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

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Progress in Energy and Combustion Science

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Review

Trends in bioconversion of lignocellulose: Biofuels, platform chemicals

& biorefinery concept
Vishnu Menon, Mala Rao*
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411-008, India

a r t i c l e i n f o a b s t r a c t

Article history: Bioconversion of renewable lignocellulosic biomass to biofuel and value added products are globally
Received 3 August 2011 gaining significant prominence. Market forces demonstrate a drive towards products benign to natural
Accepted 14 February 2012 environment increasing the importance of renewable materials. The development of second generation
Available online 17 March 2012
bioethanol from lignocellulosic biomass serves many advantages from both energy and environmental
point of views. Biomass an inexpensive feedstock considered sustainable and renewable, is an option
Keywords:
with the potential to replace a wide diversity of fossil based products within the energy sector; heat,
Lignocellulose
power, fuels, materials and chemicals. Lignocellulose is a major structural component of woody and non-
Pre-treatment
Saccharifying enzymes
woody plants and consists of cellulose, hemicellulose and lignin. The effective utilization of all the three
Consolidated biomass processing components would play a significant role in the economic viability of cellulosic ethanol. Biomass
Lifecycle assessment conversion process involves five major steps, choice of suitable biomass, effective pretreatment,
Value-added products production of saccharolytic enzymes-cellulases and hemicellulases, fermentation of hexoses and
pentoses and downstream processing. Within the context of production of fuels from biomass,
pretreatment has come to denote processes by which cellulosic biomass is made amenable to the action
of hydrolytic enzymes. The limited effectiveness of current enzymatic process on lignocellulose is
thought to be due to the relative difficulties in pretreating the feedstocks. The present review is
a comprehensive state of the art describing the advancement in recent pretreaments, metabolic engi-
neering approaches with special emphasis on the latest developments in consolidated biomass pro-
cessing, current global scenario of bioethanol pilot plants and biorefinery concept for the production of
biofuels and bioproducts.
Ó 2012 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523
2. Structure of lignocellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524
3. Selected pretreatment categories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525
3.1. Physical pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525
3.2. Physico-chemical pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526
3.2.1. Steam explosion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526
3.2.2. Ammonia fiber explosion (AFEX) and ammonia recycle percolation (ARP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526
3.2.3. Microwave-chemical pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527
3.2.4. Liquid-hot water pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527
3.3. Chemical pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527
3.3.1. Acid pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527
3.3.2. Alkaline pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528
3.3.3. Green solvents (ionic liquids) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528
3.4. Biological pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529
3.5. Techno-economics of pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529

* Corresponding author. Tel.: þ91 20 25902228; fax: þ91 20 25902648.

E-mail address: mb.rao@ncl.res.in (M. Rao).

0360-1285/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.pecs.2012.02.002
 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550 523

4. Biocatalytic valorization of lignocellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529

4.1. Saccharification systems for cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529
4.2. Saccharification systems for hemicelluloses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531
5. Fermentation strategies & consolidated biomass processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532
5.1. Future economic performance of hydrolysis process concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535
6. Metabolic engineering approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 536
7. Alternative liquid fuels - biobutanol and 2,3-butanediol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538
8. Biorefinery perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 539
9. Lifecycle assessment of lignocellulosic ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 540
10. Current global status of lignocellulosic ethanol industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542
11. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543

1. Introduction various commercial and post-consumer waste and by-product

streams. According to the IEA report for the assesemnet of avail-
The development of second generation biofuels from lignocel- able residues in 2030, it is concluded that 10% of global residues
lulosic biomass has many advantages from energy and environ- could then yield around 155 billion lge (5.2 EJ) lignocellulosic-
mental concerns. First generation biofuel derived mainly from food ethanol or roughly 4.1% of the projected transport fuel demand in
crops creates many problems ranging from net energy losses to 2030 and 25% of global residues converted to either ethanol, diesel
greenhouse gas emission to increased food prices. The recent or syngas could contribute 385e554 billion lge (13e23.3 EJ) globally
imbalance in oil market and hike in fuel costs have initiated a global [2,3]. It is well documented that cellulosic ethanol offers greater
challenge for biofuel production from lignocelluloses. Efficient environmental benefits and sustainability; however the concern is
conversion of lignocellulosic materials to ethanol and value added the economic viability of the process. The conversion process relies
biochemicals are still to day a challenging proposition. In contrast to heavily on major technological innovations centered on effective
fossil fuels, cellulosic ethanol produced through fermentation of and low cost enzymes, feedstocks and efficient process design.
sugars is a renewable energy source. Biomass is a primary source of Globally large amount of agricultural residues are produced,
food, fodder and fibre and as a bioenergy source provides about most of which is burnt as waste disposal and small amount is used
10.2% (50.3 EJ/yr) of the annual global total primary energy supply for mulching, for fuel or as fodder. Three types of energy can be
(TPES) from a wide variety of biomass sources [1]. More than 80% of produced from lignocellulosic residues by thermochemical or
biomass feedstocks used for energy are derived from wood and biochemical processing, liquid fuels such as ethanol or pyrolysis oil,
shrubs. The remaining bioenergy feedstocks are from the agricul- gaseous fuels such as biogas (methane) and electricity (Fig. 1).
tural sector (energy crops, residues and by-products) and from Biomass is used with varying degrees of energy efficiency in various

Fig. 1. Thermochemical and biochemical processing of lignocellulosic biomass.

524 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550

sectors. Low-efficiency traditional biomass such as wood, straws, Table 1

dung and other manures are used for cooking, lighting and space Composition of representative lignocellulosic feedstocks.

heating, generally by the poorer populations in developing coun- Feedstocks Carbohydrate composition (% dry wt) References
tries. High-efficiency modern bioenergy uses more convenient Cellulose Hemicellulose Lignin
solids, liquids and gases as secondary energy carriers to generate
Barley hull 34 36 19 [12]
heat, electricity, combined heat and power and transport fuels for Barley straw 36e43 24e33 6.3e9.8 [13,14]
various sectors. High energy efficiency biomass conversion is found Bamboo 49e50 18e20 23 [15,16]
typically in the industry sector associated with the pulp and paper Banana waste 13 15 14 [17]
Corn cob 32.3e45.6 39.8 6.7e13.9 [18,19]
industry, forest products, food and chemicals [4]. The biomass
Corn stover 35.1e39.5 20.7e24.6 11.0e19.1 [20]
production costs can be combined with technological and Cotton 85e95 5e15 0 [21]
economic data for related logistic systems and conversion tech- Cotton stalk 31 11 30 [22]
nologies to derive market potentials at the level of secondary Coffee pulp 33.7e36.9 44.2e47.5 15.6e19.1 [23]
energy carriers such as bioelectricity and biofuels for transport Douglas fir 35e48 20e22 15e21 [24]
Eucalyptus 45e51 11e18 29 [16,25]
[5e7]. There are many favorable factors for the implementation of
Hardwood stems 40e55 24e40 18e25 [26,27]
lignocellulose based biofuels. Geographical location of feedstock Rice straw 29.2e34.7 23e25.9 17e19 [28,29]
sources are more evenly distributed than the fossil sources Rice husk 28.7e35.6 11.96e29.3 15.4e20 [30,31]
enabling to a large extent the security of supply. The Intergovern- Wheat straw 35e39 22e30 12e16 [29,32]
mental Panel on Climate Change in its Special Report on Emission Wheat bran 10.5e14.8 35.5e39.2 8.3e12.5 [33]
Grasses 25e40 25e50 10e30 [34,35]
Scenarios: A1, A2, B1 and B2 have developed different future land- Newspaper 40e55 24e39 18e30 [26]
use patterns. Acording to the report the largest contribution to Sugarcane bagasse 25e45 28e32 15e25 [16,36]
technical potential could come from energy crops on arable land, Sugarcane tops 35 32 14 [37]
assuming that efficiency improvements in agriculture could out- Pine 42e49 13e25 23e29 [25]
Poplar wood 45e51 25e28 10e21 [38]
pace food demand so as to avoid increased pressure on forests and
Olive tree biomass 25.2 15.8 19.1 [39]
nature areas. A range of 20e400 EJ/yr is presented for 2050, with Jute fibres 45e53 18e21 21e26 [40]
a best estimate of 250 EJ/yr and 240 to 850 EJ/yr is presented for Switchgrass 35e40 25e30 15e20 [26]
2100 [8]. The reforestation schemes can contribute significantly for Grasses 25e40 25e50 10e30 [26,27]
biomass production (8e110 EJ/yr). Although the low-yielding Winter rye 29e30 22e26 16.1 [41]
Oilseed rape 27.3 20.5 14.2 [41]
biomass production is more expensive, competition with food Softwood stem 45e50 24e40 18e25 [26,27]
production is almost absent and various co-benefits, such as Oat straw 31e35 20e26 10e15 [14]
regeneration of soils and carbon storage, improved water retention Nut shells 25e30 22e28 30e40 [42]
and protection from further erosion may also offset part of the Sorghum straw 32e35 24e27 15e21 [43,44]
Tamarind kernel 10e15 55e65 [45]
establishment costs. The potential of low-productive land is
e
powder
negligible and the rest land area is assumed to partly available, Water hyacinth 18.2e22.1 48.7e50.1 3.5e5.4 [46,47]
resulting in ranges of the geographical potential from 35 to 245 EJ/
yr for the year 2050 and to 265 EJ/yr in 2100 [8]. Biofuel production
from lignocellulosic feedstocks may even help to a certain extent to bioethanol industry, lifecycle assessment of lignocellulosic ethanol,
combat the unemployment status of rural areas. The greenhouse biorefinery concept for the development of sustainable and bio-
gas impacts of lignocellulosic ethanol production based on lifecycle based sectors (biofuels and bioproducts) and recent advances in
analyses undertaken by the US environmental protection agency. alternative biofuels such as biobutanol and 2,3-butanediol.
These analysis show lignocellulosic ethanol generates 91% less
greenhouse gases than fossil-based petrol or diesel in transport 2. Structure of lignocellulose
applications, compared with just 22% for corn-based ethanol [9].
However the assumed benefit is now under discussions, especially Lignocellulosics are the most abundant source of unutilized
due to emissions from land-use changes (LUC) [10,11]. biomass and their availability does not necessarily impact land use.
The compositions of various lignocellulosic feedstocks are Biomass in general consists of 40e50% cellulose, 25e30% hemi-
illustrated in Table 1. Biomass conversion process involves five cellulose and 15e20% lignin and other extractable components [48].
major steps, choice of suitable biomass, effective pretreatment, The effective utilization of all the three components would play
production of saccharolytic enzymes such as cellulases and hemi- a significant role in economic viability of the cellulose to ethanol
cellulases along with the accessory enzymes, fermentation of process. In nature except in cotton bolls, cellulose fibres are
hexoses and pentoses and the downstream processing. It is neces- embedded in a matrix of other structural biopolymers, primarily
sary to understand the complex structure of lignocellulose to design hemicellulose and lignin. Cellulose is a linear syndiotactic (alter-
a suitable pretreatment. Within the context of production of fuels nating spatial arrangement of the side chains) polymer of glucose
from biomass, pretreatment has come to denote processes by which linked together by b-(l / 4)-glycosidic bonds whereas hemi-
cellulosic biomass is made amenable to the action of hydrolytic cellulose is a branched heteropolymer of D-xylose, L-arabinose, D-
enzymes. Research attention has been focused extensively for over mannose, D-glucose, D-galactose and D-glucuronic acid. Lignin is
two decades to enhance the digestibility of lignocellulosic biomass composed of three major phenolic components, namely p-cou-
for the efficient conversion of cellulose to ethanol, methane and in maryl alcohol, coniferyl alcohol and sinapyl alcohol. Lignin is
the recent years to hydrogen. The present article is a comprehensive synthesized by polymerization of these components and their ratio
state-of-the-art review discussing the pretreatment strategies with varies between different plants, wood tissues and cell wall layers.
the advantages, drawbacks and techno-economics, microbial Lignin is a complex hydrophobic, cross-linked aromatic polymer
systems comprising the necessary enzymes (cellulases and hemi- that interferes with the hydrolysis process. A representative dia-
cellulases) for valorization of lignocellulose, advancements in grammatic framework of lignocellulosic biomass is illustrated in
fermentation technologies and metabolic engineering approaches Fig. 2. The cellulose chains are packed into microfibrils which are
with special emphasis on consolidated biomass processing. In stabilized by hydrogen bonds. These fibrils are attached to each
addition, the review addresses the current global scenario of other by hemicelluloses and amorphous polymers of different
 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550
Fig. 2. Diagrammatic illustration of the framework of lignocellulose.
sugars as well as other polymers such as pectin and covered by
lignin. The cellulose microfibrils which are present in the
hemicellulose-lignin matrix are often associated in the form of
bundles or macrofibrils. The molecules of individual microfibrils in
crystalline cellulose are packed so tightly that not only enzymes but
even small molecules like water cannot enter the complex frame-
work. Some parts of the microfibrils have a less ordered, non-
crystalline structure referred to as amorphous region [49]. The high
molecular weight and ordered tertiary structure make natural
cellulose insoluble in water. The crystalline regions of cellulose are
more resistant to biodegradation than the amorphous parts.
Cellulose with low degree of polymerization (DP) will be more
susceptible to cellulolytic enzymes. The isolation and derivatiza-
tion/dissolution of cellulose are crucial steps in determining cellu-
lose DP [50]. In general plant cell walls are subdivided as primary
(PW) and secondary (SW) walls. The distribution of cellulose,
hemicellulose and lignin varies considerably among these layers.
The secondary wall is composed of SW1, SW2 and SW3 where SW2
is usually thicker than the others and contains the major portion of
cellulose. The middle lamella, which binds the adjacent cells, is
almost entirely composed of lignin [51].
The major impediments towards development of an economi-
cally viable technology for biodegradation of cellulose are the
association with lignin and hemicellulose, crystallinity, DP and
surface area. During the biocatalytic valorization of lignocellulosic
substrate, a residual fraction survives the attack. This fraction
absorbs a significant amount of the original enzyme and restricts
the use of these enzymes on added, fresh substrate [52]. Most
potential cellulosic substrates for bioconversion are heavily ligni-
fied. Thus, most of the cellulose in nature is unsuitable for
bioconversion unless effective and economically viable procedures
(pretreatments) are developed to remove or modify lignin.
3. Selected pretreatment categories
The crucial step in the production of biofuels from lignocellu-
losic biomass is pretreatment. Pretreatment affords the solubili-
zation or separation of the major components of biomass ie
cellulose, hemicellulose and lignin and thus render the digestibility
of lignocellulosic material. The choice of pretreatment should
consider the overall compatibility of feedstocks, enzymes and
525
organisms to be applied. Pretreatment is not only costly in its own
right but has a pervasive impact on the cost of virtually all other
biological processing operations, including those preceding
pretreatment, the handling of the liquid stream generated, the
processing of the solids from pretreatment, waste treatment, and
potential production of co-products. To implement successfully the
bioethanol production process, the first impediment to be resolved
is the efficient removal of lignin and hemicellulose through a cost
effective pretreatment process.
During the past few decades, several approaches have been used
for developing low cost pretreatments for generating sugar syrups
from cellulose and hemicellulose [53]. Different pretreatment studies
published in literature are described in terms of the mechanisms
involved, advantages and disadvantages, and economic assessment.
Pretreatments for lignocellulosic biomass include biological,
mechanical, chemical methods and various combinations thereof
[54e56]. The choice of the optimum pretreatment process depends
on the feedstocks and its economic assessment and environmental
impact. There are a number of reports on pretreatment options for
various biomass types. Table 2 illustrates some of the most promising
pretreatment categories that can be commercialized for the biofuel
industry. However, none of those can be declared outstanding as each
pretreatment has its intrinsic advantages and disadvantages. An
effective pretreatment is characterized by several criteria: avoiding
size reduction, preserving hemicellulose fractions, limiting formation
of inhibitors due to degradation products, minimizing energy input,
and being cost-effective. Except for these criteria, several other factors
are also needed to be considered, including recovery of high value-
added co-products (e.g., lignin and protein), pretreatment catalyst,
catalyst recycling, and waste treatment [57]. When comparing
various pretreatment options, all the above mentioned criteria should
be comprehensively considered as a basis. At the moment efforts on
the production of ethanol from lignocellulose is growing rapidly and
by analyzing the industrial scenario in this field more knowledge can
be gained on the applied pretreatment methods.
3.1. Physical pretreatment
Most of the lignocellulosic biomass requires some of the
mechanical processing for size reduction. Several pretreatment
methods such as milling, irradiation (using gamma rays, electron
526 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550

Table 2
Most promising pre-treatment technologies.

Method of Sugar Inhibitor Byproduct Reuse of Applicability Equipment Success at Advantages Limitations &
pre-treatment yield formation generation chemicals to different cost pilot scale disadvantages
feedstock’s
Mechanical L Nil No No Yes H Yes Reduce cellulose crystallinity High Power consumption
than inherent biomass
energy
Mineral acids H H H Yes Yes H Yes Hydrolysis of cellulose and Hazardous, toxic and
hemicellulose. alters lignin corrosive
structure
Alkali H L H Yes Yes Nil Yes Removal of lignin and Long residence time,
hemicellulose, increases irrecoverable salts formed
accessible surface area
Liquid hot H H L No e e Yes Removel of hemicellulose Long residence time,
water making enzymes less lignin removal
accessible to cellulose
Organosolv H H H Yes Yes H Yes Hydrolyze lignin and Solvents needs to
hemicellulose drained, evaporated,
condensed and reused
Wet oxidation H or L Nil L No e H e Removal of lignin, dissolves e
hemicellulose and causes
cellulose decrystallization
Ozonolysis H L H No e H No Reduces lignin content, Large amount of ozone
no toxic residues required
CO2 explosion H L L No e H e Hemicellulose removal, Does not modify lignin
cellulose decrystallization,
cost-effective
Steam H H L e Yes H Yes Hemicellulose removal Incomplete destruction
explosion and alteration in lignin of ligninecarbohydrate
structure matrix
AFXE H L e Yes e H e Removal of lignin and Not efficient for biomass
hemicellulose with high lignin content
Ionic liquids H/L L e Yes Yes e e Dissolution of cellulose, Still in initial stages
increased amenability
to cellulase

H:- High and L:- Low.

beam, microwave radiations etc) and extrusion are commonly used
to improve the enzymatic hydrolysis or biodegradability of ligno-
cellulosic materials. Improved hydrolysis results due to the reduc-
tion in crystallinity and improved mass transfer characteristics
from reduction in particle size. The energy requirements for
physical pretreatments are dependent on the final particle size and
reduction in crystallinity of the lignocellulosic material. In most
cases where the only option available for pretreatment is physical,
the required energy is higher than the theoretical energy content
available in the biomass. These methods are expensive and likely
will not be used in a full-scale process.
3.2. Physico-chemical pretreatment
Pretreatments that combine both chemical and physical
processes are referred to as physicoechemical processes. The most
important processes of this group includes: - steam explosion,
catalyzed (SO2 or CO2) steam explosion, ammonia fiber explosion
(AFEX), liquid hot water, microwave-chemical pretreatment [20,58].
3.2.1. Steam explosion
In steam explosion the biomass is treated with high-pressure
saturated steam, and then the pressure is suddenly reduced, which
makes the materials undergo an explosive decompression. Steam
explosion is typically initiated at a temperature of 160e260  C (cor-
responding pressure, 0.69e4.83 MPa) for several seconds to a few
minutes before the material is exposed to atmospheric pressure. The
biomass/steam mixture is held for a period of time to promote
hemicellulose hydrolysis, and the process is terminated by an
explosive decompression [59e61]. The process causes hemicellulose
degradation and lignin transformation due to high temperature, thus
increasing the potential of cellulose hydrolysis. A steam explosion
process using sugarcane bagasse as substrate was developed where
the individual constituents were separated as pure cellulose, hemi-
cellulose and lignin [62]. The steam-explosion pretreatment process
has been a proven technique for the pretreatment of different
biomass feedstocks. It is able to generate complete sugar recovery
while utilizing a low capital investment and low environmental
impacts concerning the chemicals and conditions being implemented
and has a higher potential for optimization and efficiency [63].
The difference between ‘steam’ pretreatment and ‘steam explo-
sion’ pretreatment is the quick depressurization and cooling down
of the biomass at the end of the steam explosion pretreatment,
which causes the water in the biomass to ‘explode’. During steam
pretreatment parts of the hemicellulose hydrolyze and form acids,
which could catalyze the further hydrolysis of the hemicellulose.
This process, in which the in situ formed acids catalyze the process
itself, is called ‘auto-cleave’ steam pretreatment. The role of the
acids, is probably however not to catalyze the solubilization of the
hemicellulose, but to catalyze the hydrolysis of the soluble hemi-
cellulose oligomers [64]. Steam explosion and thermal pretreat-
ments are widely investigated for improving biogas production
from different dedicated materials such as forest residuals [65] and
wastes of e.g. activated sludge [66,67], cattle manure [68] or
municipal solid wastes. However, there are several investigations on
combining “thermal” pretreatment with addition of bases such as
NaOH, which usually give a better result than individual thermal or
chemical pretreatment [69,70]. The process of steam explosion was
demonstrated on a commercial scale at the Masonite plants [71].
3.2.2. Ammonia fiber explosion (AFEX) and ammonia recycle
percolation (ARP)
Ammonia fiber explosion is a physicoechemical pretreatment
process in which lignocellulosic biomass is exposed to liquid
 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550
ammonia at high temperature and pressure for a period of time, and
then the pressure is suddenly reduced. The AFEX process is very
similar to steam explosion. In a typical AFEX process, the dosage of
liquid ammonia is 1e2 kg of ammonia/kg of dry biomass, the
temperature is 90  C, and the residence time is 30 min. AFEX
pretreatment can significantly improve the fermentation rate of
various herbaceous crops and grasses [72]. The AFEX technology has
been used for the pretreatment of many lignocellulosic materials
including alfalfa, wheat straw, and wheat chaff. AFEX pretreatment
results in the decrystallization of cellulose, partial depolymerization
of hemicellulose, removal of acetyl groups predominantly on
hemicellulose, cleavage of ligninecarbohydrate complex (LCC)
linkages, lignin CeOeC bond cleavage, increase in accessible surface
area due to structural disruption, and increased wettability of the
treated biomass [73,74]. The AFEX process demonstrates attractive
economics compared to several leading pretreatment technologies
based on a recent economic model [75] for bioethanol from corn
stover. An advantage of AFEX is that the ammonia used during the
process can be recovered and reused. Also, the downstream pro-
cessing is less complex compared to other pretreatment processes.
Over 90% hydrolysis of cellulose and hemicellulose was obtained
after AFEX pretreatment of bermudagrass (approximately 5% lignin)
and bagasse (15% lignin). Thus, AFEX is not a very efficient tech-
nology for lignocellulosic biomass with relatively high lignin content
such as woods and nut-shells [76]. Furthermore, ammonia must be
recycled after the pretreatment to reduce the cost and protect the
environment. However both the ammonia cost and the cost of
recovery processes drive up the cost of the AFEX pretreatment [77].
Another type of process utilizing ammonia is the ammonia recy-
cles percolation (ARP) method. In this process, aqueous ammonia
(10e15 wt %) passes through biomass at elevated temperatures
(150e170  C) with a fluid velocity of 1 cm/min and a residence time of
14 min, after which the ammonia is recovered [78]. In the ARP
method, the ammonia is separated and recycled. Under these
conditions, aqueous ammonia reacts primarily with lignin and causes
depolymerization of lignin and cleavage of ligninecarbohydrate
linkages. The ammonia pretreatment does not produce inhibitors
for the downstream biological processes, so a water wash is not
necessary [79]. Generally, AFEX and ARP processes are not differen-
tiated in the literature, although AFEX is carried out in liquid
ammonia and ARP is carried out in an aqueous ammonia solution
(10e15%). The ammonia fiber explosion pretreatment simulta-
neously reduces lignin content and removes some hemicellulose
while decrystallizing cellulose. It can have a profound effect on the
rate of cellulose hydrolysis [80]. The cost of ammonia and especially
the recovery process drives the AFEX pretreatment expensive.
3.2.3. Microwave-chemical pretreatment
The microwave/chemical pretreatment resulted in a more effec-
tive pretreatment than the conventional heating chemical pretreat-
ment by accelerating reactions during the pretreatment process
[81,82]. Zhu et al. [82] examined three microwave/chemical
processes for pretreatment of rice straw e microwave/alkali, micro-
wave/acid/alkali and microwave/acid/alkali/H2O2 e for its enzymatic
hydrolysis and for xylose recovery from the pretreatment liquid. They
found that xylose could not be recovered during the microwave/alkali
pretreatment process, but could be recovered as crystalline xylose
during the microwave/acid/alkali and microwave/acid/alkali/H2O2
pretreatment. The enzymatic hydrolysis of pretreated rice straw
showed that the pretreatment by microwave/acid/alkali/H2O2 had
the highest hydrolysis rate and glucose content in the hydrolyzate.
3.2.4. Liquid-hot water pretreatment
In liquid hot water pretreatment (LHW), pressure is utilized to
maintain water in the liquid state at elevated temperatures
527
[83e86]. Biomass undergoes high temperature cooking in water
with high pressure. LHW pretreatment has been reported to have
the potential to enhance cellulose digestibility, sugar extraction,
and pentose recovery, with the advantage of producing prehy-
drolyzates containing little or no inhibitor of sugar fermentation
[87]. The sugar-enriched prehydrolyzates can be directly fermented
to ethanol. It has been shown to remove up to 80% of the hemi-
cellulose and to enhance the enzymatic digestibility of pretreated
biomass materials such as corn fiber and sugarcane bagasse [20].
Perez et al. [88] used LHW to pretreat wheat straw and obtained
maximum hemicellulose-derived sugar recovery of 53% and enzy-
matic hydrolysis yield of 96%. Perez et al. [89] continued to optimize
process variables (temperature and residence time) in LHW
pretreatment of wheat straw and achieved 80% and 91% xylose
recovery and enzymatic hydrolysis, respectively. LHW reduces the
need for neutralization of liquid streams and conditioning chem-
icals since acid is not added [20,84,87]. Additionally, biomass size
reduction is not needed because the particles are broken apart
during pretreatment; therefore, LHW appears attractive for large
scales [90]. A difference between the LHW and steam pretreatment
is the amount and concentration of solubilized products [64]. In an
LHW pretreatment the amount of solubilized products is higher
while the concentration of these products is lower compared to
steam pretreatment [91]. This is probably caused by the higher
water input in LHW pretreatment compared to steam pretreat-
ment. The yield of solubilized (monomeric) xylan is generally also
higher for LHW pretreatment; though this result diminishes when
the solid concentration increases, because (monomeric) xylan is
then further degraded by hydrolytic reactions to, for example,
xylose and furfural [92].
3.3. Chemical pretreatments
Chemical pretreatments were originally developed and have
been extensively used in the paper industry for delignification of
cellulosic materials to produce high quality paper products. The
possibility of developing effective and inexpensive pretreatment
techniques by modifying the pulping processes has been considered.
Chemical pretreatments that have been studied to date have had the
primary goal of improving the biodegradability of cellulose by
removing lignin and/or hemicellulose, and to a lesser degree
decreasing the degree of polymerization (DP) and crystallinity of the
cellulose component. Chemical pretreatment is the most studied
pretreatment technique among pretreatment categories. The
various commonly used chemical pretreatments includes: acid,
alkali, organic acids, pH-controlled liquid hot water and ionic liquids.
3.3.1. Acid pretreatment
Acid pretreatment involves the use of concentrated and diluted
acids to break the rigid structure of the lignocellulosic material. The
most commonly used acid is dilute sulphuric acid (H2SO4), which
has been commercially used for a wide variety of biomass
typesdswitchgrass [93,94], corn stover [95,96], spruce (softwood)
[97], and poplar [98,99]. Dilute sulphuric acid has traditionally been
used to manufacture furfural [100] by hydrolyzing the hemi-
cellulose to simple sugars, such as xylose, which continues to
convert into furfural. Other acids have also been studied, such as
hydrochloric acid (HCl) [101], phosphoric acid (H3PO4) [102,103],
and nitric acid (HNO3) [104]. Due to its ability to remove hemi-
cellulose, acid pretreatments have been used as parts of overall
processes in fractionating the components of lignocellulosic
biomass [102]. Acid pretreatment (removal of hemicellulose) fol-
lowed by alkali pretreatment (removal of lignin) results in rela-
tively pure cellulose. This chemical pretreatment usually consists of
the addition of concentrated or diluted acids (usually between 0.2%
528 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550
and 2.5% w/w) to the biomass, followed by constant mixing at
temperatures between 130  C and 210  C. Depending on the
conditions of the pretreatment, the hydrolysis of the sugars could
take from a few minutes to hours [105e111]. Generally, the
pretreatment should promote high product yields in a subsequent
enzymatic hydrolysis and fermentation operations with minimum
cost. Recently it has been demonstrated that the dilute acid pre-
hydrolysis can achieve high reactions rates in short time and
significantly improve cellulose hydrolysis [112]. However,
pretreatment operating conditions must be tailored to the specific
chemical and structural composition of the various sources of
biomass. Despite continuing interest in the kinetic mechanism of
solid-phase acid-catalyzed hydrolysis for several type of biomass,
little attention has been given to incorporating those kinetic
models into the plant process modeling [113].
3.3.2. Alkaline pretreatment
Alkaline pretreatment involves the use of bases, such as sodium,
potassium, calcium, and ammonium hydroxide, for the pretreat-
ment of lignocellulosic biomass. The use of an alkali causes the
degradation of ester and glycosidic side chains resulting in struc-
tural alteration of lignin, cellulose swelling, partial decrystallization
of cellulose [114e116], and partial solvation of hemicellulose
[116,117]. Sodium hydroxide has been extensively studied for many
years, and it has been shown to disrupt the lignin structure of the
biomass, increasing the accessibility of enzymes to cellulose and
hemicellulose [118e121]. Another alkali that has been used for the
pretreatment of biomass is lime. Lignocellulosic feedstocks that
have been shown to benefit from this method of pretreatment are
corn stover, switchgrass, bagasse, wheat, and rice straw
[80,122e124]. Sun and coworkers [125] studied the effectiveness of
different alkaline solutions by analyzing the delignification and
dissolution of hemicellulose in wheat straw. They found that the
optimal condition obtained was using 1.5% sodium hydroxide for
144 h at 20  C, which resulted in 60% release of lignin and 80%
release of hemicellulose. The conditions for alkaline pretreatment
are usually less severe than other pretreatments. It can be per-
formed at ambient conditions, but longer pretreatment times are
required than at higher temperatures. The alkaline process involves
soaking the biomass in alkaline solutions and mixing it at a target
temperature for a certain amount of time. A neutralizing step to
remove lignin and inhibitors (salts, phenolic acids, furfural, and
aldehydes) is required before enzymatic hydrolysis. Recently, Zhao
and coworkers [120] showed the effectiveness of sodium hydroxide
pretreatment for hardwoods, wheat straw, switchgrass, and soft-
woods with less than 26% lignin content. A recent approach to lime
pretreatment eliminates the solideliquid separation step after
neutralization by neutralizing the lime with carbon dioxide before
hydrolysis resulting in 89% glucose recovery from leafstar rice straw
[123]. Park and coworkers [123] also used this method to test SSF
which resulted in an ethanol yield that was 74% of the theoretical
value using a mixture of Saccharomyces cerevisae and Pichia stip-
itis. The advantage of lime pretreatment is that the cost of lime
required to pretreat a given quantity of biomass is lowest among
alkaline treatments. For example, in 2005, the estimated cost of
materials was $70/ton hydrated lime compared to $270/ton fertil-
izer grade ammonia and $320/ton for 50 wt% NaOH and 45 wt%
KOH [80]. Though lime pretreatment is energy intensive, CaCO3 can
be recovered by precipitation with CO2 after solideliquid separa-
tion [126].
3.3.3. Green solvents (ionic liquids)
Recently a new class of solvent has emerged referred as ionic
liquids/green solvents. These solvents are often fluid at room
temperature, and consist entirely of ionic species. They have many
fascinating properties which make them of fundamental interest to
all chemists, since both the thermodynamics and kinetics of reac-
tions carried out in ionic liquids are different to those in conven-
tional molecular solvents. In general, ionic liquids consist of a salt
where one or both the ions are large, and the cation has a low
degree of symmetry [127]. These factors tend to reduce the lattice
energy of the crystalline form of the salt, and hence lower the
melting point [128]. Ionic liquids come in two main categories,
namely simple salts (made of a single anion and cation) and binary
ionic liquids (salts where equilibrium is involved). Ionic liquids
have been described as designer solvents [129], and which means
that their properties can be adjusted to suit the requirements of
a particular process.
Very recently ionic liquids have been confirmed to be efficient
for dissolution of lignocellulosic materials, such as cellulose, wood,
or wheat straw, switchgrass, corn stover [130e134]. Using 1-butyl-
3-methylimidazolium chloride (BMIMCl) for pretreatment, Dadi
et al. [135] found that the initial enzymatic hydrolysis rate and yield
of pretreated Avicel-PH-101 were increased by 50- and 2-fold in
comparison with untreated Avicel. Kuo and Lee [136] also observed
that the 1, 3-N-methylmorpholine- N-oxide (NMMO) pretreated
sugarcane bagasse has 2-fold higher enzymatic hydrolysis yield as
compared to untreated bagasse. Nguyen et al. [137] reported the
combined use of ammonia and ionic liquid ([Emim]Ac) for the
recovery of bio-digestible cellulose from rice straw. The treatment
exhibited a synergy effect for rice straw with 82% of the cellulose
recovery and 97% of the enzymatic glucose conversion. The cellu-
losic materials regenerated from ILs were found essentially amor-
phous and porous and were much more prone to degradation by
cellulases [133,135]. Furthermore, increased rate of cellulose
hydrolysis via cellulase in ionic liquids could lead to increased
production of fermentable sugars that can be converted into fuels.
In addition, ionic liquids involved processes are less energy
demanding, easier to operate, and more environmentally friendly
than current dissolution processes [138e140].
However, more in-depth research involving environment
friendly IL is needed to explore pretreatment green route for
resolving the challenge of ionic liquid application [139e141]. As
a result, recent efforts have been focused on exploration of enzyme
and environment-friendly ionic liquids [142], because green
solvent is promising for commercial application [143]. Therefore, it
is a key point to evaluate the biocompatibility of ionic liquids and
select suitable IL for pretreatment process [144]. Application of
ionic liquids has opened new ways for the efficient utilization of
lignocellulosic materials in such areas as biomass pretreatment and
fractionation. Binders and Raines [134] have reported a high-
yielding chemical process for the hydrolysis of cellulose and corn
stover using 1-ethyl-3-methylimidazolium chloride ([EMIM]Cl)
and obtained 90% yield of glucose from cellulose and 70e80% yield
of sugars from untreated corn stover. Ion exchange chromatog-
raphy allows the recovery of the ionic liquid and delivers sugar
feedstocks that support the vigorous growth of ethanologenic
microbes. However, there are still many challenges in putting these
potential applications into practical use, for example, the high cost
of ILs, regeneration requirement, lack of toxicological data and
knowledge about basic physico-chemical characteristics, action
mode on hemicellulose and/or lignin contents of lignocellulosic
materials and inhibitor generation issues. Consequently complete
solvent recovery will be required to make biomass processing with
IL economical. Further extensive research is required to address
such challenges. Recently Tadesse and Luque [145] have compared
the environmental impact of different ionic liquids for the
conversion of carbohydrates into useful biofuel intermediates with
their inherent advantages for biomass valorisation processes in
terms of unique and tuneable physico-chemical properties.
 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550
3.4. Biological pretreatment
Biological pretreatment employs wood degrading microorgan-
isms, including white-, brown-, soft-rot fungi, and bacteria to
modify the chemical composition and/or structure of the ligno-
cellulosic biomass so that the modified biomass is more amenable
to enzyme digestion. Although it has so far attracted little attention
due to many inherent limitations, biological pretreatment has
multiple benefits. Increasing understanding of termites and fungal
systems has provided insights for developing more effective
pretreatment technologies to realize these benefits. Fungi have
distinct degradation characteristics on lignocellulosic biomass. In
general, brown and soft rots mainly attack cellulose while
imparting minor modifications to lignin, and white-rot fungi are
more actively degrade the lignin component [80]. Present research
is aimed towards finding those organisms which can degrade lignin
more effectively and more specifically. White-rot fungi were
considered the most promising basidiomycetes for bio-
pretreatment of biomass and were the most studied biomass
degrading microorganisms [146]. The biological pretreatment
appears to be a promising technique and has very evident advan-
tages, including no chemical requirement, low energy input, mild
environmental conditions, and environmentally friendly working
manner [147,148]. However, its disadvantages are as apparent as its
advantages since biological pretreatment is very slow and requires
careful control of growth conditions and large amount of space to
perform treatment. In addition, most lignolytic microorganisms
solubilize/consume not only lignin but also hemicellulose and
cellulose. Therefore, the biological pretreatment faces tech-
noeconomic challenges and is less attractive commercially [149].
3.5. Techno-economics of pretreatments
Research is focused on converting biomass into its constituents
in a market competitive and environmentally sustainable approach.
Obstacles in the existing pretreatment processes include insuffi-
cient separation of cellulose and lignin, formation of by-products
that inhibit ethanol fermentation, high use of chemicals and/or
energy, and considerable waste production. Only few pretreatment
methods have been reported for techno-economic analysis. These
include steam explosion, liquid hot water, AFEX, lime and dilute
acid pretreatments. Most of the techno-economic studies are based
on process simulations for bioethanol production from lignocellu-
losic biomass to produce mainly sugar monomers (glucose, xylose,
arabinose, and mannose) and acid-soluble lignin, but using
conversion fractions at fixed operating conditions. Some studies are
based on batch kinetics, where it has revealed that the main factors
affecting the acid pretreatment are the type of biomass, the type of
acid, the feed acid concentration, the reaction time and the reaction
temperature. So that kinetic modeling and the operating conditions
of the pretreatment unit play an important role in the design,
development, and operation of the complete process of bioethanol
production. Process modeling and simulation is critical and decisive
for the well design of the pretreatment process of lignocellulosic
biomass [113].
Efficient and economical pretreatment for one feedstock may
not translate to an efficient process for another biomass. Eggeman
and Elander [149] carried out an economic analysis of different
pretreatment technologies (dilute acid, hot water, AFEX, ARP and
lime) for the Biomass Refining Consortium for Applied Funda-
mentals and Innovation (CAFI) as part of an initiative of the United
States Department of Agriculture. Each pretreatment process was
embedded in a full bioethanol facility model and an economic
analysis done. The same feedstock (corn stover) for the different
pretreatment strategies was used in the analysis, and the resulting
529
solid and fluid streams after pretreatment were characterized, and
gathered data were used for the material and energy balances.
Their conclusion was that the low-cost pretreatment options are
often counterbalanced by the higher costs to recover catalysts/
solvents and the higher costs of ethanol product recovery. Thus,
there is little difference in the projected economic performance of
the different pretreatment options. However, it was also clearly
stated that further process improvements such as identification of
optimum enzyme blends for each pretreatment approach and
conditioning requirements of the hydrolyzates may lead to greater
differentiation of projected process economics. Sendich and Dale
[150] used updated parameters and ammonia recovery configura-
tions in the model of Eggeman and Elander and calculated the cost
of ethanol production using AFEX. They found that the minimum
ethanol selling price reduced from $1.41/gal to $0.81/gal.
A brief survey of minimum ethanol selling price (MESPs) from
recent technoeconomic studies of biochemical cellulosic ethanol
production revealed due to differences in feedstock cost, process
assumptions, and co-product values, all of which vary considerably
across the studies [151]. For example, the analysis by Laser et al. [92]
assumes a fairly low feedstock cost and very high yields (indicating
aggressive process assumptions) as well as improved economies of
scale (if such a high feed rate can be sustained), while also receiving
positive revenue from higher-value co-products such as protein and
hydrogen. Furthermore, this study assumes a consolidated bio-
processing (CBP) approach, whichdalthough less developed than
separate saccharification and co-fermentationdcould further
improve economics by reducing enzyme costs. Conversely, studies
on the high end of the MESP range such as Kazi et al. [152] assumed
higher feedstock costs while achieving much lower ethanol yields.
They have reported the comparison of techno-economic analysis of
several process technologies (dilute acid, 2-stage dilute acid, hot
water, AFEX) for the production of ethanol from lignocellulosic
biomass, based on a 5e8 year time frame for implementation.
Economic analysis was performed for an nth plant to obtain total
investment and product value. It was observed that the product
value was lowest ($1.36/liter of gasoline equivalent) for dilute acid
pretreatment as compared to other pretreatments studied. Sensi-
tivity analysis showed that the product value is most sensititve to
feedstock cost, enzyme cost and installed equipment cost. The
product value for the pioneer plant model with dilute-acid
pretreatment is $2.30/liter of gasoline equivalent and the esti-
mated total capital investment was more than double the nth plant
cost. Compared to NREL’s 2002 design report (upon which many of
its cost inputs were based), the Klein-Marcuschamer study’s base
case assumes much longer batch times for saccharification and
fermentation (thus higher associated capital costs), higher enzyme
costs, and a lower carbohydrate fraction in the feedstock (contrib-
uting to lower yields) [153].
4. Biocatalytic valorization of lignocellulose
4.1. Saccharification systems for cellulose
Over the past decades, a great amount of research interest and
effort has been generated in the area of enzyme systems for ligno-
cellulose bioconversion. Cellulases play a significant role in the
enzymatic process by catalyzing the hydrolysis of cellulose to
soluble fermentable sugars. Cellulases are synthesized by fungi,
bacteria and plants. A list of microorganisms producing cellulases
are enlisted in Table 3. At least, three major type of cellulase activ-
ities are believed to be involved in cellulose hydrolysis based on
their structural properties: endoglucanases or 1,4-b-D-glucan- 4-
glucanohydrolases (EC 3.2.1.4), exoglucanases, including 1,4-b-D-
glucan glucanohydrolases (also known as cellodextrinases) (EC
530 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550

Table 3
Production of cellulases using microorganisms.

Microorganism Method Enzyme activities References

FPase CMCase b-glucosidase

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

3.2.1.74) and 1,4-b-D-glucan cellobiohydrolases (cellobiohy-
drolases) (EC 3.2.1.91), and b-glucosidases or b-glucoside glucohy-
drolasess (EC 3.2.1.21) [174e176]. Cellulolytic systems can be
associated into multienzymatic complexes (called cellulosomes) or
unassociated as individual enzymes. In both cases, enzymes have
a modular structure. The unassociated enzymes consist generally of
a catalytic domain responsible for the hydrolysis reaction and of
a cellulose-binding domain (CBD) mediating binding of the
enzymes to the substrate. The two domains are joined by a linker
peptide (Pro/Ser/Thr-rich), which must be sufficiently long and
flexible to allow efficient orientation and operation of both
domains. The cellulosomal enzymes are bound noncovalently to the
cellulosome-integrating protein, which carries a CBD [177]. The
more inclusive term CBM (carbohydrate-binding module) has
evolved to reflect the diverse ligand specificity of these modules.
Many CBMs have now been identified experimentally, and several
hundred putative CBMs can be further identified on the basis of
amino acid similarity. Similar to the catalytic modules of glycoside
hydrolases, CBMs are divided into families based on amino acid
sequence similarity. There are currently 64 defined families of CBMs
[178] and display substantial difference in ligand specificity. In total,
the three-dimensional structures of 36 different CBMs are now
known, many of which were determined in the last few years (PDB
database). CBMs have three general roles with respect to the func-
tion of their cognate catalytic modules: (i) a proximity effect, (ii)
a targeting function and (iii) a disruptive function. The CBM has
been considered as the limiting factor in hydrolysis. CBMs play
a pivotal role in degradative enzymes that mediate the recycling of
photosynthetically fixed carbon in the biosphere. Understanding
the structural basis by which CBMs bind to their target ligands
provides novel insights into the mechanisms of
carbohydrateeprotein recognition. The harnessing of this infor-
mation to inform strategies designed to manipulate carbohydrate-
recognition through the use of ligands that act as agonists or
antagonists will be of considerable biotechnological importance not
only within an industrial context, but also in the generation of novel
pharmaceuticals that are designed to modify cellecell signaling and
hostepathogen recognition [179]. Despite the information available
on these enzyme systems and on the structure of plant cell walls,
application of this knowledge to cellulose degradation has met with
limited success. This may be attributed to at least two factors: (i) the
inherent complexity and heterogeneity of native cellulose, and (ii)
our limited understanding of the basic hydrolysis processes [180].
Therefore, an understanding of the molecular mechanisms under-
lying cellulose degradation in combination with new and superior
enzymes may facilitate increased usage of this valuable renewable
resource [181].
The most frequently reported source of cellulases is the fungus
Trichoderma reesei, the most studied cellulolytic microorganism
during the last 60 years. Among the various microorganisms capable
of synthesizing cellulase enzymes, T. reesei produces an extracel-
lular, stable, and efficient cellulase enzyme system [182,183].
However, the low-glucosidase activity of the enzyme system from
T. reesei leads to incomplete hydrolysis of cellobiose in the reaction
mixture and, as a result, to serious inhibition of the enzymes [77].
Majority of reports on microbial production of cellulases utilizes
submerged fermentation technology (SmF) and the widely studied T
reesei is also grown in liquid media. Appropriate monitoring and
handling of cultures are still major factors in submerged fermenta-
tion. The major technical limitation in fermentative production of
cellulases remains the increased fermentation time with low
productivity. Solid state fermentation (SSF) for production of
cellulases is gaining as a cost effective technology, not only for the
production of enzyme however for the bioconversion of lignocel-
lulosic biomass employing cellulolytic microbes. Even though there
are reports on SSF for production of cellulases, commercial scale
production is still using the proven technology of SmF.
An eco-friendly approach towards the hydrolysis of poly-
saccharides to monomeric sugars is by the use of enzymes, i.e.,
cellulases and hemicellulases. The hydrolysis of cellulose by cellu-
lolytic enzymes has been investigated intensively since the early
1970s, with the objective of developing a process for the production
of ethanol. Even though soluble substrates have been developed for
measuring endoglucanase and b-glucosidase activities there are
very few substrates available for the estimation of exoglucanase
activity. The hydrolysis data from soluble substrates cannot yield
useful information on the hydrolysis of insoluble substrates. The
enzymatic hydrolysis of the lignocellulosic biomass is preceeded by
 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550
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 irre-
versibly binding to hydrolytic enzymes. Therefore, the removal of
the lignin can dramatically increase the hydrolysis rate [54].
Enzymatic hydrolysis methods have shown distinct advantages
over acid based hydrolysis methods; the very mild process condi-
tions give potentially higher yields, the utility cost is low (no
corrosion problems), Therefore this is the method of choice for
future wood-to-ethanol processes. The factors influencing enzy-
matic hydrolysis can be divided into substrate related factors and
enzyme related factors. The relationship between structural
features of cellulose and rates of enzymatic hydrolysis has been the
subject of extensive study and several reviews have been published
[176,184]. Most desired attributes of cellulases for lignocellulose
bioconversion are the complete hydrolytic machinery, high specific
activity, high rate of turn over with native cellulose/biomass as
substrate, thermostability, decreased susceptibility to enzyme
inhibition by cellobiose and glucose, selective adsorption on
cellulose, synergism among the different enzymes and ability to
withstand shear forces [185]. These parameters are fulfilled
through protein engineering approaches, over expression tech-
niques and developing optimal enzyme cocktails and conditions for
hydrolysis. The hydrolytic efficiency of a multi-enzyme complex for
lignocellulose saccharification depends both on properties of
individual enzymes and their ratio in the multi-enzyme cocktail
[186]. The ideal cellulase complex must be highly active on the
intended biomass feedstock, able to completely hydrolyze the
biomass, operate well at mildly acidic pH, withstand process stress,
and be cost-effective [48]. To improve the yield and rate of the
enzymatic hydrolysis, research has been focused on optimizing the
hydrolysis process, enhancing the cellulase activities, optimizing
the reaction conditions, enzyme and substrate cocktail composi-
tion, enzyme recycling and recovery strategies. The yield and initial
rate of enzymatic hydrolysis of the cellulose is affected mainly by
the substrate concentration. Realistic methods must be based on
physically and chemically relevant industrial substrates. 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
[187]. 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 [188]. Research is also focused on
enzymes that can tolerate both acid and heat which may contribute
towards the improvement of lignocellulosic biomass processing.
These enzymes are produced naturally by extremely thermophilic
microbes/extremophiles [189,190]. Despite intensive research over
the few past decades, the enzyme hydrolysis step remains as
a major techno-economic bottleneck in lignocellulose biomass-to-
ethanol bioconversion process.
4.2. Saccharification systems for hemicelluloses
Hemicellulases facilitate cellulose hydrolysis by exposing the
cellulose fibers, thus making them more accessible. 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 poly-
saccharide, interdomain linkers, and dockerin modules that
mediate the binding of the catalytic domain via cohesionedockerin
interactions, either to the microbial cell surface or to enzymatic
531
complexes such as the cellulosome [191]. Based on the amino acid
or nucleic acid sequence of their catalytic modules hemicellulases
are 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 [192]. Enzymes that hydrolyse
mannan have been largely neglected, even though it is an abundant
hemicellulose, therefore the application of mannanases for cata-
lysing the hydrolysis of b-1,4 mannans is as important as the
application of xylanases.
The two main glycosyl hydrolases depolymerising the hemi-
cellulose backbone are endo-1,4- b-D-xylanase and endo-1, 4-b-D-
mannanase [193]. Endo-1,4-b-xylanase cleaves the 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 [194].
Initially, the main hydrolysis products are b-D-xylopyranosyl olig-
omers, but at a later stage, small molecules such as mono-, di- and
trisaccharides of b-D-xylopyranosyl may be produced [195]. These
enzymes are produced by fungi, bacteria, yeast, marine algae,
protozoans, snails, crustaceans, insect, seeds, etc. [196]. Table 4 lists
the microorganisms producing different hemicellulases [27]. Fila-
mentous 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 [195].
Aspergillus niger, Humicola insolens, Termomonospora fusca, T. reesei,
Trichoderma longibrachiatum, T. koningii have been used as indus-
trial 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 indus-
tries, or xylitol production [196,197]. Several investigations so far
have indicated that xylanases are usually inducible enzymes [198],
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 b-D-xylopyranosyl residues
added to the culture medium during growth [193, 196 and 200].
However, constitutive production of xylanase has also been
reported [199,200]. Catabolite repression by glucose is a common
phenomenon observed in xylanase biosynthesis [192]. 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 b-xylanase, b-xylosidase, and
enzymes such as a-L-arabinofuranosidase, a-glucuronidase, acetyl
xylan esterase, and hydroxycinnamic acid esterases that cleave side
chain residues from the xylan backbone. All these enzymes act
cooperatively to convert xylan to its constituents [193]. 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 [201]. The enzymes
arabinosidase, a-glucuronidase and acetyl xylan esterase act in
synergy with the xylanases and xylosidases by releasing the
substituents on the xylan backbone to achieve a total hydrolysis of
xylan to monosaccharides [202].
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-b-mannanases (EC 3.2.1.78) and
exoacting b-mannosidases (EC 3.2.1.25) is required to cleave the
polymer backbone. Additional enzymes, such as b-glucosidases (EC
3.2.1.21), a-galactosidases (EC 3.2.1.22) and acetyl mannan esterases
532 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550

Table 4
Microorganisms producing hemicellulases & accessory enzymes.

Enzyme Organism Substrate Specific activity (mmol min1 mg1)

Feruloyl esterase Clostridium stercorarium Ethyl ferulate 88
b-1,4-xylosidase Thermoanaerobacter ethanolicus o-nitrophenyl-b-D-xylopyranoside 1073
Exo-b-1,4-mannosidase Pyrococcus furiosus p-nitrophenyl-b-D-galactoside 31.1
Endo-b-1,4-mannanase Bacillus subtilis Galactoglucomannan/glucomannans/mannan 514
a-Larabinofuranosidase Clostridium stercoarium alkyl-a-arabinofuranoside/aryl-a-arabinofuranoside/ 883
Larabinogalactan/L-arabinoxylan/
methylumbelliferyl-a-Larabinofuranoside
a-Glucuronidase Thermoanaerobacterium 4-O-methyl-glucuronosyl-xylotriose 9.6
saccharolyticum
a-Galactosidase Escherichia coli raffinose 27,350
Endo-galactanase Bacillus subtilis arabinogalactan 1790
b-Glucosidase Bacillus polymyxa 4-nitrophenyl-b-D-glucopyranoside 2417
Acetyl xylan esterase Fibrobacter succinogenes Acetylxylan/a-naphthyl acetate 2933
Feruloyl esterase Aspergillus niger Methyl sinapinate 156
Endo-1,4-b-xylanase Trichoderma longibrachiatum 1,4-b-D-xylan 6630
b-1,4-xylosidase Aspergillus nidulans p-nitrophenyl-b-D-xylopyranoside 107.1
Exo-b-1,4-mannosidase A. niger b-D-Man-(1e4)-b-D-GlcNAc-(1e4)-b-DGlcNAc- AsneLys 188
Endo-b-1,4-mannanase Sclerotium rolfsii Galactoglucomannan/mannans galactomannans/ 380
glucomannans/
Endo-a-1,5-arabinanase A. niger 1,5-a-L-arabinan 90.2
a-L-arabinofuranosidase A. niger 1,5-a-L-arabinofuranohexaose/1,5-a- L-arabinotriose/ 396.6
1,5-L-arabinan/a-Larabinofuranotriose
a-Glucuronidase Phanerochaete chrysosporium 4-O-methyl-glucuronosyl-xylobiose 4.5
a-Galactosidase Mortierella vinacea melibiose 2000
Endo-galactanase A. niger 6593
b-glucosidase Humicola insolvens (2-hydroxy methylphenyl)-b-Dglucopyranoside 266.9
Acetyl xylan esterase Schizophyllum commune 4-methylumbelliferyl acetate/4-nitro phenyl acetate 227

are required to remove side chain sugars that are attached at various
points on mannans [203e205]. The property of mannanolysis is
widespread in the microbial world. A vast variety of bacteria, acti-
nomycetes, yeasts and fungi are known to be mannan degraders
[206,207]. Mannanases of microbial origin have been reported to be
both induced as well as constitutive enzymes and are usually being
secreted extracellularly [203]. Although a number of mannanase-
producing bacterial sources are available, only a few are commer-
cially exploited as wild or recombinant strains, of these, the
important ones are: Bacillus sp., Streptomyces sp., Caldibacillus cel-
lulovorans, Caldicellulosiruptor Rt8B, Caldocellum saccharolyticum
[208e210].
Many endoglucanases have been reported to hydrolyze xylo-
glucan as a substrate analog [211], however few endo-b-1,4-
glucanases have high activity toward xyloglucan, with little or no
activity towards cellulose or cellulose derivatives [212,213]. They
have been assigned a new EC number (EC 3.2.1.151) and designated
as xyloglucanase, xyloglucan hydrolase (XGH), or xyloglucan-
specific endo-b-1,4-glucanases (XEGs) belonging to families 5, 12,
44, and 74, according to a recent classification of glycoside hydro-
lases (GHs) available at http://afmb.cnrs-mrs.fr/CAZY/ [214e216].
They represent a new class of polysaccharide degrading 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 microorgan-
isms [217e227]. 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
[217] and oligoxyloglucan reducing- end-specific xyloglucanobio-
hydrolase from Aspergillus nidulans [224], whereas the endo-type
enzymes hydrolyze xyloglucan polymer randomly. In addition,
XEG74 from Paenibacillus sp. KM21 and Cel74A from T. reesei have
been reported to have endo-processive or dual-mode endo-like and
exo-like activities [218,222]. Gloster et al. [228] have reported the
three dimensional structures in ligand free and xyloglucan-
oligosaccharide complexed formed from two distinct xylogluca-
nases from glycosyl hydrolyase families GH5 (Paenibacillus pabuli
XG5) and GH12 (Bacillus licheniformis XG12). The structure of
Clostridium thermocellum xyloglucanases, Xgh74A in both apo and
ligand-complexed forms reveals the structural basis for xyloglucan
recognition and degradation [229]. The complete hydrolysis of
galactoxyloglucan requires an accessory enzyme, b-galactosidase
which cleaves the galactose residue attached to the branched
xylose moiety in the b-D-glucopyranose backbone [230]. 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 [228].
5. Fermentation strategies & consolidated biomass
processing
The pretreated biomass can be processed using variety of
process configurations such as separate hydrolysis and fermenta-
tion (SHF), simultaneous saccharification and fermentation (SSF),
simultaneous saccharification and co-fermentation (SSCF) and
consolidated biomass processing (CBP) (Fig. 3). Process integration
reduces the capital cost [231]. SHF is a conventional two step
process where the lignocellulose is hydrolysed using the enzymes
to form the reducing sugars in the first step and the sugars, thus
formed, are fermented to ethanol in the second step using various
yeasts such as Saccharomyces, Kluveryomyces, Debaryomyces, Pichia,
Zymomonas [232] as well as their recombinants. The advantage of
this process is that each step can be carried out at its optimum
conditions. Current technology using corn stover as feedstock,
ammonia fibre expansion as the pretreatment technology and
Saccharomyces cerevisiae 424A (LNH-ST) in SHF was able to achieve
191.5 g of ethanol/Kg untreated biomass at ethanol concentration of
 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550
Multi-stage cellulosic ethanol production:
Cellulose
Native Solid/liquid Enzyme production
Plant separation
Hemicelluloses
Pentose sugar
CBP:
Biomass Modified
recalcitrance biomass
Fig. 3. Fermentation strategies and consolidated biomass processing.
40 g/L. The process is carried out without washing of pretreated
biomass, detoxification and nutrient supplementation [233].
The saccharification of the lignocellulosic biomass by the
enzymes and the subsequent fermentation of the sugars to ethanol
by yeast such as Saccharomyces or Zymomonas take place in the
same vessel in SSF. 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 [234].
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.
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 [235]. 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 tempera-
tures closer to the optimal temperature for the enzymes. However,
in all these cases saccharification of pure cellulose (e.g. Sigmacell-
50) or washed fibers, in defined fermentation medium, were
applied. SSF of cellulose with mixed cultures of different thermo-
tolerant yeast strains have also been carried out [236].
Running SSF in fed-batch mode does not necessarily give higher
ethanol yields than running in batch mode, but when the enzyme-
adding method is suitable, similar or slightly higher ethanol yields
could be obtained in fed batch SSF compared with batch mode. The
appropriate feeding strategy for the enzymes in fed-batch SSF
appears to depend on the conditions during SSF [237]. Lu et al.
[238] used a fed-batch separate enzymatic hydrolysis and
fermentation approach to achieve high ethanol yields (49.5 g/L) at
a 30% cumulative insoluble solids loading. This study also showed
that washing the pretreated material results in enhanced conver-
sion of cellulose due to removal of inhibitory compounds. A
strategy for achieving high solids concentrations is to utilize
alternative bioreactor designs for lignocellulose processing. At high
solids loadings (15%), mixing via conventional shaking and stir-
ring is ineffective [239]. Several novel mixing modes, including
533
Enzyme Hexose
fermentation
hydrolysis
Ethanol
Enzyme Pentose
hydrolysis fermentation
CBP microbes
Biomass cellulolytic Ethanol
ethanologen
gravitational tumbling in roller bottle reactors (RBRs) [240,241],
horizontal rotating shaft with paddlers [242,243], and stirring with
helical impellers [242], have been designed and applied to
saccharification or SSF under high solids loadings. Recently, Zhang
et al. [242] reported a bioreactor with a novel helical impeller for
SSF at high solids loadings of 15e30% (w/w) of steam explosion
pretreated corn stover. They found that the helical stirring system
had better performances in terms of ethanol concentration and
energy consumption, compared to a Rushton impeller stirring. At
the highest solids loading of 30%, the ethanol concentration
reached 40.0 and 64.6 g/L after 72 h SSF process, at enzyme dosages
of 7 and 30 FPU/g dry mass, respectively. Mixing energy
consumption was 58.6% of the total thermal energy of the ethanol
produced. A recombinant S. cerevisiae strain expressing the b-
glucosidase gene from Humicola grisea was used for ethanol
production from three different cellulosic sources by SSF. Initially,
an enzymatic pre-hydrolysis step was done with a solid: liquid ratio
of 1:4, and an enzyme loading of 25 FPU.g1 of cellulosic substrate.
Using sugarcane bagasse pretreated cellulignin, crystalline cellu-
lose and carboxymethyl cellulose, 51.7 g L1, 41.7 g L1 and
13.8 g L1 of ethanol was obtained, respectively, at the end of 55 h
of fermentation. The highest ethanol productivity (0.94 g L1 h1)
was achieved using sugarcane bagasse pretreated cellulignin [243].
There is scarcity of literature from SSF experiments in which
hemicelluloses have been used together with thermotolerant
strains. Menon et al. [244] 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 36 h for OSX and 48 h 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. The techno-economic study compares several
process technologies for the production of ethanol from lignocel-
lulosic material, based on a 5e8 year time frame for implementa-
tion [152]. Wringen et al. [245] have performed the techno-
economic evaluation of producing ethanol from softwood by SHF
and SSF and have discussed the bottleneck issues. The main focus of
534 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550
the techno-economic study conducted by Sassner et al. [246] was
on the pretreatment and the SSF steps for three lignocellulosic
substrates (spruce, salix and corn stover). Sensitivity analyses of
important process parameters showed their relative effects on the
production cost and on the potential for cost reduction for each raw
material. The study clearly demonstrates the importance of a high
ethanol yield and the necessity of utilizing the pentose fraction for
ethanol production to obtain good process economy, especially
when using Salix or corn stover. Furthermore, a less energy-
demanding process, here mainly achieved by increasing the dry
matter content in SSF, reduces the capital cost and results in higher
co-product credit, and therefore has a significant effect on the
overall process economy.
Simultaneous saccharification and co-fermentation (SSCF) has
been recognized as a feasible option for ethanol production from
xylose-rich lignocellulosic materials. Simultaneous saccharification
of cellulose and hemicellulose and co-fermentation of end products
glucose and xylose is reported to be carried out by genetically
engineered microbes. A combination of enzyme and substrate
feeding was shown to enhance xylose uptake by a recombinant
xylose-fermenting strain of S. cerevisiae (TMB3400) and increase
overall ethanol yield in SSCF of steam-pretreated wheat straw. This
is conceptually important for the design of novel SSCF processes
aiming at high-ethanol titers [247]. Zhang and Lynd [248] have
reported the SSCF of waste paper sludge to ethanol using two
recombinant xylose-fermenting microbes: Zymomonas mobilis 8b
and S. cerevisiae RWB222. S. cerevisiae RWB222 produced over 40 g/
L ethanol with a yield of 0.39 g ethanol/g carbohydrate on paper
sludge at 37  C, while similar titers and yields were achieved by
Z. mobilis 8b at 30  C. Enzyme kinetics and yeast sugar uptake rates
for a recombinant xylose utilizing strain of S. cerevisiae, TMB3400,
were determined in a real hydrolyzate medium. The total xylose
uptake could be increased from 40% to as much as 80% by
controlling the enzyme feed [249]. Synthetic biology approach for
co-fermentation of hexose and pentose sugars has been success-
fully demonstrated by incorporating two mutants of E. coli in
a single system e one capable of utilizing only glucose, while the
other capable of utilizing only xylose as a carbon source. In this
study, catabolite repression due to the presence of glucose was
made irrelevant by the use of two strains, since one cannot utilize
glucose at all. Furthermore, the authors demonstrate by fed-batch
experiments that the system robustly adapts to fluctuations in
the feed stream, i.e., cultures actually grow in concert with the
varying feed composition [250]. Another promising alternative for
simultaneous utilization of pentoses and hexoses is the conversion
of xylose into xylulose by xylose isomerase or oxidoreductase. The
fermentation of xylulose begins by its intracellular phosphorylation
to xylulose-5-phosphate, which enters the pentose phosphate
pathway and eventually forms ethanol. Yeasts like S. cerevisiae and
Schizosaccharomyces pombe are capable of fermenting xylulose to
ethanol, along with glucose [251].
Lignocellulosic bioprospecting involving enzymes or microbial
systems commonly includes three major biologically mediated
processes: production of saccharolytic enzymes (cellulases and
hemicellulases), hydrolysis of cellulose and hemicellulose to
monomeric sugars, fermentation of hexose and pentose sugars.
These three biotransformations occurring in a single process
configuration is called as consolidated biomass processing (CBP).
CBP offers the potential for lower cost and higher efficiency than
processes featuring dedicated cellulase production. This result from
avoided costs for capital, substrate and other raw materials, and
utilities associated with cellulase production. In addition, several
factors support the possibility of realizing higher hydrolysis rates,
and hence reduced reactor volume and capital investment, using
CBP. These include enzymeemicrobe synergy as well as the use of
thermophilic organisms and/or complexed cellulase systems. CBP
requires a microbial culture that combines properties related to
both substrate utilization and product formation. Desired substrate
utilization properties include the production of a hydrolytic
enzyme system allowing high rates of hydrolysis and utilization of
resulting hydrolysis products under anaerobic conditions with
a practical growth medium. Desired product formation properties
include high product selectivity and concentrations. A cellulolytic
culture with this combination of properties has not been described
to date. Development of microorganisms for cellulose conversion
via CBP can be pursued according to two strategies. The native
cellulolytic strategy involves naturally occurring cellulolytic
microorganisms to improve product-related properties such as
yield and tolerance. The recombinant cellulolytic strategy involves
engineering noncellulolytic microorganisms that exhibit high
product yields and tolerance so that they become able to utilize
cellulose as a result of a heterologous cellulase system [176]. Both
CBP organism development strategies involve very large challenges
that will probably require a substantial sustained effort to over-
come. The feasibility of CBP will be fully established only when
a microorganism or microbial consortium is developed that
satisfies the requirements. Jin et al. [252] have evaluated the
performance of CBP organism Clostridium phytofermentans (ATCC
700394) on AFEX-treated corn stover (AFEX-CS). At optimal
conditions with 0.5% (w/w) glucan loading of AFEX-CS,
C. phytofermentans hydrolyzed 76% of glucan and 88.6% of xylan
in 10 days. These values reached 87% and 102% of those obtained by
SSCF using commercial enzymes and S. cerevisiae 424A. Ethanol
titer for CBP was found to be 2.8 g/L which was 71.8% of that yielded
by SSCF (3.9 g/L). Fundamental and applied topics relevant to CBP
are comprehensively reviewed in 177, 255 and 256.
Process integration plays a major role in efficient process design.
When several operations can be performed in a same single unit,
the possibilities for improving the performance of the process are
higher. Reactionereaction integration has been proposed for the
integration of different biological transformations taking place
during ethanol production [255]. This type of integration can be
done between different steps of ethanol production process
involving chemical transformations as well. In this sense, the
integration of detoxification step with fermentation can play an
important role in their intensification and in the reduction of
ethanol production costs. Palmqvist and Hahn-Hägerdal [256]
propose a way to implement this kind of integration consisting in
carrying out the detoxification in the same vessel in which
fermentation is to be accomplished just before the cultivation
process. Separation is the step where major costs are generated in
process industry. Therefore, reactioneseparation integration could
have the highest impact on the overall process in comparison with
homogeneous integration of processes (reactionereaction,
separationeseparation) [257]. The development of technologies
for separationeseparation integration has been linked to the
development of the different involved unit operations and to new
approaches for process intensification. The examples of
separationeseparation integration in the case of ethanol produc-
tion mostly correspond to integration of the conjugated type, i.e.,
when integrated processes are carried out in different equipments
closing the flowsheet by fluxes or refluxes [258]. In this context, the
application of saline extractive distillation and membrane tech-
nologies for the integration of several configurations for separation
and dehydration of ethanol should be highlighted [259]. Similarly,
the integration of different chemical and biological processes for
the complete utilization of the feedstocks should lead to the
development of big “biorefineries” that allow the production of
large amounts of fuel ethanol and many other valuable co-products
at smaller volumes, improving the overall economical effectiveness
 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550
of the conversion of a given raw material. Integration opportunities
may provide the ways for a qualitative and quantitative improve-
ment of the process to make it techno-economical and eco-friendly.
5.1. Future economic performance of hydrolysis process concepts
Lignocellulosic ethanol is expected to be commercialized
during the next decade as renewable energy for transport. The
techno-economic analysis for commercial stage evaluates the
competitiveness with first generation bioethanol and gasoline.
During few decades, researchers are analyzing the techno-
economic status of second generation biofuels. The logistics of
providing a competitive, all-year-round, supply of biomass feed-
stock to a commercial-scale plant is challenging, as is improving
the performance of the conversion process to reduce costs [259].
The biochemical route, being less mature, probably has a greater
cost reduction potential than the thermo-chemical route, but here
a wider range of synthetic fuels can be produced to better suit
heavy truck, aviation and marine applications. Continued invest-
ment in research and demonstration by both public and private
sectors, coupled with appropriate policy support mechanisms, are
essential if full commercialisation is to be achieved within the next
decade. After that, the biofuel industry will grow only at a steady
rate and encompass both 1st- and 2nd-generation technologies
that meet agreed environmental, sustainability and economic
policy goals [260].
Although conversion of cellulosic biomass to ethanol has been
studied for decades, the uncertainty of techno-economic feasibility,
particularly at large scale production, prohibits commercialization
of such processes. Besides the relatively high cost of some pro-
cessing stages (i.e. pretreatment and enzymatic hydrolysis), the
cost of feedstocks share a large portion of operating costs. During
the late 19th and early 20th century techno-economic evaluation of
lignocellulosic ethanol involves two major studies by NREL in
association with other US research institutes and universities
[261,262]. The NREL 2002 report projects that for a production scale
of 2000 ton of feedstock per day, at $30/ton corn stover, accounts
for 31.3% of the overall operating costs [263]. At a larger scale of
5000 tons of corn stover per day and a higher corn stover price of
$40/ton, feedstock costs were estimated to account for 71.8% of the
operating costs with advanced bioconversion processes [254]. On
the other hand, using seasonally harvested feedstocks, such as
agricultural wastes and energy crops, also raises questions of
obtaining year-long supply or feedstock storage for large scale
production. Therefore, lower feedstock costs along with achieving
high yields of ethanol can result in significant improvements in the
economics of cellulosic ethanol.
The techno-economic evaluation strategies have been changed
from 2002, with the launch of the Biomass Program of the US
Department of Energy (DOE). Since 2007, the design of this program
has acquired a clear strategic goal with the aim of the public
authorities to reduce the use of gasoline by 20% by 2017 and produce
35  109 L of renewable and alternative fuels in 2017 [259]. DOE has
launched a multi-year program plan (MYPP) and is updated every
two years, including so far 2004 [263], 2005 [264], 2007 [265], 2009
[266], 2011 [267]. The estimation of the ethanol program cost target
(EPCT) in 2012 for the biochemical ethanol is based on the reference
scenario by the Energy Information Administration (EIA, 2009)
which forecasts the wholesale price of gasoline in 2012 at US$ 2.62/
gal gasoline (US$ of 2007). Assuming a conversion factor of
0.67 gallon gasoline per gallon of ethanol, the EPCT is set at US$ 1.76/
gal ethanol (US$ of 2007). The contribution made by feedstock
production to the ethanol production cost increases from one MYPP
to the other due to progress in understanding and estimating the
feedstock production and logistics [259].
535
Sassners et al. [246] compare the techno-economic performance
of conversion of lignocellulosic-to-ethanol based on three different
feedstocks, a softwood (spruce), a hardwood (salix) and an agri-
cultural residue (corn stover). The process consists of SO2-catalysed
steam explosion pre-treatment and SSF. The process capacity of the
ethanol plants is supposed to be 200,000 dry tons of biomass per
year. The process parameters are adjusted to experimental data and
adapted to each feedstock. Enzymes are assumed to be purchased
while the yeast is produced on-site. The authors find the following
energy efficiencies for ethanol output: 25% (salix), 25% (corn stover)
and 31% (spruce) for the base case and the maximum energy effi-
ciencies in the range of 52e53% for salix, 55% for corn stover and
56% for spruce. The economic evaluation consists in estimating
annual production cost including annulaised capital cost using 7%
interest rate and 15 years depreciation period, and annual opera-
tion costs. For the base case the annual production cost significantly
varies i.e. US$0.69/l ethanol (spruce), 0.86 (corn stover) and 0.87
(salix). For alternative cases the cost estimates were 0.66 (spruce),
0.67 (corn stover) and 0.72 (salix). Wingren et al. [268] perform
a techno-economic evaluation of SSF-based softwood-to-ethanol,
with the objective to compare the impact of various downstream
configurations. The base case consists in conversion of wood chips
of spruce to ethanol. The conversion process and the economic
evaluation use the same approach as in Sassners et al. [246]. The
production cost (US$/l) varies between 0.546 for the mechanical
vapour recompression option to 0.591 for the base case. The case of
anaerobic digestion results in 0.549 (US$/l) production cost. A
techno-economic evalution of the spruce-to-ethanol process, based
on SO2 catalysed steam pretreatment followed by SSF, has been
performed using the commercial flow-sheeting program Aspen
PlusTM [269]. Various process configurations of anaerobic diges-
tion of the stillage, with different combinations of co-products,
have been evaluated in terms of energy efficiency and ethanol
production cost. Anaerobic digestion of stillage showed a signifi-
cantly higher overall energy efficiency (87e92%) based on the
lower heating values, the production cost varied between 4.00 and
5.27 Swedish kronor per liter (0.38e0.50 euro/l).
A comprehensive techno-economic analysis was performed for
conversion of cellulosic feedstock (Tall Fescue) to ethanol using
some of the common pretreatment technologies: dilute acid, dilute
alkali, hot water and steam explosion [270]. Detailed process
models incorporating feedstock handling, pretreatment, simulta-
neous saccharification and co-fermentation, ethanol recovery and
downstream processing were developed using SuperPro Designer.
Projected ethanol yields were 252.62, 255.80, 255.27 and 230.23 L/
dry metric ton biomass for conversion process using dilute acid,
dilute alkali, hot water and steam explosion pretreatment tech-
nologies respectively. Price of feedstock and cellulose enzymes
were assumed as $50/metric ton and 0.517/kg broth (10% protein in
broth, 600 FPU/g protein) respectively. Capital cost of ethanol
plants processing 250,000 metric tons of feedstock/year was $1.92,
$1.73, $1.72 and $1.70/L ethanol for process using dilute acid, dilute
alkali, hot water and steam explosion pretreatment respectively.
Ethanol production cost of $0.83, $0.88, $0.81 and $0.85/L ethanol
was estimated for production process using dilute acid, dilute
alkali, hot water and steam explosion pretreatment respectively.
The results demonstrated the importance of addressing the trade-
offs in capital costs, pretreatment and downstream processing
technologies.
Lynd et al. [271] indicates that there is no capital or operation
expenditures required for the enzyme production within the CBP
process. Similarly, part of the substrate is not deviated to enzyme
production. According to the projections, the reduction of
production costs due to an advanced configuration involving CBP is
three times greater than the reduction related to the scale of
536 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550

economy of the process and ten times greater than the reduction
associated with a lower cost of the feedstock. This diminish is
accomplished, thanks to the reduction of more than eight times in
the cost of biological conversion [271]. The comparative cost of
ethanol production by CBP and SSCF (featuring a dedicated cellu-
lase production) processes are simulated assuming aggressive
performance parameters intended to be representative of mature
technology [253]. Their results indicated that the total biological
processing cost for ethanol production reaches 18.9 ¢/L, taking into
account the 9.90 ¢/gal ethanol for dedicated cellulase production
and 9 ¢/gal for SSCF which is more than fourfold larger than the
4.2 ¢/gal projected for CBP. Hamelinck et al. [272] have analysed the
techno-economic performance of promising conversion concepts
for the short-, middle- and long-term systems. The current avail-
able technology, which is based on dilute acid hydrolysis, has about
35% efficiency (HHV) from biomass to ethanol. The overall effi-
ciency, with electricity co-produced from the not fermentable
lignin, is about 60%. Improvements in pre-treatment and advances
in biotechnology, especially through process combinations can
bring the ethanol efficiency to 48% and the overall process effi-
ciency to 68%. They estimated the current investment costs at
2.1 kh/kWHHV (at 400 MWHHV input, i.e. a nominal 2000 tonne
dry/day input). A future technology in a 5 times larger plant
(2GWHHV) could have investments of 900 kh/kWHHV. A combined
effect of higher hydrolysis-fermentation efficiency, lower specific
capital investments, increase of scale and cheaper biomass feed-
stock costs (from 3 to 2 h/GJHHV), could bring the ethanol produc-
tion costs from 22 h/GJHHV in the next 5 years, to 13 h/GJ over the
10e15 year time scale, and down to 8.7 h/GJ in 20 or more years. In
De Vries et al. [273](based on the analyses of Hoogwijk et al. [8]), it
is indicated that the biofuel production potential around 2050
could lay between about 70 and 300 EJ fuel production capacity
depending strongly on the development scenario. Around that
time, biofuel production costs would largely fall in the range up to
15 U$/GJ, competitive with equivalent oil prices around 50e60 U$/
barrel. The process efficiency and potential technical advances and
challenges along with cost information (USD2005/GJ) for several
bioenergy processes for biofuel production are summarized in
Table 5. The relatively higher production cost of ethanol is the main
obstacle to be resolved. Process engineering plays a central role for
the generation, design, analysis and implementation of technolo-
gies improving the indexes of global process. Undoubtedly, process
intensification through integration of different phenomena and
unit operations as well as the implementation of consolidated
bioprocessing of different feedstocks into ethanol (that requires the
development of tailored recombinant microorganisms), will offer
the most significant outcomes during the search of the efficiency in
fuel ethanol production. Additionally, the intensification of bio-
logical processes implies a better utilization of the feedstocks and
the reduction of process effluents improving the environmental
performance of the proposed configurations [255].
6. Metabolic engineering approaches
One of the strategies for improving the economics and yields of
lignocellulosic bioethanol process is optimization of metabolic
pathways through genetic modifications for effective manipulation
of metabolic capabilities of microbes. The exploitation of the
diverse metabolic pathways leading to energy-rich, fuel-like
hydrocarbons opens up a path to develop renewable fuels which
are beyond the restrictions of bioethanol and plant-derived bio-
diesel [274]. The increasing number of sequenced genomes
collected globally, including potential energy crops and cellulolytic
microbes will be significant for cellulosic biofuel production [275].
Considerable progress has been made to understand the induction
mechanism of cellulases by lactose in Trichoderma ressei, which
involves an alternative D-galactose metabolism pathway. With the
recent availability of the complete genome sequence, T ressei has
entered the post-genomic era and the knowledge in advancement
of metabolic engineering approaches will enhance the probability
of obtaining new hyper active mutants strains. Trichoderma sp.
dominantly occupies ecological niches and a better understanding
of signal transduction pathways initiating and/or modulating, may
help to develop new strategies for improving cellulase gene
expression [276].
Metabolic redirection is an alternative strategy, i.e., redirec-
tion of the central metabolism of the ethanologenic organism by
gene knockout to block undesirable metabolic pathways, in
order to divert the carbon flow of pyruvate from organic acids to
ethanol production [277,278]. The yeast S. cerevisiae, rarely
possess native pathways to efficiently ferment both hexoses and
pentoses [279]. Escherichia coli is the most convenient starting
point for engineering microbial catalysts for biofuel production,
owing to the wealth of genetic and metabolic knowledge avail-
able [280]. E. coli can actively metabolize a wide range of
substrates, including hexoses and pentoses [281], but its hexose
metabolism is inferior to that of Z. mobilis, an obligate ethano-
logenic bacterium [282]. As a metabolic enhancement strategy,
E. coli has been genetically engineered for cofermentation of all
constitutive sugars from lignocellulose by importing the highly
efficient fermentation pathway for ethanol production from
Z. mobilis [283].

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

Process Feedstock Efficiency and process economics, Potential technical advances and Production cost by
Eff. ¼ energy product/biomass challenges 2030 (USD2005/GJ)
energy component costs in USD2005/GJ
SHF Barley straw Steam explosion, enzyme hydrolysis, ethanol System integration, high solids, 30 (Finland)
fermentation, High solids 15% decrease toxicity for fermentation from pilot data
SSF Bagasse Standalone plant 370 L/t dry (ethanol) Mechanical harvest improvements 6e15
þ 0.56 kWh/L ethanol (electricity) sugarcane residues (occurring)
Corn stover Dilute acid hydrolysis, 260 million L/yr Pretreatment process integration, 15.5 (US)
enzyme cost nth plant, future
Lignocellulosic various Eff. Kg/L ethanol (poplar, Miscanthus, Process integration, project a 25% 18e22 (2020)
Eff. 35% ethanol þ 4% power switchgrass, corn stover, wheat: 3.7, 3.2, 2.6, reduction in operation cost by
2.6, 2.4). Plant size 1500 to 1000 t/day 2025 and 40% by 2035
SSCF Lignocellulosic Eff.w39% ethanol þ 10% power Efficient 5 carbon conversion 25e27
technology, advanced enzymes, 28e35
thermotolerant microbes
CBP Eff.w49% for wood and 42% for straw Lignin engineering facilitates 15.5 future
(ethanol) þ 5% power cellulose access, develop
efficient CBP microbes
 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550
Plant genetic engineering technology offers great potential to
reduce the cost of lignocellulosic biofuel. All necessary cell wall
degrading enzymes such as cellulases and hemicellulases could be
produced within the crop biomass. However, research is needed to
determine the stability of the biological activity of extracted plant-
produced hydrolysis enzymes. Studies also need to be focused on
increasing the titre of enzyme production and biological activity of
the heterologus enzymes. The plant genetic engineering
approaches could be applied to modify lignin amount and/or
configuration in order to reduce the needs for expensive pretreat-
ment technologies. Recent advances in the understanding of lignin
composition, polymerization and regulation have revealed new
opportunities for the rational manipulation of lignin in future
bioenergy crops, augmenting the previous successful approach of
manipulating lignin monomer biosynthesis. Furthermore, recent
studies on lignin degradation in nature may provide novel
resources for the delignification of dedicated crops and other
sources of lignocellulosic biomass [278]. Recently Fu et al. [284]
have showed that genetic modification of switchgrass can
produce phenotypically normal plants that have reduced thermal-
chemical (180  C), enzymatic, and microbial recalcitrance. Down-
regulation of the switchgrass caffeic acid O-methyltransferase gene
decreases lignin content modestly, reduces the syringyl:guaiacyl
lignin monomer ratio, improves forage quality, and, most impor-
tantly, increases the ethanol yield by up to 38% using conventional
biomass fermentation processes. The down-regulated lines require
less severe pretreatment and 300e400% lower cellulase dosages for
equivalent product yields using simultaneous saccharification and
fermentation with yeast. Furthermore, fermentation of diluted
acid-pretreated transgenic switchgrass using C. thermocellum with
no added enzymes showed better product yields than obtained
with unmodified switchgrass. Therefore, this apparent reduction in
the recalcitrance of transgenic switchgrass has the potential to
lower processing costs for biomass fermentation-derived fuels and
chemicals significantly. Finally, future research on the upregulation
of cellulose and hemicellulose biosynthesis pathway enzymes for
increased polysaccharides will also have the potential to increase
cellulosic biofuel production [285]. The genes necessary for the
biosynthesis of cellulose and hemicellulose are identified by func-
tional genomics and mutant studies. However the complete
biosynthetic pathway is not yet well studied. Although research has
been tremendously focused on plant genetic engineering for bio-
fuel production, this science is still premature. The major challenge
is to develop efficient genotype-nonspecific genetic engineering
systems in feedstock crops.
Advancements in the fields of metabolic engineering, synthetic
biology, and systems biology have further increased the ability to
successfully implement and analyze different strategies to engineer
microbes and plants for the production of a broad range of novel
biofuels through the exploitation of various metabolic pathways.
The continued development of synthetic biology tools that both
reduce the time required to make genetic constructs as well as
increasing their predictability and reliability should greatly
improve metabolic engineering techniques for the effective
production of a wide variety of fuels [286] and chemicals [287]. For
the biomass-derived fuel production, the pathway design is
underway to expedite the hydrolytic conversion from cellulose to
monosaccharides and the subsequent fermentation to obtain C3/C4
alcohols or fatty acids. For successful construction of synthetic
organisms, a hybrid approach has been commonly practiced: based
on the alternate routes and enzymes found in multiple natural
organisms, one may develop efficient transport systems, improve
the hosts’ tolerance against toxic intermediates, optimize carbon
flux to the desired fuels, and establish robust strains that fit mass
production settings [288]. When exposed to a mixture of carbon
537
sources, wild-type E. coli exhibits a specific hierarchy for their
utilization. As long as they are present in sufficient amounts in the
growth medium, sugars such as glucose, sucrose, and fructose
repress the synthesis of enzymes necessary for the transport and
metabolism of less favorable carbon sources such as xylose, arabi-
nose, maltose, and lactose. The development of recombinant E. coli
strains capable of efficiently utilizing sugar mixtures is essential for
the large scale production of biofuels from lignocellulosic materials
[289]. While the efforts to coutilize hexose and pentose sugars in
E. coli through the inactivation of the phosphotransferase system
(PTS) were successful, the existence of other, less understood
regulatory systems that prioritize the sequential metabolism of
sugar mixtures provides another avenue to engineer E. coli strains
for the simultaneous consumption of hexose and pentose sugars.
Fermentative pathways have been utilized to engineer E. coli for the
production of a variety of alcohols, while several non-fermentative
pathways have been engineered to enable the production of bio-
fuels such as higher chain alcohols from the amino acid biosyn-
thetic pathways and novel fuels derived from the fatty acid and
isoprenoid biosynthetic pathways. In contrast to the native E. coli
pathway, the pathway for ethanol production in Zymomonas
mobilis is a two-step process in which pyruvate decarboxylase
(PDC) converts pyruvate directly to acetaldehyde and CO2 and
alcohol dehydrogense (ADH) then converts acetaldehyde into
ethanol. Expression of Z. mobilis pdc and adhB genes (encoding for
pyruvate decarboxylase and alcohol dehydrogenase, respectively)
in E. coli, via a plasmid bearing an artificial pet (production of
ethanol) operon, resulted in much higher ethanol yields, with
ethanol accounting for 95% of the fermentation products [290].
Another recent study developed a minimal E. coli cell for efficient
ethanol production from hexoses and pentoses using elementary
mode analysis to dissect the metabolic network into its basic
building blocks [291]. Analysis and construction of a minimal E. coli
cell expressing the Z. mobilis pdc and adhB genes, resulted in the
conversion of glucose or xylose to ethanol at theoretical yields.
However, recent studies have found that succinate and acetate
were also produced during glycerol fermentation and therefore
represented competing by-products whose synthesis decreases the
yield of ethanol [292,293]. Therefore, metabolic engineering
strategies were implemented in order to minimize by-product
formation through the disruption the genes encoding fumarate
reductase (frdA) and phosphotransacetylase (pta), key enzymes in
the synthesis of succinate and acetate, respectively [294]. Arabinose
is another pentose sugar obtained upon deconstruction of biomass.
Recently, a mutant yeast strain which anaerobically converts
arabinose to ethanol in batch fermentation was reported [295]. This
strain was obtained by introducing the bacterial pathway for
arabinose utilization from Lactobacillus plantarum, overexpressing
S. cerevisiae genes encoding the nonoxidative pentose phosphate
pathway enzymes, and subsequent evolutionary engineering. An
ethanol yield of 0.43 g/g carbohydrate consumed and a specific
ethanol production rate of 0.29 g/g/h from arabinose as the sole
carbon source were achieved. Peralta-Yahya and Keasling [296]
have reported the heterologus expression of the clostridial C3eC4
biosynthetic pathway for the production of isopropanol and 1-
butanol in E. coli and S. cerevisiae. The highest reported produc-
tion of isopropanol by the engineered E.coli is 4.9 g/L compared to
Clostridium (1.8 g/L). The butanol yield obtained from the engi-
neered S. cerevisiae (2.5 mg/L) was two orders-of-magnitude lower
than that obtained by E. coli (550 mg/L). It should be noted that all
these engineering processes will shift toward more and more de
novo design and synthesis, and the pace will heavily depend on the
standardization and quantitative characterization of the biological
parts using miniaturized technologies, such as single-molecule
gene sequencing, gene synthesis on a DNA microchip, and the
538 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550
multifunctional technology found in microenvironments. Finally,
new tools for better de novo design of synthetic pathways need to
be developed. Several databases, such as BNICE (Biochemical
Network Integrated Computational Explorer) [297] and ReBiT
(Retro-Biosynthesis Tool) [298], have already been established to
facilitate identification of enzymes to construct a complete
synthetic pathway for producing target compounds. It is important
to establish guidelines, such as redox balance, energy production,
and thermodynamic feasibility, to screen among these enormous
pathways for the optimal routes [299].
7. Alternative liquid fuels - biobutanol and 2,3-butanediol
Biobutanol or Biobased butanol fuel is second generation alco-
holic fuel with a higher energy density and lower volatility as
compared to ethanol. 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 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 [300]. Butanol’s application as
a replacement for gasoline will outpace ethanol, biodiesel and
hydrogen when its safety and simplicity of use are seen. Butanol’s
application for the Department of Defense as a clean-safe
replacement for batteries when used in conjunction with fuel cell
technology is seen as an application for the future. Disposable
canisters made of PLA that carry butanol to be reformed and used to
generate electricity for computers, night vision and stealth equip-
ment can be easily disposed of [301]. Using fermentation to replace
the chemical process for 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 [302], which are released from wood and
agricultural residues upon hydrolysis, to produce ABE. Parekh et al.
[303] produced ABE from hydrolysates of pine, aspen, and corn
stover using Clostridium acetobutylicum P262. Similarly Marchal
et al. [304] used wheat straw hydrolysate and C. acetobutylicum,
while Soni et al. [305] used bagasse and rice straw hydrolysates and
Clostridium saccharoperbutylacetonicum to convert these agricul-
tural wastes into ABE. Qureshi et al. [306] have studied the
production of butanol from corn fibre hydrolysate using Clos-
tridium beijerinckii BA101. Sun and Liu [307] have reported the
production of butanol from sugar maple hemicellulose hydrolysate
using C. acetobutylicum ATCC824. As compared to the existing
strains of C. beijerinckii BA101, C. acetobutylicum PJC4BK and C.
acetobutylicum P260, which produces a total ABE on the order of
25e33 g/L [306,308e310], development of more robust and effi-
cient second generation cultures are essential for the economic
viability of biobutanol production. Improvements in yields and
a better understanding of the butanol toxicity are among the
potential benefits of engineering the butanol production pathway
into a well known host such as E.coli or S. cerevisiae [311]. The
highest butanol productivity observed in E. coli was 1.2 g L1 over
a 60-h period, with a carbon yield that was 15% of the theoretical
maximum of 0.41 g butanol g1 glucose [312]. Although the latter
value is significantly lower than in native Clostridia, it represents
a promising starting point for further engineering.
Research on the production of butanol from other agricultural
residues including corn stover, barley straw and switchgrass has
steadily progressed. Use of several product-recovery technologies
such as liquideliquid extraction, gas stripping, perstraction, and
pervaporation has been successfully applied in laboratory-scale
bioreactors [313]. It is expected that these recovery technologies
will play a major role in commercialization of this fermentation
[306]. The production of biobutanol from biomass has led to the re-
examination of ABE fermentation, including strategies for reducing
or eliminating butanol toxicity to the culture and for manipulating
the culture to achieve better product specificity and yield. Advances
in integrated fermentation and in situ product removal processes
have resulted in a dramatic reduction of process streams, reduced
butanol toxicity to the fermentating microbes, improved substrate
utilization and overall improved bioreactor [310]. Recently butanol
is a potential gasoline replacement that can also be blended in
significant quantities with conventional diesel fuel. These efforts
have transitioned to research focused on the development of viable
methods for the production of an array of oxygenated and fully
saturated jet and diesel fuels from butanol [301].
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 of many synthetic polymers
and resins [314]. Butanediol is produced during oxygen-limited
growth, by a fermentative pathway known as the mixed acid-
butanediol pathway [315]. The 2,3-BD pathway and the relative
proportions of acetoin and butanediol serve to maintain the
intracellular NAD/NADH balance under changing culture condi-
tions. 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 [316]. Hexose and pentose can be
converted to 2,3-BD by several microorganisms including Aero-
monas [317], Bacillus [318], Paenibacillus [319], Serratia, Aerobacter
[320], Enterobacter [321] and also Klebsiella [322e327]. 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 [317,328], molasses [329], water hyacinth
[330] and Jerusalem artichoke tubers [331] 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 [332]. An industrial
medium containing urea as a sole nitrogen source, low levels of
corn steep liquor and mineral salts as nutrition factors to retain
high 2,3-butanediol production through co-fermentation of
glucose and xylose (2:1, wt/wt) by Klebsiella oxytoca was developed
to yield 0.428 g/g of 2,3-BD, which was 85.6% of theoretical value
[326]. Cheng et al. [327] have reported production of 2,3-
butanediol from corn cob hydrolysate by fed batch fermentation
using K. oxytoca. A maximal 2,3-butanediol concentration of 35.7 g/
l was obtained after 60 h of fed-batch fermentation, giving a yield of
0.5 g/g reducing sugar and a productivity of 0.59 g/h/l.
There are numerous reports on ABE fermentation processes
from the last 30 years, which together accomplishes advancements
in butanol production. In recent years several economic studies
have been performed on the production of butanol from corn (dry
corn and wet corn milling) whey permeate, and molasses
[309,333]. In these studies it was determined that the distillative
recovery of biobutanol from the fermentation broth is not
economical when compared with butanol derived from the current
petrochemical route. Nevertheless, studies employing C. beijerinckii
BA101, C. acetobutylicum P260, hydrolyzed fiber-rich distillers
dried grains and solubles (DDGS) and wheat straw suggest that
commercial production of biobutanol from agricultural byproducts/
 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550
wastes is drawing closer. Increased butanol concentrations,
productivity, and yields were achieved by the establishment of high
cell density continuous cultures by cell immobilization or by cell
recycling together with cell bleeding, fed-batch culture integrated
with in-situ butanol recovery system, and butanol production from
butyric acid in living cells [334]. The enzymes responsible for
butanol production should be characterized in order to deepen our
understanding of the thermodynamics and kinetics of the meta-
bolic pathway. Industrial biobutanol production currently lags
behind production of bioethanol and biodiesel however significant
amount of progress has been made on the development of new
process technologies, including studies with high-productivity
reactors and energy-efficient product-recovery systems. Recently,
DuPont (US) and BP (UK) announced their plans to invest in bio-
butanol production research. It is anticipated that the first plants
would operate on sugar or corn starch; however, it is likely that
agricultural waste would become a potential substrate in the near
future. The use of lignocellulosic substrates in combination with
developed process technologies is expected to make the production
of biobutanol economically viable [310,335].
8. Biorefinery perspectives
The development and implementation of biorefinery processes
is of upmost importance to meet the vision towards a sustainable
economy based on bio-resources. Biorefinery concept is to utilize
inedible lignocellulosic biomass to produce biofuels, cellulose,
hemicellulose, lignin and byproducts. Replacement of petroleum-
derived chemicals with those from biomass will play a key role in
sustaining the growth of the chemical industry. Contrary to petro-
resources which are limited in nature and composition, the bio-
resource are composed of heterogenous compounds such as
cellulose, hemicellulose, oils, lignin, starch and proteins. Each
constituent in the biomass (plant) can be functionalized in order to
produce non-food and food fractions, intermediate agro-industrial
products and synthons [336]. Thus, a complete set of specific
technologies must be developed in order to convert each fraction as
efficiently into value added products. These fractions can be used
directly as desired biochemicals or can be converted by chemical,
enzymatic, and/or microbial approaches. Conversion of these by-
products to high-value co-products will offset the cost of biofuel,
improve the economy of lignocellulose biorefinery, minimize the
waste discharge, and reduce the dependence of petroleum-based
products. The biorefinery will offer new economic opportunities
for agriculture and chemical industries by the production of
a tremendous variety of chemicals, transportation fuels, and energy
[337]. To develop technologically sustainable bio-refinery routes,
the entire chain of biomass production, i.e., from breeding, culti-
vation, and harvest, its (pre)treatment, and conversion to products
should be considered. The relatively low energy content, season-
ality and discrete geographic availability of biomass feedstocks
have been noted as barriers to the large volume demands for
energy and fuel [338]. In addition, it will be shown that small scale
(pre)processing of the biomass may be advantageous over large-
scale processing [339]. Conceptually, a biorefinery would apply
hybrid technologies from different fields including polymer
chemistry, bioengineering and agriculture [340].
The U.S. Department of Energy (DOE) identified high-volume
commodity chemicals that could be produced from biomass and
can serve as starting materials for many chemical products via
biological processes [341]. Ethanol is perhaps the most widely
known example of a bio-based chemical produced worldwide;
however there is a range of bioproducts produced and used in
a variety of industrial applications. These fall into several generic
categories such as naturally occurring carbohydrate polymers, fats
539
and oils from plant origin, terpene based materials, chemical
products of carbohydrate containing materials, fermentation
products of carbohydrate containing sources. Cellulosic plant
materials are used as fuel, lumber, mechanical pulps and textiles.
Purified cellulose is currently used to make wood-free paper,
cellophane, photographic film, membranes, explosives, textile
fibres, water-soluble gums, and organic-solvent-soluble polymers
used in lacquers and varnishes. The principal cellulose derivative is
cellulose acetate a biodegradable, which is used to make photo-
graphic film, acetate rayon, various thermoplastic products, and
lacquers [342]. Lyocell is the first new textile fibre, commercially
produced from a solvent spinning process. Specialty chemicals can
be made using fermentation and enzymatic processes. Several
commodity chemicals are already produced by fermentation,
including glutamic acid (w1.7 billion kg/year worldwide), citric
acid (w1.6 billion kg/year), and lysine (w850 million kg/year)
(346). In addition to these commodity chemicals, commodity
polymers may also be produced using biologically produced
monomers combined with classical chemical methods, as well as
through biological polymerization methods, either with isolated
enzymes or in actively growing cells. Lactic acid produced by
fermentation [344] can be converted chemically to methyl lactate,
lactide, and polylactic acid. The latter polymer commercially
available under different trade names is a fully biodegradable
replacement for polyethylene terephthalates [345]. Efficient
processes for converting biologically produced lactic and hydrox-
ypropionic acids to methacrylic and acrylic acids are being devel-
oped. Genencor and DuPont, who have developed a cost-effective
fermentative route to 1,3-propanediol, the key building block in
poly- (propyleneterephthalate), which is not readily available from
petrochemical feedstocks [346]. Fermentation by various microor-
ganisms has been used to produce succinic acid [347,348], which
may potentially replace maleic anhydride, now produced from
butane. Recently, chemical companies such as Dow Chemical
Company, Huntsman Corporation, Cargill and Archer Daniels
Midland Corporation, have begun to use glycerol as a low-cost
building block material for conversion to higher value propylene
glycol. Furthermore, Dow Chemical Company and Solvay are
exploring the use of glycerol in the production of epichlorohydrin,
which can be used in the manufacture of epoxy resins and
epichlorohydrin elastomers [343]. Due to its versatility, it is antic-
ipated that glycerol could become a substitute for many commonly
used petrochemicals [349]. A promising sugar-lignin platform
potential for the generation of value-added bioproducts under the
lignocellulosic biorefinery concept is illustrated in Table 6. The
current situation indicates that the demand for food, energy,
mobility, chemicals and materials will increase tremendously in the
near future. 80e90% of the fossil resources are used for energy
supply and mobility. Being aware that a coupling between world
markets for energy and fuels, raw materials for chemicals and
materials, feed and food and biomass exists one has to solve the
problems of raw material supply for food production as well as for
materials, fuels and energy production [336].
The effective use of biomass feedstocks, particularly lignocel-
lulosic materials in large-scale applications will evolve from inno-
vative research aimed at the development and implementation of
biorefineries e multi-step, multi-product facilities established for
specific bio-sourced feedstocks (340). In the realization of a bio-
based chemical industry two distinct approaches can be identi-
fied. In the first approach, the value chain approach, value added
compounds in biomass are identified and isolated in different
processing and (bio)conversion steps. The remaining biomass is
then transformed into a universal substrate from which chemical
products can be synthesized. In this approach it is thought that is
technologically and economically beneficial to extract valuable
540 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550

Table 6 the demand for food, energy, mobility, chemicals and materials will
Value added biochemicals potentially derived from cellulose, hemicellulose and increase tremendously in the near future. 80e90% of the fossil
lignin.
resources are used for energy supply and mobility. Being aware that
Lignocellulosic Cellulose Polymers a coupling between world markets for energy and fuels, raw
biomass Levulinic acid Succinic acid, THF, materials for chemicals and materials, feed and food and biomass
MTHF, 1,4 butanediol,
NMP, Lactones
exists one has to solve the problems of raw material supply for food
Ethanol production as well as for materials, fuels and energy production
Lactic acid Acrylic acid, [336]. Biomass feedstocks available decentrally will be more
Acetaldehyde commodious for localized biorefinery approach than the exhaustive
2,3-pentanedione,
large scale and centralized plants driven by cost intensive tech-
Pyruvic acid
3-hydroxy- nology. Depending on regional and technological boundary condi-
propanioc acid tions the most economic solution for a biorefinery may be provided
Itaconic acid 3-methyl THF, by large centralized facilities as well as by smaller decentralized
3-methyl pyrrolidone plants [350].
2,methyl-1,4- butane
diamine
Energy and GHG emission savings from bio-based polymers in
Itaconic diamide specific terms were found to be 20e50 GJ/t polymer and 1.0e4.0 t
Glutamic acid CO2eq/t polymer respectively. Bio-based polymers are attractive in
Glucuronic acid terms of specific energy and emissions savings and cannot offset
Succinic acid 2-pyrrolidones,
the additional environmental burden due to the growth of petro-
1,4- butanediol,
Tetrahydrofurane chemical polymers [351]. Within the next two decades, bio-based
Hemicellulose Xylitol polymers will not be able to compensate the environmental
Ethanol, butanol, impacts of the economy as a whole. The potential environmental
hydrogen and socio-economic effects studied concluded that while environ-
2,3-butanediol
Ferulic acid Vanillin, Vanillic acid,
mental effects in specific terms are high, effects in absolute terms
Protocatechuic acid relative to those of total industry or society are low. There is low job
Lactic acid creation potential. It must be emphasized that these relatively low
Furfural contributions have their reason in the comparatively low produc-
Chitosan
tion volumes of bio-based polymers until 2020 [352,353].
Xylo-
oligosaccharides
Lignin Syngas 9. Lifecycle assessment of lignocellulosic ethanol
Syngas products Methanol/Dimethy
ether, Ethanol, Mixed A life-cycle assessment (LCA) takes into account all resource and
liquid fuels
Hydrocarbons Cyclohexanes, higher
energy inputs required to make a product, the wastes, and the
alkylates health and ecological burdens associated with the product. In effect,
Phenols Cresols, Eugenol, performing a life-cycle assessment of the entire ethanol production
Coniferols, Syringols system (preproduction through consumption) would quantify the
Oxidized products Vanillin, vanillic acid,
total benefits as well as any drawbacks that the process might
DMSO, aldehydes,
Quinones, aromatic contain and identify opportunities for process improvement [353].
and aliphatic acids LCA techniques allow detailed analysis of material and energy fluxes
Macromolecules Carbon fibres, on regional and global scales. At the same time if not used properly,
Activated carbon, LCA can lead to incorrect and inappropriate actions on the part of
polymer alloys,
industry and/or policy makers [354]. Earlier technoeconomic or life-
polyelectrolites,
substituted lignins, cycle analyses of ethanol from lignocellulosic biomass have focused
thermosets, on energy, greenhouse gases, criteria pollutants, and economics.
composites, wood Lynd et al. [355] identified costs of production as the key barrier,
preservatives,
while acknowledging environmental and energy benefits using
Neutraceuticals/drugs,
adhesives and resins a limited life-cycle analysis that excluded pre-manufacturing
stages. Wang et al. [356] showed that corn ethanol used in
conventional internal combustion engines reduces fossil energy
chemicals and polymers from biomass rather then building these consumption between 28% and 50%, but only ethanol from ligno-
compounds from universal building blocks. It can be concluded that cellulose can achieve reductions over 90%. Similarly, Sheehan et al.
the main technological challenges to aid the economic feasibility of [357] conducted a thorough life-cycle assessment of ethanol
this approach lay in the area of biomass refining, separation tech- production from corn stover in Iowa and discovered that E85 (85%
nology and bioconversion technology. Moreover, a far reaching ethanol blend with gasoline) reduces petroleum consumption 95%
integration of food, feed and chemical industries is required as well per kilometer traveled compared to conventional gasoline, reduces
as a major investment in infrastructure. The second approach, the ozone precursor emissions, reduces total fossil energy consumption
integrated process chain approach, follows the analog of the and greenhouse gas emissions by 102% and 113%, respectively, but
petrochemical industry. In this scheme a ”universal” substrate is increases emissions of CO, NOx, and SOx. However, none of these
first transformed into universal building blocks, based on which studies investigated the effects of process improvements on life-
chemical products are produced. In this approach it is thought that cycle impacts. Kemppainen and Shonnard [353] have studied the
it is economically and technologically beneficial to build chemicals LCA approach to evaluate to evaluate the environmental impacts of
in highly integrated production facilities. The main technological a lignocellulosic biomass-to-ethanol process using two different
challenges for this approach lay in the high-efficient trans- regional biomass options: timber and recycled newsprint. Readers
formation of biomass into commonly known building blocks for the are referred to various monographs on LCA for potential use of
petrochemical industry [337]. The current situation indicates that lignocellulosic feedstocks in bioethanol production [354,358e360]
 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550
which elaborates various factors (type of biomass, ethanol conver-
sion technologies, utilization of produced ethanol, system
boundary, byproduct allocation and reference system) affecting the
outcome of the analysis. Mu et al. [361] have explored the potentials
of the biochemical and thermochemical conversion pathways,
different technological scenarios were modeled, including current,
2012 and 2020 technology targets, as well as different production/
co-production configurations. The modeling results suggest that
biochemical conversion has slightly better performance on green-
house gas emission and fossil fuel consumption, but that thermo-
chemical conversion has significantly less direct, indirect, and
lifecycle water consumption. Also, if the thermochemical plant
operates as a biorefinery with mixed alcohol coproducts separated
for chemicals, it has the potential to achieve better performance
than biochemical pathway across all environmental impact cate-
gories considered due to higher co-product credits associated with
chemicals being displaced. The results from this work serve as
a starting point for developing full life cycle assessment model that
facilitates effective decision-making regarding lignocellulosic
ethanol production.
Biofuels production as a strategy to mitigate global warming is
currently under debate due to the influence of including direct and
indirect land-use changes in the GHG balance of biofuels. Increased
demand for biofuels in order to partially substitute fossil fuels in
different parts of the world will require the use of a significant
amount of biomass. Energy crops production on current land and
use of biomass in a given region can induce displacement of
activities and land-use changes elsewhere. This effect is known as
indirect land-use change (ILUC). Due to changes in the carbon stock
of the soil and the biomass, ILUC has consequences on the GHG
balance of a biofuel that are not presently considered in the eval-
uation of the environmental merits of biofuels. Significant changes
in land-use are expected to occur in biofuel producing countries
and their consequences may affect global markets. Greenhouse gas
(GHG) emissions reduction is one of the main drivers of biofuels
development. However, this assumed benefit is now under
discussion, especially due to emissions from land-use change (LUC).
Therefore some countries and regions worldwide being experi-
enced biofuels blending policies (e.g. the EU, the UK, Switzerland,
the Netherlands, State of California in the US) have defined a carbon
conservation criterion (i.e. an emissions reduction target) that must
be fulfilled in order to trade and supply biofuels. Thus biofuel
importing countries should select production regions that satisfy
this criterion. Recent studies have highlighted both positive and
negative environmental and socioeconomic effects of bioenergy
and the associated agriculture and forestry LUC [362,363]. Like
conventional agriculture and forestry systems, bioenergy can
exacerbate soil and vegetation degradation associated with over-
exploitation of forests, too intensive crop and forest residue
removal, and water overuse [364,365]. Diversion of crops or land
into bioenergy production can influence food commodity prices
and food security [366]. With proper operational management, the
positive effects can include enhanced biodiversity [367,368], soil
carbon increases and improved soil productivity [369,370], reduced
shallow landslides and local flash floods, reduced wind and water
erosion and reduced sediment volume and nutrients transported
into river systems [371]. For forests, bioenergy can improve growth
and productivity, improve site conditions for replanting and reduce
wildfire risk [372]. However, forest residue harvesting can have
negative impacts such as the loss of coarse woody debris that
provides essential habitat for forest species.
It is overly simplistic and inaccurate to view land use change
worldwide as being driven primarily by increased agricultural
production, as has been assumed [373]. There is a rich academic
literature on the subject of land use change [374,375]. According to
541
these studies, land use change is driven by three primary forces:
timber harvest, infrastructure development (e.g., road building),
and agricultural expansion. Any one of these variables taken alone
explains less than 20% of documented land use changes worldwide.
Taken together, they explain over 90% of observed cases of land use
change. Agricultural expansion alone is therefore seldom the
reason for land use change. Thus it is arbitrary and unreasonable to
assume that all land use change worldwide is driven primarily by
agricultural expansion. Whatever the final result of the ongoing
debate about the validity and limits of the indirect land use change
analysis, both direct and indirect LUC analyses depend on a number
of variables and assumptions [376].
Studies of environmental effects, including those focused on
energy balances and GHG emission balances, usually employ
methodologies in line with the principles, framework, require-
ments and guidelines in the ISO 14040:2006 and 14044:2006
standards for Life Cycle Assessment (LCA) [4]. LCA studies of
prospective bioenergy options involve projections of technology
performance and have relatively greater uncertainties. The way
that uncertainties and parameter sensitivities are handled across
the supply chain to fuel production significantly impacts the
results. Studies combining several LCA models and/or Monte Carlo
analysis provide bioenergy system uncertainties and levels of
confidence for some bioenergy options [377e379]. The chosen
combination of production options to satisfy a given demand of
biofuels is the result of a multi-criteria decision based on
economical, political, technological, agro-ecological, social and
environmental factors, which determined the local framework for
biofuels production. This decision will have an impact on LUC, and
therefore on the GHG emissions balance of the biofuel. Conse-
quently, reducing GHG emissions from direct (DLUC) and indirect
LUC (ILUC) is a key element to optimize the environmental
performance of a given biofuel pathway. It can be concluded that
LUC can affect GHG balances in several ways, with beneficial or
detrimental outcomes for bioenergy’s contribution to climate
change mitigation, depending on conditions and context. When
land high in carbon (notably forests and especially peat soil forests)
is converted to bioenergy, upfront emissions may cause a time lag
of decades to centuries before net emission savings are achieved.
But the establishment of bioenergy plantations can also lead to
assimilation of CO2 into soils and aboveground biomass in the short
term. Increased utilization of forest biomass can reduce forest
carbon stocks. The use of post-consumer organic waste and by-
products from the agricultural and forest industries does not
cause LUC if these biomass sources were not utilized for alternative
purposes. Bioenergy feedstocks can be produced in combination
with food and fibre, avoiding land use displacement and improving
the productive use of land. Lignocellulosic feedstocks for bioenergy
can decrease the pressure on prime cropping land. Stimulation of
increased productivity in all forms of land use reduces the LUC
pressure.
Air pollutant emissions from bioenergy production depend on
technology, fuel properties, process conditions and installed
emission reduction technologies. Compared to coal and oil
stationary applications, sulphur dioxide (SO2) and nitrous oxide
(NOx) emissions from bioenergy applications are mostly lower.
When biofuel replaces gasoline and diesel in the transport sector,
SO2 emissions are reduced, but changes in NOx emissions depend
on the substitution pattern and technology [4]. Bioenergy
production can have both positive and negative effects on water
resources. Bioenergy production generally consumes more water
than gasoline production [380,381]. However, this relationship and
the water impacts of bioenergy production are highly dependent
on location, the specific feedstock, production methods and the
supply chain element. Feedstock cultivation can lead to leaching
542 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550
and emission of nutrients that increase eutrophication of aquatic
ecosystems [363,382]. The subsequent processing of the feedstock
into biofuels and electricity can increase chemical and thermal
pollution loads from effluents and generate waste to aquatic
systems [383,384]. These environmental impacts can be reduced if
suitable equipment is installed [385,386]. Increased biomass
output for bioenergy can directly impact wild biodiversity through
conversion of natural ecosystems into bioenergy plantations or
through changed forest management. Habitat and biodiversity loss
may also occur indirectly, such as when productive land use dis-
placed by energy crops is re-established by converting natural
ecosystems into croplands or pastures elsewhere. Because biomass
feedstocks can generally be produced most efficiently in tropical
regions, there are strong economic incentives to replace tropical
natural ecosystemsdmany of which host high biodiversity values
[387]. However, forest clearing is mostly influenced by local social,
economic, technological, biophysical, political and demographic
forces [388]. The considerable soil impacts of increased biofuel
production include soil carbon oxidation, changed rates of soil
erosion, and nutrient leaching. However, these effects are heavily
dependent on agronomic techniques and the feedstock under
consideration [389]. The overall performance of bioenergy
production systems is therefore interlinked with management of
land use and water resources.
10. Current global status of lignocellulosic ethanol industry
The commercialization of cellulose to ethanol technology in the
present-day scenario is not economically viable. Currently there are
special programs in a number of countries targeting production of
biofuel such as biogas, bioethanol, biodiesel and fuel cells from
renewable resources [390]. Global production of bioethanol
increased from 17.25 billion liters in 2000 to over 46 billion liters in
2007, which represented about 4% of the 1300 billion liters of
gasoline consumed globally [391]. With all of the new government
programs in America, Asia, and Europe in place, total global fuel
bioethanol demand could grow to exceed 125 billion liters by 2020
[392]. Bioenergy ranks second (to hydropower) and accounts for 3%
in renewable primary energy production in U.S [393]. The United
States is the world’s largest producer of bioethanol fuel, accounting
for nearly 47% of global bioethanol production in 2005 and 2006
[394]. The “Biofuels Initiative” in the U.S. (US Department of Energy),
strives to make cellulosic ethanol cost-competitive by 2012 and
supposedly to correspond and account for one third of the U.S. fuel
consumption by 2030. In 2007, the U.S. president signed the Energy
Independence and Security Act of 2007 (EISA, 2007), which requires
34 billion liters of bio-fuels (mainly bioethanol) in 2008, increasing
steadily to 57.5 billion liters in 2012 and to 136 billion liters in 2022.
Companies such as POET (earlier called Broin) and Abengoa are
building refineries that can process biomass and turn it into ethanol,
while companies such as Genencor, Diversa, Novozymes and Dyadic
are producing enzymes which could enable a cellulosic ethanol
future. Brazil is the world’s largest exporter of bioethanol and
second largest producer after the United States. Ethanol from
sugarcane provides 40% of automobile fuel in Brazil and approxi-
mately 20% is exported to the U.S., EU, and other markets.
Commercial scale plants are expected to be operational by 2012.
In US, plants built with substantial federal funding totaling 12
million gallon per year are operational with an additional 21.3
million gallon of capacity- 26 new plants- under construction.
There are more than 10 ethanol biofuel facilities either in operation
or under construction in Canada [395,396]. In eastern Canada and
the U.S., corn is used as the feedstock while in western Canada
wheat is used. Many Asian countries such as China, India, Japan,
and Indonesia are also developing ethanol production capacity
[397,398]. Iogen Corp, Canada, is a developer of cellulosic ethanol
process technology. Iogen has developed a proprietary process and
operates a demonstration-scale plant in Ontario. The facility has
been designed and engineered to process 40 tons of wheat straw
per day into ethanol using enzymes from in-house enzyme
manufacturing facility. Lignol is a Canadian company which is
undertaking to construct biorefineries for the production of fuel-
grade ethanol and biochemicals from Canadian forests and vast
supplies of biomass feedstocks. Lignol has acquired and since
modified, a solvent based pre-treatment technology Lignol has
recently produced ethanol and other biochemicals from both
hardwood and softwood species representative of Canadian forests.
Ethtec is building a pilot plant in Harwood, New south Wales,
Australia which uses wood residues as a feedstock. Another plant
has been built by Queensland University of Technology near Bris-
bane. SEKAB, Sweden has developed an industrial process for
production of ethanol from biomass feedstocks, including wood
chips and sugarcane bagasse. The technology will be gradually
scaled up to commercial production in a new breed of bio-refineries
from 2013 to 2015. A $ 400 million investment programme to cover
the construction of a World scale ethanol plant and a high tech-
nology demonstration plant to advance development work on the
next generation biofuels has been announced by BP, Associated
British Foods (ABF) and DuPont. The bioethanol plant with an
annual production capacity of 420 million liters of biofuel from
wheat feedstock will be built on BP’s existing chemicals site at
Saltend, Hull, UK. In India, Praj Industries have recently started
a demonstration plant that can process 2 tonnes per day of ligno-
cellulosic feedstocks such as corn stover, corn cob, bagasse, agro
waste and wood chips for biofuel production [57]. A few companies
have operated pilot plants (Table 7), however no commercial
industrial scale plants for biofuel production is in operation. China
Resources Alcohol Corporation (CRAC) has announced their inten-
tion to construct sufficient cellulosic ethanol facilities to generate
330 million gallons of ethanol by 2012. SunOpta provided its
patented systems and technology to CRAC in September 2006 and
the plant began production of ethanol from local corn stover in
October 2006. This facility is reported to be the first cellulosic
ethanol production facility operational in the People’s Republic of
China. The SunOpta system is currently operating on a continuous
basis and steps are currently being taken to scale the SunOpta
process up to full commercial levels for use in future plants in China.
China has committed $5 billion to cellulosic ethanol production and
recently announced that they would allow no further increase in
ethanol production from starch (corn), due to the needs for starch as
food. Nippon Oil Corporation and other Japanese manufacturers
including Toyota Motors Corporation plans to set up a research body
to develop cellulose-derived biofuels. The consortium plans to
produce 1.6 million barrels per year of bioethanol by March 2014
and produce bioethanol at $0.437 per litre by 2015.
Recently research is directed towards identifying, evaluating,
developing and demonstrating different pretreatment methods
that result in efficient enzymatic hydrolysis. Several physical,
physico-chemical, chemical and biological pretreatments or their
combinations are under evaluation. Due to the high cost of enzyme
the current fuel grade ethanol produced from lignocellulosic
material is still not able to compete with gasoline. In a contempo-
rary process of lignocellulosic ethanol which is being worked out
for more than 2e3 decades is still not materialized into a viable
technology. The permissible cost of enzymes is 15e30 cents/gallon
of ethanol which is still not a reality. The lignocellulosic biomass
feedstock including pretreatment costs around 50e80 cents/gallon
of ethanol [399]. Today cellulosic ethanol still lies around approx
US$ 4 per gallon of ethanol based on best estimates [400]. The
cellulase cost has been reduced dramatically from US$5.40 per
 V. Menon, M. Rao / Progress in Energy and Combustion Science 38 (2012) 522e550 543

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

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

Abengoa Spain 4.000 t/a EtOH Demo facility, Wheat straw Acid catalysed steam As co-product, Recovered after
start-up 2009 explosion, Enzymatic distillation
hydrolysis
Inbicon Denmark 4.000 t/a EtOH Demofacility, Wheat straw Liquid hot water Solid biofuel for power-plant,
C5-molasses start-up 2009 (hydro-thermal, auto Recovered after distillation
Solid biofuel catalysed)
Iogen Canada 70.000 t/a EtOH Commercial facility, Straw (wheat, barley, oat) Modified steam explosion, For steam and electricity
start-up 2011 Enzymatic hydrolysis generation,
Recovered after enzymatic
hydrolysis
KL Energy USA 4.500 t/a EtOH Demo facility, Wood waste, cardboard Thermo mechanical For steam or electricity
operational and paper generation, or as wood pellet
since 2007 co-product,
Recovered after distillation
SEKAB Sweden 4.500 t/a EtOH Demo facility, Wood chips or sugarcane Acid pre-treatment For energy production or other
start-up 2011d bagasse uses Recovered after
(enzymatic) hydrolysis
Verenium USA 4.200 t/a EtOH Demo facility, Sugarcane bagasse, Mild acid hydrolysis Lignin-rich residue burned for
process operational energy crops, wood and steam explosion steam generation Recovered
since 2009 products and switchgrass after distillation
Range fuels Georgia 20 million gallon/ Commercial facility, Woody biomass and Thermo-chemical For energy production
annum EtOH start up at 2010 grasses process
POET LLC Emmetsburg 55 million Pilot plant facility Corn cob Biogas production
IA gallon/annum
EtOH

Sources: Biotechnology Industry Organization (BIO), companies.

gallon of ethanol to approximately 20 cents per gallon of ethanol;
further efforts are carried out to reduce the costs below 5 cents per
gallon ethanol. The hydrolysis and subsequent fermentation of
lignocellulose is complicated and the economic viability of the
process is yet to be achieved.
11. Future prospects
Although bioethanol production has been greatly improved by
new technologies there are still challenges that need further inves-
tigations. Tremendous focus is essential for developing a detailed
understanding of lignocellulose, the main structural material in
plants, from cellulose synthesis and fibril formation to a mature
plant cell wall, forming a foundation for significant advancement in
sustainable energy and materials. Characterization, understanding
and overcoming the barriers for enzymatic hydrolysis of different
raw material is essential for the development of economically
competitive processes based on enzymatic treatments.
Availability of feedstocks for biofuel production, their variability
and sustainability are major criteria’s to be addressed. Each raw
material requires a different processing and pretreatment strategy
which has to be tailored taking in to consideration their composi-
tion and susceptibility to such treatments. Physical/chemical/
physico-chemical or combinations thereof needs to be optimized
for pretreatment of each feedstocks. The pretreatment must be
advanced and appropriately integrated with the rest of the process
to achieve the complete potential of lignocellulosic ethanol.
Another major challenging area of research is to develop low cost
effective enzymes for lignocellulose saccharification of the pre-
treated biomass. Extensive research in developing new technolo-
gies for high solid handling is also a major objective. Improvements
in fermentation technology and media optimization approaches
have to be performed along with genetic engineering techniques to
improve the yield and efficiency of cellulases. Although enzyme
costs have decreased in the last few years, this is still at its pre-
mature state. The high cost of enzyme production and the
requirement of higher enzyme dosage for hydrolysis of biomass are
considered to be main hurdles for the economic viability of ligno-
cellulosic bioethanol. Synthetic biology and metabolic engineering
approaches has to be utilized for developing efficient and robust
microbes for SSF, SSCF and CBP processes. Integration of processes
for reducing the number of process steps, energy demands and re-
use of process streams to make the conversion process economical.
Advances in the cost-effective conversion of lignocellulosic
biomass are often difficult to assess accurately because of the lack of
integrated process configurations. Despite current expectations,
significant uncertainty remains on the different environmental and
sustainability factors on the performance of second generation
biofuel in a commercial scale. Tremendous R&D studies are
improving the conversion process but the issue of feedstock avail-
ability and revenue stability remain uncertain and subject to
political risks. 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. Even though
developing the technology for cost-effective motor fuel production
by 2030 is challenging, the advances in scientific understanding
necessary to achieve this goal appear realizable.
Acknowledgements
The authors acknowledge the financial support and the senior
research fellowship from CSIR Emeritus Scheme, Govt. of India
respectively.
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