [186] have

reported production of 2, 3-butanediol from corn cob hydrolysate by fed batch

fermentation using Klebsiella oxytoca. A maximal 2,3-butanediol concentration of 35.7

g/l was obtained after 60 h of fed-batch fermentation, giving a yield of 0.5 g/g reducing

sugar and a productivity of 0.59 g/h l.

4.3.2 Ferulic acid and Vanillin

Ferulic acid is the major phenolic acid of cereal grains such as wheat, triticale,

and rye [192, 193, 194]. It can exist as an extractable form, as free, esterified, and

glycosylated phenolic constituents [192] as well as an insoluble-bound occurring in the

outer layers of wheat grains [195]. Alkaline hydrolysis is reported to release ferulic acid

from the insoluble form [195, 196]. Also, enzyme preparations are used for the same

23

purpose [197]. There are numerous reports on antiradical, oxidase inhibitory,

antiinflammatory, antimicrobial,anticancer activities of ferulic acid and its derivatives in

the literature data [198-200]. Feruloyl esterases (FAEs) act synergistically with xylanases

to hydrolyze ester-linked ferulic (FA) and diferulic (diFA) acid from cell wallmaterial

and therefore play a major role in the degradation of plant biomass [201]. FAEs are used

as a tool for the release of Ferulic acid from agroindustrial byproducts such as wheat bran

[202-216], maize bran [217-219], maize fiber [210], brewer’s (or barley) spent grain

[202,220-227], sugar beet pulp [216,217,228-231], coastal bermudagrass [232], oat hulls

[233-235], jojobameal [236], wheat straw[217], coffee pulp [217] and apple marc [217].

Vanillin can be obtained by fermentation using suitable microorganisms in the stationary

growth phase [237-240]. The microbial transformation of ferulic acid is recognized as

one of the most attracting alternatives to produce natural vanillin. Bacteria belonging to

different genera are able to metabolize ferulic acid as the sole carbon source, producing

vanillin, vanillic acid and protocatechuic acid as catabolic intermediates [241,242].

Vanillin is industrially used as fragrance in food preparations, intermediate in the

productions of herbicides, antifoaming agents or drugs [243], ingredient of household

products such as air fresheners and floor polishes, and, because of its antimicrobial and

antioxidant properties [244,245], also as food preservative [246]. The much higher price

of the natural vanillin compared to the synthetic vanillin has been leading to growing

interest of the flavor industry in producing it from natural sources by bioconversion

[243,247–250]. Pseudomonas fluorescens was shown to produce vanillic acid from

ferulic acid [251,252], with formation of vanillin as an intermediate [253]. Promising

vanillin concentrations were obtained from ferulic acid by Amycolatopsis sp. [250,254]

and Streptomyces setonii [241,255,256]. However, the process development is difficult

because of the well known slow growth of actinomycetes and high viscosity of broths

fermented by them; therefore, the construction of new recombinants strains of quickly

growing bacteria able to overproduce vanillin is attractive. The biotechnological process

to produce vanillin from various agro by-products had been investigated using different

microorganisms as biocatalysts. Aspergillus niger I-1472 and Pycnoporus cinnabarinus

MUCL39533 were used in a two-step bioconversion using sugar-beet pulp [257,258],

24

maize bran [259], rice bran oil [260], and wheat bran [261]. Wheat bran [262] and corn

cob [263] is recently reported as a good substrate for biovanillin production by

Escherichia coli JM 109/pBB1.

4.3.3 Lactic acid

Lactic acid is widely used in food, pharmaceutical and textile industries. It is also

used as a source of lactic acid polymers which are being used as biodegradable plastics

[264,265]. The physical properties and stability of polylactides can be controlled by

adjusting the proportions of the L(+)- and D(−)-lactides [266]. Optically pure lactic acid

is currently produced by the fermentation of glucose derived from corn starch using

various lactic acid bacteria [267,268]. However, the fastidious lactic acid bacteria have

complex nutritional requirements [269] and the use of corn is not favoured as the

feedstock competes directly with the food and feed uses. The use of lignocellulose

biomass will significantly increase the competitiveness of lactic acid-based polymers

compared to conventional petroleum based plastics.

Lactobacillus spp. are used extensively in industry for starch-based lactic acid

production, the majority of which lack the ability to ferment pentose sugars such as

xylose and arabinose [266]. Although, Lactobacillus pentosus, L. brevis and Lactococcus

lactis ferment pentoses to lactic acid, pentoses are metabolized using the phosphoketolase

pathway which is inefficient for lactic acid production [270,271]. In the phosphoketolase

pathway, xylulose 5-phosphate is cleaved to glyceraldehyde 3-phosphate and acetyl-

phosphate. With this pathway, the maximum theoretical yield of lactic acid is limited to

one per pentose (0.6 g lactic acid per g xylose) due to the loss of two carbons to acetic

acid. Sugarcane bagasse [272], steam exploded wood [273], soybean stalk [274], corncob

molasses [275], trimming vine shoots [276] and wheat straw [270] hydrolysate are used

for lactic acid production. John et al, [277] have reported the production of lactic acid

from agro residues using Lactobacillus delbrueckii in solid state and simultaneous

saccharification and fermentation [278].

25

4.3.4 Furfural

Furfural is commercially synthesized from lignocellulosic biomass in a process

which involves acid hydrolysis of the pentosans or xylans, present in the hemicelluloses

of some agriculture residues and hardwoods into pentoses or xylose and successive

cyclodehydration of the latter to form furfural. The second step of dehydration is

comparatively slower than that of mineral acid hydrolysis. There is no synthetic route

available for the production of furfural. The potential furfural yield for typical feedstock

is expressed in terms of kg of furfural per metric ton of dry biomass. It is reported to be

220 for corncobs, 170 for bagasse, 160 for cornstalks, 160 for sunflower hulls, and

approximately 150–170 for hardwoods. Furfural, derived from renewable biomass, is

used for the production of a wide spectrum of important non- petroleum derived

chemicals. World market for furfural currently is estimated to be about 200,000 to

210,000 tpa of which about 60–62% is used for the production of furfuryl alcohol.

Furfural is well known for its thermosetting properties, physical strength, and corrosion

resistance. It is mainly used for the production of resin which accounts for 70% of the

market. Currently, commercial production of furfural suffers from technological

challenges and maintenance problems [279]. The use of conventional acid catalysts

(mineral acids such as H2SO4 or HCl) for the production of furfural poses serious

environmental problems and subsequently results in few undesirable side reactions

causing the formation of huge amounts of toxic waste [280]. Hence, there is a need to

develop ecofriendly catalytic processes to eliminate environmental corrosion as well as

waste-disposal problems. Coniochaeta ligniaria NRRL30616 metabolizes furfural and 5-

hydroxymethylfurfural (HMF) as well as aromatic acids, aliphatic acids and aldehydes.

NRRL30616 grew in corn stover dilute-acid hydrolysate and converted furfural to both

furfuryl alcohol and furoic acid [281].

4.3.5 Butanol

Butanol is a 4-carbon alcohol (butyl alcohol), an advanced biofuel, offers a

number of advantages and can help accelerate biofuel adoption in countries around the

world. It can be produced through processing of domestically grown crops, such as corn

26

and sugar beets, and other biomass, such as fast-growing grasses and agricultural waste

products. Biobutanol's primary use is as an industrial solvent in products such as lacquers

and enamels and is also compatible with ethanol blending and can improve the blending

of ethanol with gasoline. Using fermentation to replace chemical processes in the

production of butanol depends largely on the availability of inexpensive and abundant

raw materials and efficient conversion of these materials to solvents. Solventogenic

Acetone Butanol Ethanol (ABE)-producing Clostridia have an added advantage over

many other cultures as they can utilize both hexose and pentose sugars [282], which are

released from wood and agricultural residues upon hydrolysis, to produce ABE. Parekh et

al. [283] produced ABE from hydrolysates of pine, aspen, and corn stover using

Clostridium acetobutylicum P262. Similarly Marchal et al. [284] used wheat straw

hydrolysate and C. acetobutylicum, while Soni et al. [285] used bagasse and rice straw

hydrolysates and C. saccharoperbutylacetonicum to convert these agricultural wastes into

ABE. Qureshi et al, [286] have studied the production of butanol from corn fibre

hydrolysate using Clostridium beijerinckii BA101. Sun and Liu, [287] have reported the

production of butanol from sugar maple hemicellulose hydrolysate using Clostridia

acetobutylicum ATCC824.

4.3.6 Biohydrogen

Hydrogen is considered to be an ideal energy alternative for the future. Compared

with the conventional hydrogen generation process (thermochemical and

electrochemical), bio-hydrogen production processes are more environmentfriendly and

less-energy intensive. Hydrogen could be produced from renewable materials, such as

waste water, organic wastes, corn straws and waste water sludge, etc. Biohydrogen has

the potential to considerably reduce costs and environmental impact as it can be produced

with sunlight and minimal nutrients or organic waste effluents. Hydrogen (H2) producing

microorganisms can be rapidly grown in bioreactors with relatively small energy and

environmental footprints, making biohydrogen a renewable and low impact technology.

Biological hydrogen, biohydrogen, production may provide a renewable, more

sustainable alternative but has yet to reach a scale large enough for consideration in

replacing a significant portion of the hydrogen supply [288].Various hemicellulosic

27

hydrolysates from wheat straw [289,290], Corn stover [291], sugarcane bagasse [292],

Sweet sorghum [293], Corn straw [294] have been evaluated for biohydrogen production.

4.3.7 Chitosan

Chitosan, as an important kind of biomacromolecules, has protective and

supportive functions in cell walls of zygomycetes [295]. Chitosan synthesis is a complex

process, which involves changes of many intermediate metabolites and energy charges in

cells. Chitosan itself has found numerous applications in food, cosmetics and

pharmaceutical industries because of their unique properties such as nontoxicity,

biodegradability, biocompatibility, film-forming and chelating properties along with their

antimicrobial activity [296-298]. Tai et al, [299] have studied the potential of

hemicellulose hydrolysate of corn straw for the production of chitosan by Rhizopus

oryzae.

4.3.8 Xylo-oligosaccharides

Xylo-oligosaccharides are xylose-based oligomers that may have variable

proportions of substitute groups like acetyl, uronic, and phenolic acids, depending on the

lignocellulosic from which they are extracted and the process of production. Substituted

xylo-oligosaccharides have some specific characteristics that are driving research efforts

to develop applications in fields related to the food and pharmaceutical industries. They

may be used as soluble dietary fiber because of its low calorific value and acceptable

organoleptic properties. Furthermore, they are non-carcinogenic and act as prebiotics

promoting the growth of beneficial Bifidobacteria in the colon [300,301], and are

considered a possible ingredient in functional foods [302,303]. Some studies point to the

beneficial effect of xylo- ligosaccharides may have on reducing the risk of colon cancer

[304-306]. Recently, xylo-oligosaccharides extracted by autohydrolysis of bamboo have

been found to posses a cytotoxic effect on human leukemia cells [307]. Also xylo-

oligosaccharides can be used as a source of xylose for the production of xylitol, a well-

known low-calorie sweetener [308].

28

5 Recombinant approaches for ethanol and xylitol production

Recombinant DNA methods are being currently used for overexpression of

cellulases and hemicellulases for lignocellulose hydrolysis and production of bioproducts.

Saccharomyces cerevisiae and Z.mobilis have been explored for utilizing lignocellulosic

biomass by genetic manipulations. Several metabolic and evolutionary engineering

techniques have been applied to: increase substrate range, like in S. cerevisiae and Z.

mobilis; maximize ethanol production like in E. coli; supply other important traits to

improve lignocellulose conversion to ethanol and xylitol. In general, approaches involve

genetic engineering towards those specific traits, often followed by strain optimization

through adaptation. Since the molecular basis for ethanol and inhibitor tolerance is not

fully understood, random mutagenesis and evolutionary engineering have also been

applied to improve those traits. Moreover, as a result of technological developments,

systems biology approaches have been recently applied to characterise the functional

genomics of microorganisms and to evaluate the impact of metabolic and evolutionary

engineering strategies. This advanced characterisation (genomics, transcriptomics,

proteomics, metabolomics) is already contributing to better understand physiological

responses and to identify crucial targets for metabolic engineering [309-313]. Table 5

illustrates the production of xylitol and ethanol using recombinant strains.

6 Future prospects

The implementation of lignocellulosic material for the development of alternative

energy is an important aspect for the lucrative gratification of biofuel vision. For an

economically viable bioconversion process it is necessary to utilize both the cellulosic

and hemicellulosic fractions of biomass. The research focus on cellulosic ethanol is

tremendously enhanced all over the world. On the other hand, corn and other grains can

be readily hydrolyzed to glucose with high yield by low-cost enzymes. However this

causes the debate on food versus fuel. Therefore a hemicellulose based process, wherein

hemicellulose can be readily hydrolyzed to value added products is foreseen to be

significant from this aspect. The fermentation of C6 sugars by Saccharomyces is a well

29

established technology whereas the utilization of C5 sugars is a challenging area.

Extensive research on xylose fermenting yeasts such as Pachysolen tannophilus, Candida

shehatae and Pichia stiptis, has been conducted. However, low ethanol tolerance,

conversion rates and catabolite repression in xylose conversion due to glucose need to be

addressed. Another potential ethanologen is recombinant Zymomonas mobilis in view of

its ability to ferment both xylose and glucose. E. coli strain developed by Ingram’s group

deserves special recognition, as it not only ferments all five sugars (glucose, xylose,

arabinose, mannose and galactose) present in the synthetic sugar mixtures to ethanol but

also performs competently in real hydrolyzates like that of Pinus. Saccharomyces LNH-

ST is another promising recombinant capable of fermenting dilute acid treated corn fiber

hydrolyzates.

Pervasive research needs to be performed to develop an efficient cost-effective

and eco-friendly pretreatment methods, enzymes or cocktails of various enzymes for

complete conversion of hemicellulose to monomers, efficient and robust microorganisms

for fermentation of hemicellulosic hydrolysates to value added products in a cost-

competitive way. Although bioethanol production has been greatly improved by new

technologies there are still challenges that need further investigations. These challenges

include maintaining a stable performance of the genetically engineered yeasts in

commercial scale fermentation operations and integrating the optimal components into

economic ethanol production systems.

Metabolic engineering and other classical techniques such as random mutagenesis

address the further enhancement of microorganism capabilities by adding or modifying

traits such as tolerance to ethanol and inhibitors, efficient hydrolysis of

cellulose/hemicellulose, thermotolerance, reduced need for nutrient supplementation and

improvement of sugars transport. The improvement achieved in the fermentation step

with the help of metabolic engineering is just one of the aspects of an integrated process.

Keeping a realistic perspective one can conclude that several pieces still remain to be

properly assembled and optimized before an efficient industrial configuration is acquired.

30

Acknowledgements

MR acknowledges the financial support from CSIR Emeritus Scheme.

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Table 1 Composition of representative hemicellulosic feedstocks

Substrate Xylose Arabinose Galactose Mannose Rhamanose Uronic acid Reference

Agricultural and agro-industrial biomass Corn cob 28-30 3-5 1-1.2 - 1 3 15,16,17,18 Corn stover 15-25 2-3.6 0.8-2.2 0.3-0.4 - - 15,19,20 Corn stalks 26 4.1 <2.5 <3.0 - - 21 Corn fibre 21.6 11.4 4.4 - - - 22 Rice husk 17.7 1.9 - - - - 23 Rice straw 15-23 3-5 0.4 1.8 - - 22,20 Sugarcane bagasse 21-26 2.5-7 1.6 0.5-0.6 - - 24-26

Wheat straw 19-21 2.4-4 2-2.5 0-0.8 - - 27,20 Wheat bran 16 9 1 0 - - 16 Barley straw 15 4 - - - - 28 Hardwoods Birch 19-25 0.3-0.5 0.7-1.3 1.8-3.2 0.6 4-7 29,30 Maple 18-19 0.8-1 1.0 1.3-3.3 - 4.9 15,30 Eucalypt 14-19 0.6-1 1-1.9 1-2 0.3-1 2 16, 31-33 Oak 22 1 1.9 2.3 - 3 34 Aspen 18-28 0.7-4 0.6-2 0.9-2.5 0.5 5-6 27,29,30,35 Poplar 18-21 0.9-1.4 1 3.5 - 2.3-3.7 30, 36 Softwoods Spruce 5-10 1.2 1.9-4.3 9.4-15 0.3 1.5-6 29,30,37-39 Douglas fir 6 3 3.7 - - - 35 Pine 5-10 2-4 2-4 6-13 - 3-6 30

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Table 2 List of microorganisms with high specific activity (μmol.min-1.mg-1) for hemicellulases (119) ENZYME Organism Substrate Specific

activity Feruloyl esterase Clostridium

stercorarium Ethyl ferulate 88

β-1,4-xylosidase

Thermoanaerobacter ethanolicus

o-nitrophenyl-β-D-xylopyranoside

1073

Exo-β-1,4-mannosidase

Pyrococcus furiosus

p-nitrophenyl-β-D-galactoside

31.1

Endo-β-1,4-mannanase

Bacillus subtilis

Galactoglucomannan/ glucomannans/mannan

514

α-Larabinofuranosidase

Clostridium stercoarium

alkyl-α-arabinofuranoside/ aryl-α-arabinofuranoside/ Larabinogalactan/ L-arabinoxylan/ methylumbelliferyl-α-Larabinofuranoside

883

α-Glucuronidase

Thermoanaerobacterium saccharolyticum

4-O-methyl-glucuronosyl-xylotriose

9.6

α-Galactosidase Escherichia coli raffinose 27350 Endo-galactanase Bacillus subtilis arabinogalactan 1790 β-Glucosidase Bacillus polymyxa 4-nitrophenyl-β-D-

glucopyranoside 2417

Acetyl xylan esterase Fibrobacter succinogenes

Acetylxylan/ α-naphthyl acetate

2933

Feruloyl esterase

Aspergillus niger

Methyl sinapinate

156

Endo-1,4-β-xylanase Trichoderma longibrachiatum

1,4-β-D-xylan

6630

β-1,4-xylosidase

Aspergillus nidulans

p-nitrophenyl-β-D-xylopyranoside

107.1

Exo-β-1,4-mannosidase

Aspergillus niger

β-D-Man-(1-4)-β-D-GlcNAc-(1-4)-β-DGlcNAc- Asn-Lys

188

Endo-β-1,4-mannanase

Sclerotium rolfsii

Galactoglucomannan/mannans galactomannans/glucomannans/

380

Endo-α-1,5-arabinanase Aspergillus niger 1,5-α-L-arabinan 90.2 α-L-arabinofuranosidase Aspergillus niger 1,5-α-L- 396.6

53

arabinofuranohexaose/ 1,5-α- L-arabinotriose/ 1,5-L-arabinan/ α-Larabinofuranotriose

α-Glucuronidase

Phanerochaete chrysosporium

4-O-methyl-glucuronosyl-xylobiose

4.5

α-Galactosidase Mortierella vinacea melibiose 2000 Endo-galactanase Aspergillus niger 6593

β-glucosidase

Humicola insolvens

(2-hydroxymethylphenyl)-β-Dglucopyranoside

266.9

Acetyl xylan esterase

Schizophyllum commune

4-methylumbelliferyl acetate/ 4- nitrophenyl acetate

227

54

Table 3 Ethanol production from hemicellulosic hydrolysates

Substrate Pretreatment Detoxification Microorganisms Ethanol (g/L) Ethanol yield (g/g) Reference

Bagasse Steam pretreatment - Pichia Stipitis CBS

6054 19.5 0.22 141

Sunflower seed

hull Acid hydrolysis Over-liming combined

with Na2SO3 Pichia Stipitis NRRLY-

7124 11 0.32 142

Bagasse Acid Hydrolysis Over-liming or electrodialysis

Pachysolen tannophilus DW 06 21 0.35 143

Prosopsis Julifora Acid hydrolysis Over-liming Pichia stipitis NCIM

3498 7.13 0.39 144

Water hyacinth

Acid hydrolysis - Pichia stipitis - 0.425 145

Olive Tree Acid hydrolysis Over-liming and Activated charcoal Pichia stipitis - 0.42 146

Yellow poplar wood meal

Alkaline pretreatment

Acid hydrolysis Over-liming Pichia stipitis 28.7 0.48 147

Oat spelt xylan Enzymatic hydrolysis - Debaromyces hansenii 7.55 0.42

139 Wheat bran Enzymatic

hydrolysis - Debaromyces hansenii 6.42 0.41

Tamarind kernel powder Acid hydrolysis Over-liming and

Activated charcoal Debaromyces hansenii 16 0.35 148

Tamarind kernel powder

Enzymatic hydrolysis - Debaromyces hansenii 5.18 0.39 148

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Table 4 Xylitol production from detoxified hemicellulosic hydrolysates

Substrate Pretreatment Detoxification Microorganisms Xylitol (g/L) Xylitol yield (g/g) Reference

Sugarcane bagasse Acid hydrolysis Activated charcoal Candida guilliermondii 35.5 0.72 164

Eucalyptus Acid hydrolysis Activated charcoal Ion exchange resins Candida guilliermondii 32.7 0.57 169

Corn fibre Acid hydrolysis Over-liming

Activated charcoal Ion exchange resins

Candida tropicalis 129 0.43 170

Brewery spent-grain Acid hydrolysis Over-liming Debaromyces hansenii 18 0.38 171

Rice straw Acid hydrolysis Over-liming Activated charcoal

Candida subtropicalis WF79 - 0.73 172

Wheat straw Acid hydrolysis Activated charcoal Candida guilliermondii FTI 20037 24.2 0.48 173

Corn cob Acid hydrolysis Over-liming, solvent

extraction Candida tropicalis

W103 68.4 0.7 174

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Table 5 Production of xylitol and ethanol using recombinant strains Sugars Genetic manipulations Microorganism Yield and/or

productivity Reference

Xylitol Xylose + glucose Expressed P.stipitis XR L.lactis 2.7 g/l/h 314

Xylose + glycerol Disrupted XYL2 C.tropicalis 3.2 g/l/h 315

Xylose + glucose Expressed XylE and K.lactis XR, deleted xylA,yhbC-deficient

E.coli 0.81 g/l/h 316

Xylose + glucose Overexpreesed G6PDH, expreesed Xyl1, attenuated PGI activity

S.cerevisiase 0.34g (g cdw h)-1 317

Ethanol

Xylose Disrupted CRC 1 P.stipitis 0.46 g/g 318

Xylose + glucose Genomic DNA integration

Z.mobilis 0.4 g/g 319

Xylose Overexpreesed native P.stipitis XDH, xylulokinase, deleted GRE3

S.cerevisiae 0.39 g/g 320

Xylose Evolutionary engineering, increased PDH activity

E.coli 0.46 g/g 321

XylE- xylose permease; XR- xylose reductase; G6PDH- glucose-6-phoshate dehydrogenase; PGI- phosphoglucose isomerase; PDH- pyruvate dehydrogenase; XDH- xylitol dehydrogenase; CRC 1- cytochrome c

57

Mechanism of action of the hemicellulases

Figure 1 : Xylanolytic enzymes involved in the degradation of hard wood and soft wood xylan. Ac, acetyl group; Ara, α- arabinofuranose; MeGlcA, α-4-Omehtylglucuronic acid; Xyl xylose.

58

Figure 2 Microbial conversion of xylose to ethanol and xylitol

Acetyl

NADH NAD

Alcohol dehydrogenase

Pyruvate decarboxylase

Pyruvate formate lyase

Pyruvate Acetaldehyde ETHANOL

CO2

Aldehyde dehydrogenase

ATP

ADP

NADH

ATP

ADP

NAD

(TKL)

Transaldolase (TAL)

Transketolase (TKL)

D-Ribose - 5-

Glyceraldihyde-3- Phosphate

D-Fructose-6- Phosphate

NAD

NADH

D-Xylulose - 5- Phosphate

Xylose isomerase (XI)

Xylulose kinase (XK)

ADP

ATP

NAD(P) NAD(P)

Xylitol dehydrogenas

Xylose reductase (XR)

D - XYLOSE

XYLITOL

D-Xylulose

Hemicellulose Hemicellulase