Production of Cellulosic Fuels

REVIEW

Production of Cellulosic Fuels

Saikat Chakraborty • Ashwin Gaikwad

Received: 31 July 2010 / Accepted: 8 April 2011 / Published online: 1 February 2012

� The National Academy of Sciences, India 2012

Abstract Bio-fuels (fuels derived from renewable bio-

sources) are important alternatives to future energy needs.

Bio-ethanol is one such fuel. This paper reviews various

processes and techniques involved in the production of bio-

ethanol from cellulose and lignocellulose bio-mass.

Experimental details are discussed for hydrolysis & the

depolymerisation of cellulose to glucose and the sub-

sequent biological/chemical formation of glucose to pro-

duce fuel derivatives. Use of ionic liquids during chemical

fermentation is also discussed. Kinetics of the enzymatic

hydrolysis have been mathematically modeled. Limitations

of bio-ethanol and its possible replacement by bio-butanol

in future is also briefly discussed.

Keywords Bio-fuels � Bio-ethanol �Enzymatic hydrolysis � Fermentation � Ionic liquids

Introduction

Energy—its availability, supply and use—happens to be

the organizing center of modern society. Considering our

energy sources, the consumption of fossil fuel in the past

fifty years has seen a dramatic change in a way that most

developed countries are the largest consumer of the oil.

The use of fossil fuel has led to the buildup of carbon

dioxide and other green-house gases in the atmosphere,

which have, in turn, resulted in climatic changes. Thus, the

geopolitical issues such as security of oil supply, increasing

oil prices and environmental concerns of global warming

have led to a push towards decreased oil consumption. This

has led to a recent revival of interest in biofuels since some

types of biofuels may be substantially less carbon-intensive

than fossil fuels [1]. The basic concept behind the above

hypothesis is that solar energy is trapped by the photo-

synthetic tissues of plants and used to reduce and condense

the carbon dioxide in the air into polysaccharides and lig-

nin in the plant body, particularly in the cell walls. So when

plants are burned, the trapped solar energy is released as

heat that could be converted to work, and the carbon

dioxide is recycled back to the atmosphere, thus balancing

the carbon cycle in the nature. Ethanol is a cellulosic

biofuel because it can be produced from inexpensive and

abundant biomass available in nature. India has a rich

biomass resource which can be utilized for generating bio-

energy. Ethanol is an alcohol-based fuel produced by fer-

menting plant sugars. It can be made from number of

agricultural products and food wastes containing sugar,

starch or cellulose. Sugar is the cheapest source of ethanol

production. In Asia, India is the largest producer as well as

consumer of sugar, so, it is not possible to use sugarcane as

a raw material for ethanol production. Next to sugar, starch

is the dominant feedstock in the starch-ethanol industry

worldwide. In the bioethanol industry, grains such as corn,

wheat, barley are the main sources of starch. Though both

the sugar and starch materials are successfully converted

into bioethanol it has some drawbacks, they are: potential

effect on food domain, potential pressure on land use and

natural resources such as water.

To overcome these drawbacks researchers have found

out the possible route, bioconversion of lignocellulosic

material to ethanol. Lignocellulosic materials are the most

abundant source of unutilized biomass. However their

availability does not necessarily impact land use. Ligno-

cellulose is mainly composed of lignin, hemicellulose and

S. Chakraborty (&) � A. Gaikwad

Department of Chemical Engineering, Indian Institute

of Technology Kharagpur, Kharagpur 721302, India

e-mail: [email protected]

123

Proc. Natl. Acad. Sci. Sect. A Phys. Sci. (January–March 2012) 82(1):59–69

DOI 10.1007/s40010-012-0007-y

cellulose. Typical sources of lignocellulosic biomass are

woody biomass, grasses, bagasse of sugarcane or sweet

sorghum, corn stover, industrial wastes and dedicated

woody crops. A comparative production potential of some

of the feedstocks are given in Table 1. The biological

conversion of ethanol from cellulose, starch or sugar can be

achieved by pretreatment, enzymatic hydrolysis and fer-

mentation. The simultaneously saccharification and fer-

mentation (SSF) process, shown in Fig. 1, was first

introduced in the year 1977, and offers high yields and

rates for ethanol production [4–6]. Pretreatment is an

essential step to improve bioethanol yield of SSF from

mainly a lignocellulosic biomass. Production of ethanol

from lignocellulose is a very different system than that

used for corn grain and sugar cane because carbohydrates

are much more difficult to solubilize than the starch in

grain [7] and sucrose from sugar cane [8].

Lignocellulose material is very resistant to enzymatic

breakdown, requiring pretreatment to enhance the suscep-

tibility of biomass material to enzyme. Number of tech-

niques has been employed to enhance the enzymatic

hydrolysis. Steam/steam explosion [9, 10], hot water/

autohydrolysis [11, 12], alkali treatment [13], acid treat-

ment [14, 15] are some of the established methods used for

the pretreatment of lignocellulosic material. Aqueous

ammonia solution has been used for the pretreatment of

lignocellulose as it has some advantages, such as its ability

to swell cellulosic material and selectivity for undesirable

lignin [13]. Lignin is the main hindrance in enzymatic

hydrolysis of lignocellulose. It limits the rate and extent of

enzymatic hydrolysis by acting as a shield and prevents the

digestible parts of the substrate to be hydrolyzed [16, 17].

Lignin and its derivatives are toxic to microorganisms and

inhibit the rate of enzymatic hydrolysis. Low lignin sub-

strates show improved microbial activities and enzyme

efficiency which in turn lowers the enzyme requirement.

Pretreatment

Lignocellulose

The abundance, low cost and high carbohydrate content

(70–80% approximately—almost equal to the starch con-

tent of corn) makes lignocellulosic material an attractive

feedstock for enzymatic depolymerization and bioconver-

sion to ethanol. Lignocellulosic biomass consists of three

types of polymers namely cellulose, hemicellulose and

lignin. Cellulose, the most abundant polymer on the earth,

is composed of fibrous bundles of crystalline cellulose

encased in a polymer matrix of hemicellulose and lignin.

Lignin is an amorphous heteropolymer consisting of three

different phenylpropane units (p-coumaryl, coniferyl and

sinapyl alcohol) joined together by different linkages. The

amorphous heteropolymer is non-water soluble, optically

inactive and resist microbial attacks and oxidative stress

yields of relatively pure glucose syrup without generating

glucose. All these factors make it difficult to degrade [18].

Hemicellulose is a complex carbohydrate structure, con-

sisting of different polymers like pentose (xylose and

arabinose), hexose (mannose, glucose and galactose) and

sugar acids [18, 19]. It is a lower molecular weight com-

pound than cellulose, and has branches with short lateral

chains consisting different sugars which can be easily

hydrolyzed [18]. The solubility of hemicellulosic com-

pound into water starts at around 180�C under neutral

conditions [20]. The solubilization of lignocellulose com-

ponents not only depends on temperature but also on other

parameters [21] such as moisture content and pH. Major

difficulties in pretreatment are the heterogeneous compo-

sition of polysaccharide in plant cell walls and the recal-

citrant nature of the cellulosic part of the substrate. The

bundles of cellulose molecules aggregated together to form

microfibrils in which highly ordered crystalline regions,

alternate with less ordered amorphous regions, thus making

cellulose fibers very resistant to acid and enzymatic

hydrolysis.

Pretreatment is an essential process in the bioconversion

of lignocellulosic biomass. It is required for efficient

enzymatic hydrolysis of lignocellulosic material, which is

difficult to treat because of its complex physical and

chemical structure; pretreatment enhances the accessibility

of cellulose to enzymes [22]. Lignocellulosic biomass is

only partially soluble in its original form, often less than

20%, resulting in slow enzymatic hydrolysis. The biolog-

ical production of ethanol from lignocellulosic material

involves three major steps: biomass pretreatment, enzy-

matic hydrolysis and fermentation. Pretreatment is pre-

requisite step for the bioconversion of lignocellulosic

biomass to ethanol. In case of bioethanol production from

sugarcane or corn, the pretreatment is rather simple

Table 1 Feedstocks for bioethanol production and comparative

production potential [2, 3]

Feedstocks Bioethanol production potential (l/ton)

Sugar cane 70

Sugar beet 110

Sweet potato 125

Potato 110

Cassava 180

Maize 360

Rice 430

Barley 250

Wheat 340

Bagasse 280

60 Proc. Natl. Acad. Sci. Sect. A Phys. Sci. (January–March 2012) 82(1):59–69

123

compared to that for lignocellulose. For any of the biomass,

the purpose of pretreatment is to make the substrate ame-

nable to the action of enzyme so that enzymatic hydrolysis

gives high degradation products and thus lowering the cost

of hydrolysis under milder conditions.

Mechanical Pretreatment

The objective of a mechanical pretreatment is reduction of

particle size and crystallinity, which ultimately results in

increase of available surface area and reduction of degree

of polymerization (DP). Thus, the total hydrolysis yield

increases by 5–25% (depends on kind of biomass, kind of

milling and duration of milling) and reduces the technical

digestion time by 23–59%, [23, 24]. However, milling/

grinding is highly energy and capital intensive and hence

uneconomical on larger scale [25].

Ball milling not only decrystallizes lignocellulose but

also reduces its particle size. Ball milled cellulose can be

completely hydrolyzed to sugar. The effectiveness of the

milling however dependant on cellulosic source, softwood

shows the least response. Similarly other milling machin-

eries, for example, two roll milling, hammer milling, col-

loid milling, vibro-energy milling, also serve for the same

purpose [17, 26].

High energy radiation was found to enhance in vitro

digestibility as well as acid or enzymatic hydrolysis of

cellulose. Radiations are effective in breaking the lignin-

cellulose complex, which results in increase in surface area

while the effect on crystallinity of cellulose is still con-

troversial. Irradiation accompanying with milling, in the

presence of nitric salts or treatment with acid/alkali prior to

irradiation increases the digestibility of the treated mate-

rial. The amount of reducing sugar obtained by enzymatic

hydrolysis of bagasse irradiated with 100 Mrad was about

three times higher than the untreated bagasse. At 50 Mrad

crystallinity decreases while digestibility increase. Irradia-

tion at higher intensity, over 50 Mrad, is not suggested for

direct glucose production, because of further glucose

decomposition [27]. Microwave irradiation pretreatment of

ground rice straw released 2 to 4% of reducing sugars [28].

Irradiation with microwaves singly or in combination of

alkali treatment significantly accelerated the hydrolysis rate.

Chemical Pretreatment

Lignocellulosic biomass contains three main constituent:

cellulose, hemicellulose and lignin. The chemical treat-

ments for lignocellulosic biomass are so designed that they

can open up cellulose from lignin and hemicellulose cas-

ing, destroy the cellulose crystalline structure and increase

the pore size and surface area of cellulose. Considerable

attention has been devoted to agents that are responsible for

swelling of cellulose and disrupt the highly ordered crys-

talline structure. This can happen in two ways:

(1) Intercrystalline swelling caused by uptake of water

between the crystalline units, which causes a revers-

ible volume change of up to about 30%.

(2) Intracellular swelling requires a chemical agent that is

capable of breaking the hydrogen bonds of the

cellulose, which leads to unlimited swelling or

complete solution of the cellulose.

The cellulose dissolving agents fall into four groups:

strong mineral acids such as H2SO4 and H3PO4, quaternary

ammonium bases, transitional metal complexes (e.g.

CMCS) and organic solvents. Sodium hydroxide is used as

an intercrystalline swelling agent for both crystalline and

amorphous cellulose. Similarly amines and anhydrous

ammonia have been used for intercrystalline swelling.

Concentrated sulphuric acid, fuming hydrochloric acid and

metal chelating solvents are used for intracrystalline

Fig. 1 Simultaneous saccharification and Co-fermentation-SSCF

Proc. Natl. Acad. Sci. Sect. A Phys. Sci. (January–March 2012) 82(1):59–69 61

123

swelling. All the chemical pretreatment methods used for

improving enzymatic digestibility generate Hydrolysates

which is a mixture of sugars (e.g. hexose and pentose) and

lignin. The hydrolysates obtained from pretreatment pro-

cesses require detoxification, because the microorganisms

poorly withstand the inhibitory environment of lig-

nocellulose-hydrolysates [29–31]. This factor increases the

cost of pentose (xylose) fermentation. This is the main

disadvantage of chemical pretreatment.

Alkali Pretreatment

Among all the chemical treatment processes, alkali treat-

ment using bases like sodium hydroxide is most widely

used to enhance in vitro both digestibility and rates of

enzymatic hydrolysis of the lignocellulose. The success of

this method primarily depends on the amount of lignin in

the biomass [32]. The alkali treatment causes swelling,

decreases the degree of polymerization and crystallinity,

and lignin content and increases the surface area of the

substrate. The mechanism involved is the saponification of

intermolecular ester bonds cross-linking the hemicellulose

and lignin. The extent of hydrolysis increases with increase

in concentration of NaOH for pretreatment. The optimum

level range is between 5 and 8 g NaOH/100 g substrate

[33]. The digestibility of Hardwoods treated with NaOH

increases from 14 to 55% at the same time the lignin

content decreases from 24–55 to 20%. No effect of dilute

NaOH pretreatment was observed for softwoods with lig-

nin content greater than 26%.

Ammonia has also been used for pretreatment but in

general the enhancement obtained is less than that of sodium

hydroxide treatment. The benefit of ammonia in pretreat-

ment includes breakage of glucuronic acid ester cross-links,

solubilization of lignin, and disruption of crystalline struc-

ture, swelling and increase in accessible surface area of

cellulose. Wheat straw treated with 50% ammonium

hydroxide showed 20% delignification, with three fold

increase in rate of hydrolysis. An improved pretreatment

method involving two step is reported by Cheng [34]. In the

first step, steeping the lignocellulosic biomass in aqueous

ammonia at ambient temperature removed the lignin, acetate

and extractives. Second step involves dilute acid pretreat-

ment that hydrolyzed the hemicellulose fraction. The

advantage of this method is step by step removal of lignin,

hemicellulose and cellulose from biomass. It removes

80–90% of lignin in the steeping step.

Autohydrolysis/Hot-Water

In this method hot-water at around 150�C, reacts with

lignocellulosic biomass and forms acids from the solubi-

lization of acidic components in hemicellulose, such as

acetic acid, formic acid and glucuronic acid [35]. Under

hot-water treatment, hydronium ion first causes xylan

depolymerization and cleavage of the acetyl group. Then

autohydrolysis reaction takes place in which acetyl group

catalyses the hydrolysis of hemicellulose. Glucosidic

linkages in hemicellulose and beta-ether linkages in lignin

are catalyzed by acetic acid formed at high temperature

from acetyl group present in hemicellulose [9].

Steam Explosion

One of the most commonly used physiochemical pretreat-

ment method is steam explosion. In this process, ligno-

cellulosic material is treated with steam under high

pressure and temperature, followed by quick release of

pressure, causing the biomass to undergo an explosion and

shatter the structure in a popcorn-like effect. The disad-

vantage of the process is, it does not always break down all

the lignin, requires small particle size and produces com-

pounds which can inhibit subsequent fermentation. In this

method wood chips are treated with saturated steam at

210–300�C and 500–1000 psi in a reactor, usually called a

gun reactor. After a few minutes, reaction is frozen and by

sudden decompression to atmospheric pressure, the wood

is exploded into a fine powder. Enzymatic hydrolysis of

this material gives 80% of the theoretical glucose. Steam

explosion of softwood chips at 210�C and 4 min achieved a

maximum theoretical sugar yield of 50% [28].

Acid Pretreatment

Acid hydrolysis is the most prevalent pretreatment method

as it can handle a wide range of feedstocks. For higher acid

concentrations it can be carried out at temperature as low

as 30�C. For concentrated sulphuric acid, intercrystalline

swelling occurs in the concentration range of 62–70%. The

reprecipitated cellulose is then easily hydrolyzed by acid or

enzyme with high conversions. Walseth [36] used 85%

H3PO4 as a cellulose solvent and observed a tenfold

increase in extent of conversion by cellulase. H3PO4 causes

less degradation of cellulose than the other acids. A novel

lignocellulose fractionation method using concentrated

H3PO4 or acetone was recently reported by Zang et al. [37].

The main features of this method are moderate reaction

conditions (50�C and 1 atm), releases highly reactive

amorphous cellulose, hemicellulose sugars, lignin and

acetic acid and cost effective reagent recycling.

Enzymatic Hydrolysis

Cellulose is a long chain of glucose molecule, linked to one

another only with b-1-4 glycosidic bonds. The simplicity of


123

the cellulosic structure, using repeated identical bonds,

means that only a small number of enzymes are required to

degrade this material. Hydrolysis of the substrate means the

cleaving of a molecule of the substrate by adding a water

molecule. A mixture of glucose, fructose and pentose is

obtained at the end of the reaction. This reaction is catalyzed

by enzymes and it has many advantages as it can perform at

very mild conditions such as pH 4.8 and temperature

318–323 K. The yield and the maintenance cost are low

compared to alkaline and acid hydrolysis due to non-corro-

sion problems [3]. Enzymatic hydrolysis is viewed as the

most cost effective method for ethanol production in the long

run [34]. Enzymes are biological catalysts that are protein

molecules with molecular weight *106. They are produced

by living cells (animal, plant and microorganism) and are

absolutely essential as catalysts in biochemical reactions.

Enzymatic hydrolysis of cellulose is carried out by

cellulase enzyme which is highly specific. The products of

hydrolysis are usually reducing sugars including glucose.

Both bacteria and fungi can produce cellulase for the

hydrolysis of cellulosic material. These microorganisms

can be aerobic or anaerobic, mesophillic or thermophillic.

Bacteria belonging to Clostridium, Cellulomonas, Bacillus,

Thermomonospora, Ruminococcus, Bacteriodes, Erwinia,

Acetovibrio, Microbispora and Streptomyces can produce

cellulase [38]. Cellulolytic bacteria such as Clostridium

thermocellum and Bacteroides cellulosolvens produce cel-

lulases with high specific activity. Cellulases are usually a

mixture of several enzymes. The three major groups of

cellulases involved in hydrolysis are:

(1) Endoglucanase (EG, endo-1,4-D-glucanohydralase, or

EC 3.2.1.4) which attacks regions of low crystallinity

in the cellulose fiber, creating free chain ends.

(2) Exoglucanase or cellobiohydrolase (CBH, 1,4-b-D-

glucan cellobiohydrolase, or EC 3.2.1.91) which

degrades the molecule further by removing the

Cellobiose units from the free chain ends.

(3) b-glucosidase (EC 3.2.1.21) which hydrolyzes Cello-

biose to produce glucose.

During the enzymatic hydrolysis, cellulose is degraded

by the cellulases to reducing sugars that can be fermented

by yeasts or bacteria to ethanol [39].

Kinetics of Enzymatic Hydrolysis of Cellulose

The mechanism of binding of three major component of

cellulase is considered to happen in the following way:

Endoglucanase (E1) binds to cellulose molecule (Gi) to

form enzyme-substrate complex. It cuts the long polymeric

chains to produce cellobiose (G2) and glucose (G1). E1 is

inhibited by both cellobiose and glucose either competi-

tively or noncompetitively.

Okazaki and Moo-Young have first introduced the

concept and later it was revised by Zhang [40] and Lynd

[41]. The model is slightly modified to include a ternary

mixture of enzymes acting on cellulose molecules. The

activity of Endoglucanase is described as

Gi þ EG1, EG1:Gi�!kEG1

EG1þ Gi�j þ Gj: ð1Þ

Exoglucanase (CBH) forms a complex with the

nonreducing ends of the cellulose molecules and

produces cellobiose. The reaction is given by

Gi þ CBH , CBH:Gi�!kCBH

CBH þ Gi�2 þ G2 ð2Þ

(1) Finally b-glucosidase (G2) produces glucose from

cellobiose; inhibited by glucose competitively or

noncompetitively. It is given by

G2 �!kb�glucosidase

2G1 ð3Þ

All three enzymes E1, CBH and b-glucosidase act

simultaneously. Using Michaelis–Menten kinetics, we have

a series of reactions for E1 attack of a cellulose molecule

(Gi) of degree of polymerization i (DPi C 3). Assuming

non competitive inhibition,

E1þ Gi ,ks1

ks�1

E1 � Gi�!k1

E1þ Gj þ Gi�j ð4:1Þ

E1þ G2 ,kc1

kc�1

E1 � G2 ð4:2Þ

E1 � Gi þ G1 ,kG1

kG�1

E1 � Gi � G1 ð4:3Þ

E1þ G1 ,kG1

kG�1

E1 � G1 ð4:4Þ

E1 � G1 þ Gi ,ks1

ks�1

E1 � G1 � Gi ð4:5Þ

E1 � Gi þ G2 ,kc1

kc�1

E1 � G1 � G2 ð4:6Þ

E1 � G2 þ Gi ,ks1

ks�1

E1 � Gi � G2 ð4:7Þ

Note that k1, ks1, ks-1, kG1, kG-1, kc1, kc-1 are rate

constants for E1 (independent of degree of polymerization

i) in the reactions listed. Gj and Gi-j are cellulose chains

with degree of polymerization DPj and DPi-j, respectively.

The equation of continuity for species Gi from the above

set of equation is obtained as (Aniket and Chakraborty

[42])


123

d

dtGi½ � ¼ � ks1ði� 1Þ E½ �f Gi½ � þ ks�1 E1Gi½ �

þ 2k1

Xn

p¼iþ1

E1Gp

� �

p� 1� ks1ði� 1Þ E1G1½ � Gi½ �

þ ks�1 E1GiG1½ � � ks1ði� 1Þ E1G2½ � Gi½ �þ ks�1 E1GiG2½ � ð5Þ

Here, [E]f is the concentration of free E1. The

probability of breaking of chain of length p into a

smaller fraction i, is given by the term 2/p-1. It is

assumed that the rate and probability of breaking of chain

into a smaller chain of a particular size are equal with any

other size. The factor two accounts for two cases, when i is

broken down into the same fractions j and (i-j) and

correspondingly when (i-j) & j. The term (i-j) represents

the number of bonds that are available for breakage in

cellulosic chain of size i. The bonds are assumed to have

equal probability for degradation by endoglucanase. Total

concentration of endoglucanase is given by

E1½ � ¼ E1½ �fþX1

i¼1

E1Gi½ � þX1

i¼3

E1GiG1½ � þX1

3

E1GiG2½ �:

ð6Þ

Assuming quasi steady state condition for intermediate

species,

d

dtE1Gi½ �ð Þ ¼ ks1ði� 1Þ E1½ �f Gi½ �

þ ðks�1 þ k1Þ E1Gi½ � ¼ 0;ð7Þ

d

dtE1G1½ �ð Þ ¼ kG1

E1½ �f G1½ � þ kG�1E1G1½ � ¼ 0; ð8Þ

d

dtE1G2½ �ð Þ ¼ kC1

E1½ �f G2½ � þ kC�1E1G2½ � ¼ 0; ð9Þ

d

dtE1GiG1½ �ð Þ ¼ kG1

E1Gi½ � G1½ � þ kG�1E1GiG1½ �;

¼ ks1ði� 1Þ E1G1½ � Gi½ � � ks�1 E1GiG1½ � ¼ 0;

ð10Þd

dtE1GiG2½ �ð Þ ¼ kG1

E1Gi½ � G2½ � þ kC�1E1GiG2½ �;

¼ ks1ði� 1Þ E1G2½ � Gi½ � � ks�1 E1GiG2½ � ¼ 0:

ð11Þ

Rearranging Eq. 5–11, the rate equation for

endoglucanase is obtained as: For i [ 2,

d

dtGi½ �ð Þ ¼

k1 E1½ � 2P1

p¼iþ1

Gp

� ��ði�1Þ Gi½ �

!

KM1þP1

i¼3

i�1ð Þ Gi½ �f g� �

1þ G1½ �=KG1þ G2½ �=KC1

� �

ð12Þ

where KM1 ¼ ks�1þk1

ks1; KG1 ¼ kG�1

kG1and KC1 ¼ kC�1

kC1:

Similarly, for cellobiohydrolase (CBH), for i [ 2, the

rate equation is given by

d

dtGi½ �ð Þ¼ k2 E2½ � Giþ2½ � � Gi½ �ð Þ

KM2þP1

i¼3

Gi½ �f g� �

1þ G1½ �=KG2þ G2½ �=KC2

� � ;

ð13Þ

where KM2 is the Michaelis constant of CBH, and KG2 and

KC2 are dissociation constants between CBH and glucose

and cellobiose, respectively. Total rate of change of

cellulose by endo- and exo-component of cellulase is the

summation of the two rate expressions given by Eqs. 12

and 13. Therefore,

d

dtGi½ �ð Þ ¼

k1 E1½ � 2P1

p¼iþ1

Gp

� �� ði� 1Þ Gi½ �

!

KM1 þP1

i¼3

i� 1ð Þ Gi½ �f g� �

1þ ½G1�½KG1� þ

½G2�½KC1�

� �

þ k2 E2½ � Giþ2½ � � Gi½ �ð Þ

KM2 þP1

i¼3

Gi½ �f g� �

1þ ½G1�½KG2� þ

½G2�½KC2�

� � ;

for i [ 2:

ð14Þ

For i [ 2. For cellobiose (i = 2),

d

dtGi½ �ð Þ ¼

k1 E1½ �P1

i¼3

Gi½ ��

KM1 þP1

i¼3

i� 1ð Þ Gi½ �f g� �

1þ ½G1�½KG1� þ

½G2�½KC1�

� �

þ k2 E3½ � G4½ � � G2½ �ð Þ

KM2 þP1

i¼3

Gi½ �f g� �

1þ ½G1�½KG2� þ

½G2�½KC2�

� �

� k3 E3½ � G2½ �KM3 þ G2½ �ð Þ 1þ G1½ �=KG3

� � ;

ð15Þ

and for glucose (i = 1),

d

dtGi½ �ð Þ ¼

k1 E1½ �P1

i¼3

Gi½ ��

KM1 þP1

i¼3

i� 1ð Þ Gi½ �f g� �

1þ G1½ �=KG1þ G2½ �=KC1

� �

þ k2 E2½ � G4½ �� G2½ �ð Þ

KM2þP1

i¼3

Gi½ �f g� �

1þ G1½ �=KG2þ G2½ �=KC2

� �

þ k3 E3½ � G2½ �KM3 þ G2½ �ð Þ 1þ G1½ �=KG3

� � :

ð16Þ


123

Assuming that each association of cellulases with

cellulose fragments is the same, the rate of change of

number average degree of polymerization is given by

d

dt

X1

i¼1

Gi½ � !

¼ N�d

dt

1

DP

� �;

¼k1 E1½ �

P1

i¼3

i� 1ð Þ Gi½ ��

KM1 þP1

i¼3

i� 1ð Þ Gi½ �f g� �

1þ ½G1�½KG1� þ

½G2�½KC1�

� �

þk2 E2½ �

P1

i¼3

G3½ ��

KM2 þP1

i¼3

Gi½ �f g� �

1þ G1½ �=KG2þ G2½ �=KC2

� �

þ k3 E3½ � G2½ �KM3 þ G2½ �ð Þ 1þ G1½ �=KG3

� � ;

¼ k1 E1½ �Sw

KM1 þ Swð ÞIh1

þ k2 E2½ �SM

KM2 þ SMð ÞIh2

þ k3 E3½ � G2½ �KM3 þ C2½ �ð ÞIh3

ð17Þ

Here, DP ¼P1

i¼1i Gi½ �ð ÞP1

i¼1Gi½ �¼ N�P1

i¼1Gi½ �; and N� ¼

P1

i¼1

i Gi½ �ð Þ ¼

constant: Sw ¼P1

i¼1

i� 1ð Þ Gi½ �f g and

SM ¼X1

i¼3

Gi½ �;

Ih1 ¼ 1þ G1½ �=KG1þ G2½ �=KC1

� �;

Ih2 ¼ 1þ G1½ �=KG2þ G2½ �=KC2

� �;

Ih3 ¼ 1þ G1½ �=KG3

� �

Experimental

Enzymatic reactions can be carried out in batch, continuous

or loop reactors. For reactions to happen, a co-operative

action is needed between the so called endoglucanase,

exoglucanase and b-glucosidase. Crystalline and amor-

phous or treated substrates show different affinity to bind

themselves to enzymes and thereby yield products that

depends on the crystallinity index, Degree of Polymeriza-

tion (DP), particle size etc. Cellulosic substrate mixed in a

buffer solution can be stirred for 1–2 h in a shaker before

allowing the enzymes to react. Cellulase extracted from T.

reesei having molecular weight in the range of

48,000–52,000 Da has to be maintained in the optimal

temperature range of 40–50�C to be in active state

throughout the hydrolytic reactions. The total reducing

sugar can be measured using DNS assay prepared by using

standard procedures given in the literature. It can also be

measured using refractometer which measures total

concentration of reducing sugars in the solution based on

the solution refractive index [43]. Recently, enzymatic

reactions in ionic liquid are spotted and recognized for

enzymatic biotransformation. These solvents can be

designed with different combination of cations and anions

allowing the possibility of tailoring reaction solvents with

highly specific properties beneficial for the particular

reaction to happen [44].

Some studies have suggested that the substrates pre-

treated with ionic liquids achieve faster saccharification.

Substrates are pretreated in ionic liquids over a period of

time and regenerated substrates are enzymatically reacted.

Substrates treated with Ionic Liquids are found to have

58–75% lower crystallinity than the original substrate [15].

Since the regenerated substrate has more accessible surface

area, a better adsorption of enzymes on the substrate pro-

tects enzymes from thermal denaturation and thus allowing

reactions to happen at slightly higher temperature

(*60�C), and are much faster. Reactions can be completed

in 24 h at 60�C instead of 48 h at 50�C. 1-butyl-3-meth-

ylimidazolium chloride ([BMIM]Cl), 1-allyl-3-methylimi-

dazolium chloride ([AMIM]Cl), ([EMIM] OAc) are

important ionic liquids [45].

Fermentation

Biological Fermentation

Many organisms grow without using the electron transport

chain. The generation of energy without the electron

transport chain is called fermentation. It is basically a

chemical transformation in which fermentable sugars,

especially glucose, can be converted to other valuable

products such as fructose, ethanol, numerous organic acids

and other by products through biochemical conversion by

microorganism. The degradation of carbohydrates by

microorganisms is followed by glucolytic or Embden-

Myerhof-Parnas pathways. In the ethanol production, car-

bohydrates are reduced to pyruvate with the aid of nico-

tinamide adenine dinucleotide (NADH); ethanol is the end

product [46–48].

Production of ethanol from all the three discussed bio-

masses e.g. lignocellulose, sugar cane and corn are dif-

ferent since the solubilization of carbohydrate from

lignocellulosic biomass is very difficult compared to corn

and sugar cane [7]. The processes can be classified as,

SHF

In this particular process enzyme production, enzymatic

hydrolysis and fermentation is performed sequentially in


123

separate vessels. The separated process is called Separate

Hydrolysis and Fermentation.

DMC

Direct Microbial Conversion combines all three major

processes (enzyme production, hydrolysis and fermenta-

tion) in one step. The process is cost effective because of

less number of unit operations, savings of large expenses

incurred due to sophisticated pretreatment techniques [49,

50]. However yield of ethanol is very low; forms several

metabolic by-products and the organisms usually suffer

from low ethanol tolerance.

SSF

Simultaneous saccharification and fermentation combines

hydrolysis of the substrate and fermentation in one step

[46, 51]. At the end of SSF very low levels of cellobiose

and glucose can be seen in the reactor because of their

immediate consumption by microorganisms. This reduces

the cellulase inhibition, which in turn increases sugar

production rates, yields and concentrations and reduces

enzyme loading requirements. It can be seen that the SSF

process offers a lot of advantages in comparison with other

processes mentioned above. So a detailed discussion about

this process is given below.

Simultaneous Saccharification and Fermentation (SSF)

SSF is a process in which both saccharification and fer-

mentation are carried out simultaneously in one vessel.

This process was first introduced in 1977, and it gives high

yield and rapid rates for ethanol production [4]. For ethanol

production from cellulose, the glucose released by cellulase

enzyme (from T reesei) is simultaneously converted to the

end product by the microorganism. Substrate inhibition on

enzyme is thus prevented. Other advantages are the

potential for use of low enzyme loadings and reduced

potential of microbial contamination. Since the optimum

temperature for both saccharification and fermentation is

different, usually thermotolerant yeast such as Kluyver-

omyces marxianus is used.

Bioconversion of lignocellulosic material involves the

saccharification of both the cellulose and hemicellulose.

Most microorganisms converts glucose to ethanol but this

is not the case for xylose or mannose which is mainly

found in hemicellulose. Fermentation of pentose sugar

xylose into ethanol is essential for the economical process

development.

Hydrogenation

Traditional microorganisms used for ethanol fermentation,

Saccharomyces cerevisiae and Zymomonas mobilis, do not

metabolize pentose.

Only in last few decades researchers have been able to

efficiently ferment pentose into ethanol either by using xylose-

fermenting yeast or recombinant microorganisms [52, 53].

One of the best microorganisms currently available for

fermentation of mixed sugar streams is ethanologenic

Escherichia coli strain K011 [54]. This strain ferments glu-

cose, xylose and arabinose with good ethanol yield and

productivity, and has a high tolerance of common hydroly-

sate inhibitors, e.g. acetate [55]. Two strains of the recom-

binant E. Coli strains have been studied in detail: strain

ATCC 11303 (pLOI297), in which the foreign genetic ele-

ments are plasmid-borne and KO11 in which the foreign

genetic elements are integrated into the host chromosome.

An ethanologenic xylose fermenting Z. mobilis strain has

also been developed, but detailed information on fermenta-

tion performance characteristics is not yet available [56].

In terms of metabolism, xylose is transported across the

cell membrane where it is converted to xylulose-5-phos-

phate (X-5-P). It is then converted to pyruvate by the way

of pentose phosphate (PP) and Embden-Meyerhof-Parnas

(EMP) or Entner–Doudoroff (ED) pathways, as shown in

Fig. 2. In PP cycle, X-5-P is metabolized to glucolytic

intermediates such as glyceraldehyde-3-phosphate and

fructose-6-phosphate. These compounds are then converted

to pyruvate via EMP (or ED) pathway. The pyruvate is then

converted to ethanol via an acetaldehyde intermediate by

the sequential action of pyruvate decarboxylase and alco-

hol dehydrogenase (ADH) enzymes [5, 46]. In this scheme,

a minimum of 3 mol of xylose are required to produce

5 mol of ethanol. The theoretical yield according to this

stoichiometry is 0.51 g-ethanol/g-xylose. The hydrolysis

and fermentation reactions are:

C12H22O11 þ H2O �!Maltase in yeast2C6H12O6;

C6H12O6 �!Zymase in yeast2C2H5OH þ 2CO2:

Chemical Fermentation

In chemical fermentation, the hydrolysed product i.e. glu-

cose is treated chemically and the steps involved are

chemical treatment, extraction using solvent and dehydra-

tion. 5-HMH, Levulinic acid, Tetrahydrofurfuryl alcohol

and 2-furaldehyde are the main products. Chemical fer-

mentation can be carried out in two ways to convert glu-

cose to final product. These are (i) hydrogenation (ii)

dehydration.


123

In hydrogenation, glucose in ionic liquid is taken and

catalyzed with acids such as sulphuric acid or HCl. The

reaction is conducted in a high pressure reactor where

pressure is maintained at 920 psi and temperature is kept at

120�C. The total reaction time is 12–24 h under the

maintained conditions.

As the reaction progresses glucose gets reduced and

forms sorbitol. Sorbitol then loses three molecules and

forms five membered ring structures, i.e. 5-HMF. The

mixture is then extracted using suitable solvent. Raney

Nickel gave maximum yield of 5-HMF. The hydrogenation

reaction is as given in Fig. 3.

Dehydration

The second process for glucose conversion to useful

products is dehydration. Unlike in the case of hydrogena-

tion, dehydration does not require adverse conditions for

the reaction to happen. In this, a mixture of glucose, ionic

liquid and catalyst is prepared and allowed to react at 90�C

under atmospheric pressure (see Fig. 4).

Further, 5-HMF in presence of catalyst and hydrogen

gas produces 2,5-di methyl furan which is a gasoline

derivative [57]. Reaction is continued for 3 h and the final

mixture is extracted using suitable solvents such as diethyl

ether or acetone. In this 5-HMF is obtained directly unlike

the previous one where sorbitol gets converted to 5-HMF.

In some cases byproduct 5(chloro) methyl furfural is

obtained because of the chloride ion present in the ionic

liquid which replaces hydroxide ion present in 5-HMF.

Conclusions

This paper reviews the various processes and techniques

involved in the production of bioethanol from cellulosic

and lignocellulosic biomass. The different pretreatment

methods for obtaining cellulosic substrate from

Fig. 2 The Embden-Meyerhof-Parnas pathway of anaerobic conver-

sion of glucose to ethanol

Hydrogenation:

Glucose Sorbitol 5-HMF 5-MF

Fig. 3 Products formed by hydrogenation of glucose in ionic liquid

H2SO4

-3H2O

Glucose 5-HMF 5-(chloro) methyl

Fig. 4 Products formed from dehydration of glucose in ionic liquid


123

lignocellulosic biomass are presented, followed by the

experimental and theoretical analysis of enzymatic hydro-

lysis for the depolymerization of cellulose to glucose, and

the subsequent biological/chemical fermentation of glucose

to produce fuel derivatives.

The substrate (cellulose) can be replaced by other pre-

treated substrates such as PASC, BC, CMC, or filter paper

and cotton for faster and better yield.

The experiments can be extended to demonstrate the

individual contribution of cellulase components and syn-

ergistic studies can be done. Chemical fermentation pro-

cesses are carried out in presence of ionic liquids because

of the unique characteristics the latter possesses. Detailed

mathematical modeling is used to quantify the detailed

kinetics of the enzymatic hydrolysis, and the rate expres-

sion for cellulose, cellobiose and glucose for non-com-

petitive inhibition [5, 42, 58].

While the advantages of replacing fossil fuels by bio-

ethanol have been emphasized earlier in this paper, we

need to point out its drawbacks too. The major problem

results from ethanol’s solubility in water, which leads to a

substantial energy cost for distillation and difficulties in its

transportation via pipelines. Recently, bio-butanol is being

considered as an alternative to bioethanol because of the

former’s low vapor pressure as well as its ability to

dehydrate spontaneously at less than 10% solution and to

reduce the risks of explosions when added to gasoline-

ethanol mixtures. The coming years may see the replace-

ment of bioethanol by other types of cellulosic fuels such

as bio-butanol, which could also be produced by fermen-

tation of sugars from biomass.

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