Renewable and Sustainable Energy Reviews 73 (2017) 798–820

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Renewable and Sustainable Energy Reviews

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Status of availability of lignocellulosic feed stocks in India: Biotechnological MARK

strategies involved in the production of Bioethanol
⁎
Gentela Jahnavi, Govumoni Sai Prashanthi, Koti Sravanthi, Linga Venkateswar Rao
Department of Microbiology, Osmania University, Hyderabad, Telangana State 500 007, India

A R T I C L E I N F O A BS T RAC T

Keywords: Bioethanol, as a biofuel has got an exceptionally noticeable role in the world. It is a solution to the overwhelming
Lignocellulosic biomass reliance on petroleum based products, in terms of energy security, impact on environment and climatic change
Pretreatment caused by vehicular pollution. There are many setbacks associated with the usage of petrol based resources.
Acid hydrolysis India, being agriculturally enriched, produces bountiful amounts of residues after harvest of the crop. Taking
Enzymatic hydrolysis
this into consideration, the review manly explains about the availability of agroresidues in India and
Genetic engineering
Consolidated bioprocessing
emphasizing the requirement for pretreatment and saccharification methods like acidic and enzymatic
Simultaneous saccharification and techniques. The mode of action of these treatment methods on lignocellulosic wastes were also discussed.
fermentation We also focused on the role of microorganisms in fermentation and the need for genetic engineering approach,
with a detailed discussion on CBP and SSF of lignocellulosic material to ethanol. Finally, this article concludes
with a brief investigation on biofuels from algae, an emerging technology for third generation biofuels.

1. Introduction availability scenarios and issued the recommendations to expand the

Bioethanol is the cutting-edge technology to petroleum since it is
produced from renewable and sustainable biomass, generates less
emissions than fossil fuels, produces no net CO2 and is compatible
with current fuel distribution infrastructure. India is a nation with an
impetus in uplifting view point towards renewable energy innovations
and focusing on the utilization of renewable sources to supplement its
energy necessities. For the last 20 years, Governments all over the
world are involved and have been participating actively in identifying,
developing and commercializing the technology to produce a substitute
to transportation fuels [1], since vehicular pollution is evaluated to
have amplified eight times over the last two decades. Most of the air
pollution is contributed specially by automobiles. So, it is indeed a very
emergent need for the countries to substitute fossil fuels with the bio
based fuels using feed stocks. India along with other nations is very
keen to develop bio fuel technology because of the ascent in oil costs
and fuel emissions have become a great threat to the nations while the
country also has an aspiration to diminish the green house gas
emissions. In April 2003, the planning commission released a report
on bio fuels, where India further strengthened its bioethanol program
[2], the report analyzed various blending targets, price and feedstock
industry to meet 5–10% blends of bio ethanol. Later the 11th five-year
plan (2007–2012) suggested to increase the mandatory bioethanol
blending to 10% once E5 blends were put in place across the country
[3] The E10 blending target remains in effect and will be scaled up to
E20 by 2017, as proposed by the country’s recently enacted National
Policy on Biofuels. India’s energy security would remain vulnerable
until alternative fuels produced from renewable feedstock are devel-
oped to substitute petro-based fuels [4]. India is one among other
nations with a separate ministry which is involved in the development
of renewable energy resources [5]. The Ministry of Petroleum &
Natural Gas (MoPNG), Government of India mandated 5% ethanol
blend in gasoline by the oil marketing companies (OMCs). The 5%
blending was initiated after consulting with the key stakeholders at the
state and central government levels, including the society for Indian
automobile and major sugar manufacturers [6]. This was put up in nine
Indian states and four union territories with effect from January 2003,
through its ambitious ‘Ethanol Blending Program’ (EBP). A Committee
was constituted in July 2002 by the planning commission and the final
report was released in July 2003. The report encouraged India to
develop and gradually move towards the use of biofuels [7]. In a
nation-wide survey conducted by IMRB employed by NIIST, sponsored

Abbreviations: GHG, Green house gases; IMRB, Indian Market Research Bureau; NIIST, National Institute of Inter Disciplinary Science and Technology; TIFAC, Technology
Information, Forecasting and Assessment Council; IIT, Indian Institute of Technology; TERI, Tata Energy Research Institute; ICRISAT, International Crops Research Institute for the
Semi-Arid Tropics
⁎
Corresponding author.
E-mail address: vrlinga@gmail.com (L.V. Rao).

http://dx.doi.org/10.1016/j.rser.2017.02.018
Received 30 September 2015; Received in revised form 23 November 2016; Accepted 3 February 2017
1364-0321/ © 2017 Published by Elsevier Ltd.
G. Jahnavi et al.
by TIFAC, Government of India has reported the availability of biomass
residues which account for about 80% generated mostly by crops [8].
India has long been into lignocellulosic to ethanol research with the
innovative efforts of Biochemical Engineering Research Centre, IIT,
Delhi. In 1980, they instituted a demonstration for production of 50 L
bioethanol/day. Rice straw was used as feed stock for this process with
an ethanol yield of 230 L/ton. [9]. In a study conducted by Arvind Lali
(DBT-ICT Centre for Energy Biosciences, Institute of Chemical
Technology, India) the current situation in India is that the land per
capita is less than Canada, USA, Brazil and Australia and to resolve few
intricacies involved in the production, marginal lands must be used to
develop suitable crop varieties. The agroresidues can be used comple-
tely and judiciously. In response to the Government’s initiatives on
Make in India and Swachh Bharat Abhiyan, the Department of
Biotechnology, Ministry of Science and Technology supported DBT-
ICT Centre for energy biosciences at ICT Mumbai, and has successfully
demonstrated for the first time the production of ethanol from
lignocellulosic biomass. India’s first 2G or Cellulosic Ethanol (alcohol)
demonstration plant with a capacity of 10 t/day was set up on 22nd of
April 2016 at India Glycols Ltd., Kashipur, Uttarakhand. Using this
technology and plant, any biomass feedstock like wheat straw, rice
straw, bagasse, cotton stalk, bamboo, etc. will be converted to alcohol
in less than 24 h, and if effectively operated and scaled-up will make
India a global technology provider in the field of renewable resources
and results in the reduction of carbon-emissions other than affecting
significant savings in import of unrefined petroleum. The DBT-ICT
Centre has developed designs of plants which can convert 250 t/day
and 500 t/day of biomass. The Department of Biotechnology is
confident that this technology with the lowest capital and operating
costs would allow 2G-Alcohol to be produced and sold at competitive
price. http://www.dbtindia.nic.in/india%E2%80%99 s-first-cellulosic-
alcohol-technology-demonstration-plant-inaugurated/.
In our earlier review by Chandel et al. [10] have tried to explain the
availability of agrowastes in Andhra Pradesh since it is agriculturally
enriched state. The current review focuses on the availability of the
agricultural residues in India, the strategies involved in the bioconver-
sion of these wastes to bioethanol and engineering the genetic makeup
in yeasts and a note on fermentation methodologies like consolidated
bioprocessing and simultaneous saccharification and fermentation.
1.1. Molasses to ethanol in India
The question posed to us is that why country needs to make use of
lignocellulosic feed stocks. In India bioethanol is primarily produced
from molasses, a by-product of sugar production. Total quantity of
molasses produced in India enables about 11.6% bio ethanol blending.
Molasses alone is not adequate for achieving the target of 20% blending
in 2017 [11]. To meet this target there is a need for the use of other
lignocellulosic based feed stocks. In 2003-04, India faced drought that
finally lowered the production of sugar cane crop which consequently
lead to the decrease in using molasses to 6.75 Mt (usually 10 Mt) and
therefore the manufacturing of ethanol declined to 1518 million liters
[12], this eventually resulted in the shoot up in the price of molasses.
These circumstances made India to import ethanol and molasses in
2003-04. According to Kaushik Ranjan Bandyopadhyay [7], there was
revitalization in cane production in 2005–07 and this has driven the
government to resuscitate the 5% blending in November 2006. In
October 2008, the government moved towards the target of 10%
blending which did not emerge out to be successful. In the study
conducted by Joseph B Gonsalves [13], on the assessment of biofuel
industry in India, it was stated that one ton of sugarcane yields about
100 kg sugar and 40 kg molasses from which 10 L of ethanol could be
recovered and the use of sugarcane juice results in the yield of 70 L of
ethanol from 1ton of sugar cane. Because of the increasing population,
there will be higher dependence on sugar and for which million
hectares of land is required for cultivation of sugarcane crops for
799
Renewable and Sustainable Energy Reviews 73 (2017) 798–820
Table 1
World fuel ethanol production (2014).
Source: RFA analysis of public and private estimates, http://ethanolrfa.org/pages/
World-Fuel-Ethanol-Production#sthash.gzTnTMl1.dpuf
Country Ethanol (Millions of Gallons)
United States 14,300
Brazil 6190
Europe 1445
China 635
Canada 510
Thailand 310
Argentina 160
India 155
Rest of the world 865
higher yields. It becomes even more difficult to increase the area
because of other competing crops. Thus, there will be a burden on
sugar industry to meet this demand.
1.2. Production of ethanol: International scenario
Brazil, China and the US are the leading bioethanol producers in
the world. The production of ethanol in the US was 39×109 L using
corn as the major substrate and Brazil produced 30×109 L of ethanol
using sugarcane [14]. As per the report given by GAIN [15], Brazil is
the largest producer of sugarcane and ethanol. Nearly 590 million tons
of sugarcane was harvested in 2015, and a large part of this harvest
produced 2.34 billion liters of ethanol approximately. By 2016, an
increase in 5% ethanol production is predicted compared with that in
2015, with the exportation of > 1.3 billion liters. The Government of
Canada has consented to diminish the GHG’s by 6% between 2008 and
2012 in its Kyoto convention [16]. The production of ethanol in
countries in 2014 is given in Table 1.
1.3. Fossil fuels: a serious threat to the environment
The use of fossil fuels continuously to meet most the world’s energy
demand is threatened by increasing the amount of carbon dioxide
(CO2) and carbon monoxide (CO) in the atmosphere released by
burning of fossil fuels. As a concern for global warming, a search for
renewable energy sources that reduce CO2 emissions becomes a matter
of widespread attention. Global warming causes harmful and devastat-
ing effects on earth. Over the last 150 years, there is more than 25%
increment in the amount of CO2 released due to burning of fossil fuels
(http://opinion.bdnews24.com/2016/10/09/why-fossil-fuel-
companies-shouldnt-be-allowed-in-cop-22/). There is an emergent
need to replace the fossil fuels with lignocellulosic biofuels. These
major pollutants like CO, CO2, sulfur oxide (SO), nitrogen oxide (NO),
hydrocarbon compounds, lead (Pb) and suspended particulate matter
(SPM) emitted from vehicles cause harmful effects on health and
environment. The organic lead emitted from cars, gets easily absorbed
and results in serious health hazards. For this reason, use of leaded
petrol in the developed countries has been banned [17]. In
metropolitan cities 95% of all CO emissions are from automobiles. It
is very much lethal to humans if the concentration of CO exceeds
approximately 750 ppm. (http://www.pollutionissues.com/A-Bo/Air-
Pollution.html#ixzz3eXIBaAsL). In one of his talks, former Indian
president Dr. A.P.J. Abdul Kalam has suggested to substitute the use of
fossil fuels with biodiesel and bioethanol, where India ought to reduce
the dependency on fossil fuel usage and imports and must adopt
alternative methods such as mandatory ethanol usage in the future
http://www.psgtech.edu/ Abdulkalamvisit.php.
2. Availability of agro residues in India: an overview
India, being agriculturally enriched, has copious amounts of agro
G. Jahnavi et al. Renewable and Sustainable Energy Reviews 73 (2017) 798–820

Table 2 2.2. Prosopis juliflora

Area wise production of various lignocellulosic feedstocks.
It is the noxious and invasive weed found in many countries and
Substrate Area Production Ref.
has wide range of applications as a fuel wood and do not compete with
Wheat straw 31.47 MHA 86.53 MMT [18] food production and is very tough to remove. It is widely distributed in
Sweet sorghum 5.0 MHA 4.41 MMT [18] states like Telangana, Andhra Pradesh, Delhi, Haryana, Karnataka,
Cotton stalks 11.9 MHA 26.4 Million 480 lb. bales [18]
Madhya Pradesh, Maharashtra, Punjab, Rajasthan, Tamilnadu and
Prosopisjuliflora 24.72 m2/HA 2.5 tons of wood/HA/year [19,20]
Water hyacinth NA 60–100 t/HA/year [21] Uttar Pradesh. The average amount of an area (basal area) occupied by
Banana stem 803/HA 29,725MT/HA * P. juliflora is 24.72 m2 per hectare [19].
Lantana camara 13 MHA NA #

MHA- Million Hectares, MMT- Million metric tonnes, HA- Hectare, NA-Not available.
#
* http://nhb.gov.in/area-pro/NHB_Database_2015.pdf http://www.conservationindia.
org/articles/lantana-in-india-a-losing-battle.
residues like wheat straw, banana stem, sugarcane bagasse, sunflower
stalk, sweet sorghum, weeds like Saccharum spontaneum, Typha
latifolia, Eicchornia crassipes, Prosopis juliflora, Lantana camara
which can be utilized to produce bioethanol. To reduce food vs. fuel
debate and to make the production process economically feasible with
petroleum-based fuels; other cheaper and renewable raw materials
must be used. This review explains about the availability of some of the
lignocellulosic wastes in India. The area wise availability of lignocellu-
losic biomass in India (Table 2) and an Illustration depicting the state
wise availability of agroresidues is given in Fig. 1.
2.1. Wheat straw
In India, Uttar Pradesh shares a highest percentage of 33%,
followed by Punjab, Haryana, Madhya Pradesh and Bihar. According
to Misra et al. [22], these states together contribute more than 85% of
total wheat production in India and by the year 2020, the quantity of
wheat straw available will amplify up to 120 million tons, registering a
growth of 6%. A large quantity of wheat straw is being burnt by farmers
in northern part of India contributing to serious air pollution problems.
2.3. Eicchornia crassipes
Water hyacinth (Eicchornia crassipes) is the most predominant,
persistent and troublesome free floating aquatic weed in India. Water
hyacinth has stretched over 200,000 ha of water surface in the country
[23]. West Bengal, Kashmir and Rajasthan are the major water
hyacinth growing states in India [21]. The massive growth of water
hyacinth in water generates lot of difficulty for fishing and Irrigation
and are very tough to exterminate.
2.4. Lantana camara
It is a notorious weed which has imposed a great threat to land
productivity, grazing livestock, biodiversity and consequently to the
overall ecology [24]. It is abundantly available biomass and can be used
to produce value added products such as ethanol. It is found to be an
obstacle where it competes with the agricultural lands in India, in the
states like Jammu and Kashmir, Telangana, Andhra Pradesh and
Orissa [http://www.conservationindia.org/articles/lantana-in-india-a-
losing-battle].
2.5. Banana stem
Banana is the most important fruit crop of India having great
socioeconomic significance. Many states in India cultivate banana. In
India, farmers usually discard the residue generated from banana

Fig. 1. State wise availability of agroresidues in India.

800
G. Jahnavi et al.
plantations is usually burnt in the fields causing serious environmental
problems. The main residuals of banana crops are leaves and pseudo
stem, both containing high levels of holocellulose [25]. The authors
current ongoing research in on bioethanol production using banana
pseudo stem.
2.6. Sweet sorghum
Sweet sorghum stalk is an impending source of raw material for
commercial ethanol production. Sweet sorghum does not compete with
food, feed or fodder production thereby meeting the biofuel program’s
vision of not compromising on food security [26]. It can also grow in
clay and loamy soils and can tolerate high alkalinity and salinity [27].
The major sweet sorghum producing states mainly include Andhra
Pradesh, Telangana and Maharashtra [18]. ICRISAT (located in
Hyderabad, India) is already into research in using sweet sorghum
for bioethanol technology.
2.7. Cotton stalks
Cotton is one of the major crops of India and the country occupies
first place among cotton growing countries of the world. [28]. Cotton
plantation is widely distributed in states like Haryana, Rajasthan,
Punjab, Madhya Pradesh, Gujarat, Orissa, Maharashtra, Andhra
Pradesh, Tamilnadu and Karnataka. Cotton stalk is one of the
important by products of cotton crop [18]. The stalks are treated as
waste and burnt in the fields immediately after the harvest of the crop.
This may even effect the environment in terms of pollution which is not
desirable.
These lignocellulosic wastes are subjected to pretreatment, which is
followed by hydrolysis (both acid and enzyme) the hydrolysate thus
obtained is further subjected to ethanol fermentation using suitable
microorganism. A general schematic representation: conversion of feed
stocks to bioethanol is given in Fig. 2.
3. Pretreatment
Cellulose and hemicellulose are the key components in the ligno-
cellulosic substrates. These polymers are further hydrolyzed into
monomers. Cellulose is crystalline that makes the structure complex
and is difficult to hydrolyse while the hemicellulose exists in amor-
phous form and can be easily degraded into its constituent sugars. The
digestibility of the cellulose and hemicellulose in lignocellulosic sub-
strates is usually influenced by certain factors like lignin content,
cellulose crystallinity and particle size and greatly improved by
pretreatment [29]. According to Bishnu Joshi et al. [30] pretreatment
Fig. 2. General schematic representation of feed stock conversion to bioethanol.
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Renewable and Sustainable Energy Reviews 73 (2017) 798–820
is considered as one of the major rate limiting steps in bioethanol
production. Pretreatment is an effective, challenging and fundamental
step for successful hydrolysis and downstream operations. It mainly
involves the process of delignification where in the feed stocks need to
be processed further to make the substrate amenable for hydrolysis.
Pretreatment technology includes physical, chemical, physicochemical,
and biological pretreatment [31]. According to Taherzadeh and Karimi
[32], a chemical pretreatment of lignocellulosic material should remove
maximum lignin with minimum sugar loss i.e. not more than 5% and
on subsequent saccharification should hydrolyse maximum holocellu-
lose. There are various reports on pre-treatment methodologies, few of
which are found to be ideal for biochemical conversion of lignocellu-
losic residues to bioethanol. Advantages and limitations of various
pretreatment strategies is given in Table 3.
3.1. Physical pretreatment
3.1.1. Milling
Smaller pieces of lignocellulosic biomass are usually attained by this
process. The goal of pretreatment is to reduce the particle size and
cellulose crystallinity, this reduction in the size increases the available
specific surface with a reduction of the degree of polymerization (DP)
[43]. The reduction in particle size below 40 mesh shows slight effect
on the yield and rate of hydrolysis of biomass [44].
3.1.2. Extrusion
It is a promising physical pretreatment strategy for the conversion
of biomass to ethanol. The materials are exposed to heating, blending
and shearing that result in physical and chemical changes. The speed of
the screw and barrel temperature disrupts the structure of lignocellu-
lose resulting in defibrillation, fibrillation and shortening of the fibers
that makes the substrate amenable for enzymatic hydrolysis [45]. The
use of enzymes as catalysts during extrusion is being contemplated as a
potential approach to produce bioethanol.
3.1.3. Irradiation
Irradiation is the most promising physical method that involves
ultrasonic waves, microwaves, γ-rays, and electron beam and enhances
the effect of saccharification [35].
3.1.3.1. Gamma rays. These high-energy radiations have deeper
penetration power with high energy photons which are produced by
the decay of atomic nuclei as they return from high to low energy state
(“gamma decay”). Cobalt-60 and Cesium-137 results in the production
of gamma rays while undergoing self-disintegration [46]. From the
sealed source, the radiations travel at the speed of light and bombard
the biomass. Carbon, hydrogen and oxygen atoms in the biomass
polymers absorb equal energy. The structure of biomass in the
lignocellulosic polymers is reformed by cross-linking and molecular
scission [47]. The depolymerization of the constituents of cell wall
occurs with the high dosage of gamma rays. This degradation further
increases the digestibility of organic matter. The digestibility in soft
wood occurs with 40 kGy dosage of irradiation while it was 90 kGy
dosage in hardwood [48].
3.1.3.2. Electron beam. In electron beam irradiation, the target
material is exposed to highly charged stream of electrons. The most
commonly used device for electron beam irradiation is the “Electron
beam accelerator”. The electrons are emitted from the source as a
stream or from an “electron gun.” The accelerator speeds up the
electrons [35]. Henniges et al. [49], reported that, the interaction of
high energy electron beam results in the depolymerization of cellulose
because of chain scission. The oxidizing affects of electron beams
results in the chemical modification of polymers. The hydrogen bonds
G. Jahnavi et al. Renewable and Sustainable Energy Reviews 73 (2017) 798–820

Table 3
Advantages and limitations of various pretreatment strategies.

Pretreatment method Advantages Disadvantages Ref.

Ball milling Increases the surface area of biomass; decrease in crystallinity and
degree of polymerization of cellulose; no chemical requirement;
functional groups are not generated
Wet disk milling reduction in particle size, increase in surface area, pore volume,
reduced crystallinity index of cellulose, no release of fermentation
inhibitors
Irradiation does not involve use of solvents in large quantities, recovery or
recycling, downstream steps of cooling and neutralization after
biomass pretreatment are not required
Alkaline Pretreatment Saponification of intermolecular ester bonds cross-linking
hemicellulose and lignin. Decrease in the degree of polymerization of
cellulose and causes swelling of cellulose leading to an increase in its
internal surface area
Ozonolysis Low generation of inhibitory furfural and HMF (which might hinder
following downstream stages). Selective lignin degradation with
minimal effects on cellulose and hemicellulose. Operation at ambient
temperature and pressure
Organosolv easy process, possibility of recovery of solvent and the effectiveness
of organic solvent to break the internal bonding of lignin with
hemicellulose
Hydrothermal reduces the downstream pressure by making cellulose more
pretreatment accessible to the enzymes, minimizes the formation of degradation
products, catalyst is not needed
Ionic liquids achieve high sugar yields with low biomass loading, mild process
conditions, makes cellulose to amorphous, when combined with
other methods can efficiently process a wide range of lignocellulosic
feedstocks
Steam explosion hemicellulose solubilization and lignin transformation; cost-effective
technique; high yield of cellulose and hemicellulose in a two-stage
process
AFEX increases accessible surface area, removes lignin and hemicellulose
to an extent; does not produce inhibitors for downstream processes
Biological low impact on the environment, increased yield of the product, mild
pretreatment reaction conditions, few side reactions, less energy demand,
decreased reactor necessities to resist pressure and corrosion and
reduces the formation of inhibitors since there are degradation of
sugar compounds
High energy requirement and is not economically feasible [33,34]
High energy consumption [34]
During technology development, the commercial implementation is [35]
a costly affair, safety regulations are to be followed while using
radiations to avoid health hazards associated
irrecoverable salts and incorporation of salts into the biomass during [29,36]
the pretreatment reactions makes the alkaline pretreatment
challenging issue, high cost of catalyst
Highly reactive, flammable, corrosive and toxic characteristics of [37]
ozone, leading to potentially dangerous processes. cooling systems
are required for exothermic processes
expensive, so they should be recovered as much as possible, solvents [38]
need to be drained from the reactor, evaporated, condensed, and
recycled, this causes an increase in energy consumption
LHW pretreatment pattern selected for a certain lignocellulosic [39]
biomass and combined with other pretreatments would overcome its
disadvantages of high water consumption and energy input
Expensive, recovery of solvents besides, as they are recycled and [40]
reused, the efficiency of the ILs for pretreatment decreases
Partial destruction of hemicellulose, incomplete disruption of the [41]
lignin-carbohydrate matrix; generation of compounds inhibitory to
microbes
not efficient for biomass with high lignin content, high cost of [41]
ammonia, recycling of ammonia
Longer duration time [42]

between cellulose chains are broken causing reduction in crystallinity
making it more amorphous. The electron beam irradiation singly or in
combination with other methods can be developed for production of
bioethanol [35]. Microwaves.
Microwaves are electromagnetic waves which have been widely
employed because of its high heating efficiency and ease in operation.
The frequency of microwaves is in the range of 300–300 GHz [50]. The
residence time of microwave irradiation ranges from 5 to 20 min. It
results in the degradation of lignin and hemicelluloses and changes the
ultra structure of cellulose with an increase in the enzymatic suscept-
ibility of lignocellulose [51]. The concept of heating mechanism makes
microwaves a favoured method over conventional heating. The forma-
tion of hot spots occurs within lignocelluloses because of heating that
causes an explosive effect in the recalcitrant structure of lignocellulose
rendering its disruption [35].
materials [53]. However, certain chemicals and conditions of alkaline
pretreatment might result in the formation of inhibitory compounds
therefore optimization of pretreatment conditions is necessary to make
substrate accessible to hydrolysis. Use of alkaline pretreatment is on its
rise and is found to be an emerging technology involving use of non-
corrosive chemicals like ammonia, sodium hydroxide, sodium carbo-
nate, calcium hydroxide (lime) etc. The most frequently used reagents
for alkaline pretreatment include sodium, potassium, calcium and
ammonium hydroxides. To carry out pretreatment, different chemicals
as delignifying agents should be investigated [29] as the choice of
pretreatment method also has a significant impact on bioethanol
production. Among these, sodium hydroxide has received the greatest
attention due to its outstanding delignification capacity which is
essential to achieve high biomass digestibility [54], high reaction rate,
high ethanol production and very less release of inhibitors [55].

3.2.2. Sodium hydroxide

3.2. Chemical pretreatment
3.2.1. Alkaline pretreatment
The lignocelluloses are treated with chemicals that ultimately may
change the crystallinity of cellulose with a modification in hemicellu-
lose and lignin removal [33]. Alkali pretreatment causes the solubiliza-
tion of lignin and hemicellulose with an increase in the digestibility of
cellulose when compared with acid or hydrothermal processes [52].
The alkaline pretreatment is usually performed at room temperature
and may even range from minutes to days. The advantage of this
treatment over acid is that it causes less degradation of sugars which
seems to be more effective on agricultural residues than on wood
It is one of the strongest base catalysts, with a great degree of
enzymatic hydrolysis when compared with other alkaline treatments.
Kumar et al. [53] has reported that, the treatment with sodium
hydroxide was found to increase the digestibility of hardwood from
14% to 55% with a decrease in lignin content from 24 to 55–20%. It
was found to attack lignin-carbohydrate complexes; cleaving the ether
and ester bonds and is also effective in cleavage of ester and carbon-to-
carbon (C–C) bonds in lignin molecules (ferulic acid). During the
pretreatment reaction using NaOH, the hydroxide ion (OH_) and
sodium ion (Na+) are dissociated and with an increase in the
concentration of hydroxide ion, the rate of the hydrolysis reaction
increases accordingly [56,57]. NaOH causes swelling of cellulose and

802
G. Jahnavi et al.
increases the internal surface of cellulose with a decrease in the degree
of polymerization and crystallinity [33].
3.2.2.1. Ammonia. Ammonia pre-treatment is also a preferred
alkaline treatment, as it is easy to recover, non-corrosive and non-
toxic [56]. It results in the modification of cellulose and hemicellulose
structures, removal or modification of lignin with an increase in surface
area or pore size. In aqueous form, it causes the swelling of biomass
with a significant change in the morphology. As other alkaline
pretreatments, it also affects the degree of crystallinity of cellulose.
Ammonia pretreatment opens the structure of the lignocellulosic
biomass, making it accessible to enzymatic hydrolysis [58].
3.2.2.2. Calcium hydroxide. Calcium hydroxide also known as lime
leads to the increase in crystallinity index with the removal of
amorphous substances such as lignin. The removal of lignin causes
the reduction in non-productive adsorption sites for enzymes thereby
increasing cellulose accessibility [59]. The acetyl groups from
hemicellulose are removed which reduces steric hindrance of
enzymes and thereby enhancing the digestibility of cellulose [36].
The use of lime was successful at temperatures from 85 to 150 °C and
for 3–13 h [59]. Lime pretreatment has lower cost and requires less
safety measures compared to NaOH or KOH pretreatments and can be
easily recovered from hydrolysate by reaction with CO2 [60]. Although
lime pretreatment is not as effective as that of ammonia or sodium
hydroxide, it is still in use as it is simple and economically feasible
method which makes it attractive [61].
3.2.3. Ozonolysis
Ozone (O3) is one of the strongest oxidizing agents (E0=2.07 V,
25 °C) and ozonolysis is a promising oxidative pretreatment for lignin
degradation with low effect on the hemicellulose and cellulose contents
[62]. Ozone is readily soluble in water (110 mg/L, 25 °C). Ozone reacts
with olefinic, aromatic and phenolic compounds because of their
electron density. The removal of lignin by O3 reaction increases the
release of sugars during enzymatic hydrolysis. The main inhibitory
compounds formed during ozonolysis are the short-chain carboxylic
acids. The effect of moisture and the reactor model are the key
parameters for efficiency of ozonolysis. The consumption of ozone
depends on several process parameters such as the design of reactor,
moisture content, particle size, pH, reaction time, ozone/air flow and
ozone concentration. The optimization of process parameters is
necessary to achieve high yields with economic feasibility. [37].
3.2.4. Organosolv
Organosolv uses numerous organic or aqueous solvent mixtures
including methanol, ethanol, acetone, ethylene glycol and tetrahydro-
furfuryl alcohol, to solubilize lignin and make cellulose accessible for
enzymatic hydrolysis. When compared with other chemical pretreat-
ment methods organosolv process results in the recovery of relatively
pure lignin as a by-product [63]. To break hemicellulose bonds, the
mixtures of organosolv are combined with acid catalysts like HCl,
H2SO4, oxalic or salicylic acids [64]. The process of organosolv is
carried out at 150–200 oC. This process removes lignin effectively and
results in the solubilization of hemicellulose by hydrolyzing the ether
and 4-O-methylglucuronic acid ester bonds between lignin and hemi-
cellulose and partially hydrolyzing glycosidic bonds in cellulose [63].
3.2.5. Hydrothermal pretreatment
In hydrothermal pretreatment system, the lignocellulosic materials
are exposed to water at elevated temperatures, usually 100–250 °C.
The time of pretreatment varies from few minutes to several hours. In
some cases, biomass is taken into a closed oxygen-free reactor by
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pressurizing inert gases (e.g., N2 or He) or reducing gases (e.g., H2 or
CO) [65,66]. The solid-liquid ratio will be below 20% [64]. Few
researchers used catalysts in this system, such as alkali and alkaline
earth metals MgCl2, CaCl2, KCl, NaCl [67] and metal chlorides (AlCl3,
CrCl3, FeCl3, ZnCl2) [68] and acid like H2SO4 [69], to improve the
conversion of biomass during hydrothermal pretreatment. The crystal-
linity is changed by disrupting inter- and intra-hydrogen bonding of
cellulose chains [70]. It is difficult to degrade the cellulose below
200 °C [71]. The degradation of hemicellulose leads to the disruption of
hydrogen bond between the celluloses [70] therefore decomposition of
cellulose bundles is not as difficult as earlier. The reduction of
hemicellulose favors enzymatic hydrolysis and fermentation. If the
temperature is more than 150 °C, the hemicellulose is reported to be
firstly solubilized [72]. Lignin is a complex and amorphous structure,
where the cellulose and hemicellulose are tightly bound to the lignin by
covalent bonds. Hydrothermal pretreatment leads to the cleavage,
resulting in depolymerization and repolymerization of lignin [73].
The depolymerization is by the cleavage of β-O-4’ linkages and ester
bonds and repolymerization by acid-catalyzed condensation are the
predominant reactions [74].
3.2.6. Ionic liquids
Ionic liquids (ILs) as a pretreatment technology is gaining interest
these days and used in the release of fermentable sugars for biofuel
production from lignocellulosic biomass. ILs are organic salts usually
composed of organic cations and either organic or inorganic anions.
For categorizing ILs, four groups of cations are mainly used: quatern-
ary ammonium, N-alkylpyridinium, N-alkyl-isoquinolinium and 1-
alkyl-3-methylimidazolium [75]. The interaction of ILs with biomass
depends on combination of anion and cation and the biomass
components (cellulose, hemicelluloses and lignin). The mechanism by
which ILs interact with lignin and hemicelluloses is less understood
[76]. The treatment of lignocellulose with ILs leads to the swelling of
cell walls, the breakdown of intermolecular hydrogen bonding interac-
tions, enhanced surface area and a reduction in the crystallinity of
cellulose [76,77] all of which make the cellulose more accessible to
water.
3.2.7. Co-solvent enhanced lignocellulosic fractionation (CELF)
CELF is a recently developed method which uses tetrahydrofuran
THF (boiling point-66 °C), and is a renewable co-solvent. CELF causes
fractionation of biomass into three streams: glucan enriched solids
which are highly reactive, xylose and other hemicellulosic components
in the liquid stream at near theoretical yields, an ultra-pure stream of
lignin, with > 80% original lignin removed and recovered [78,79].
CELF reduces the recalcitrance of biomass with high titers of ethanol
with low enzyme loadings of 2 mg protein/g glucan. Nevertheless, the
recovery and recycling of THF is vital in terms of scalability and
economic feasibility of the technology. The pretreated solids from
CELF was applied with simultaneous saccharification and fermentation
with the glucan loading of 11 wt%, that lead to the ethanol titer of
58.8 g/L at 89.2% yield with an enzyme loading of 15 mg-protein
glucan in raw corn stover in only 5 days. The corn stover solids
obtained from dilute acid pretreatment was subjected to SSF at 10 wt%
glucan loadings achieved an ethanol titer and yield of 47.8 g/L and
73.0%, respectively in 20 d with an additional prehydrolysis of 18 h at
50 °C. SSF of CELF pretreated corn stover resulted in the final ethanol
titers consistently above 50 g/L, that corresponds to ethanol yield of
80% [79].
3.2.8. γ-valerolactone (GVL)
GVL is a safe and renewable solvent, which could be derived from
lignocelluloses and served as a food additive. Therefore, currently it has
been found to be an ideal and efficient organic solvent used in biomass
refinery [80]. GVL is miscible with water which has low melting point
(31 °C) and low vapor pressure [81]. To increase the release of sugars
G. Jahnavi et al.
from lignocellulosic biomass and to avoid the formation of inhibitors,
this is usually a preferred pretreatment to attain high cellulosic
conversion efficiency and recovery of lignin that can be achieved
simultaneously. In this method hemicelluloses and lignin will be
removed because of the existences of acid water and GVL thus making
the residue rich in cellulose content and is easily hydrolyzed to glucose
using enzymes. The advantage of this method is the use of low dosage
of acid concentration (0.1%) [82]. Li Shuai et al. [83], pretreated
hardwood with 80% GVL, 20% water solvent system at mild tempera-
ture of 120 °C and acid loading of 75 mM H2SO4. It was found that 96–
99% of original cellulose was retained with the removal of 80% of
original lignin. As the temperature and acid concentration is low the
sugar degradation was found to be negligible. This finally led to the
recovery of 99% of the original glucan and 96% of the original xylan.
GVL can be easily recovered and recycled by CO2 extraction. GVL leads
to increased digestabilities when compared with THF or ethanol. Miao
Wu et al. [82] reported a novel and mild biomass pretreatment method
using GVL/H2O system. Mild acid hydrolysis was done using γ-
valerolactone/ water system integrated with enzymatic hydrolysis.
The treatment was maintained at 170 °C for 1 h and the different
ratios of GVL/H2O mixture were set at 90:10, 80:20, 70:30 and 60:40.
This method enhanced the rate of enzymatic hydrolysis of pretreated
samples in cotton stalks by two-fold and over 92.6% of glucose was
recovered with the mixture of 80/20 GVL/H2O.
3.2.9. Switchable butadiene sulfone pretreatment
Butadiene sulfone (BS) or sulfolene (C4H6O2S) is a β,γ-unsaturated
cyclic sulfone (M.pt- 65 °C) [84], which in a reversible reaction, has the
ability to “switch” from solvent to gaseous 1,3-butadiene and sulfur
dioxide [85] that helps BS below 100 °C and the gases above 100 °C.
Sulfur dioxide forms in situ sulfurous acid in the presence of water [86]
which acts as catalyst in the deconstruction of lignocellulosic biomass
[87] resulting in the disruption of ester bonds linking the hemicellulose
and lignin with the delocalization of the amorphous structure of
hemicellulose and subsequent acid hydrolysis [88]. The advantage of
BS over other traditional acid/alkali/ ionic liquid based pretreatments
is its scalability and recyclability. When compared with ionic liquids
and solvents, use of BS is inexpensive at lab scale [89]. In a study
conducted by Atilio de Friasa and Hao Feng [90], the pretreatment of
Miscanthus giganteus, a C4 perennial grass and energy crop by
switchable butadiene sulfone as a new one-step chemical approach at
90–110 °C for 6–30 h, resulted in the xylan removal of up to 91% into
the liquid phase via bronsted acid catalysis with the activation energy
to be 89 kJ mol−1. Butadiene sulfone brings lignin solubilization of up
to 58%.
3.3. Physico-chemical pretreatment
3.3.1. Steam explosion
Steam explosion, also known as autohydrolysis, used for any
lignocellulosic biomass. It causes disruption in crystallinity of cellulose,
delignification and easy hydrolysis of hemicelluloses [91]. It replaces
chemical reagents and is an environment-friendly approach that
enhances the enzymatic hydrolysis of cellulosic biomass [92]. The high
pressure saturated steam (0.69–4.83 MPa) and temperature (160–
260 °C) are used to treat wet biomass during a short period (from
seconds to few minutes) and then the pressure is suddenly dropped,
this drop-in pressure disrupts the lignocellulosic wall structure releas-
ing hemicellulose and lignin and exposing cellulose fibers, which favors
the digestibility by enzymes and releases glucose during hydrolysis
[93]. At such high temperatures, water acts as acid and there by
contributes to hydrolysis of hemicellulose into liquid phase. As the
conditions of seam explosion pretreatment becomes more severe, the
condensation and repolymerization reactions takes place between the
decomposition products of hemicelluloses, leading to the formation of
pseudo-lignin, as observed in dilute acid pretreatment [94]. Factors
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affecting steam explosion pretreatment include residence time, tem-
perature, biomass size, and moisture content [95]. Addition of H2SO4
or SO2 or CO2 (0.3–3% w/w) results in the decrease in time and
temperature of reaction, improvement in hydrolysis rate, decrease in
the production of inhibitory compounds, complete removal of hemi-
celluloses [96].
3.3.2. Ammonia fiber explosion
AFEX is a physico-chemical pretreatment method in which ligno-
cellulosic biomass is treated with liquid ammonia at relatively moder-
ate temperature (90–100 °C) for a period of 30–60 min followed by a
rapid release in pressure [97]. The liquid ammonia used causes
swelling and physical disruption of biomass fibers leading to partial
decrystallization of cellulose and reduces lignin fraction. The digest-
ibility of lignocellulosic biomass is increased by removing the acetyl
groups by deacetylation process [98]. Ammonia loading, temperature,
water loading, blow down pressure, time and numbers of treatments
are the parameters that affect AFEX process [99]. Unlike most acidic
and alkaline pretreatments, AFEX pretreated biomass is readily
hydrolyzable and fermentable without detoxification or supplementa-
tion of external nutrients [100]. Acetamide and acetic acid is formed
with the removal of acetyl groups from hemicellulose but it is also
reported that AFEX removes least amount of acetyl groups from
lignocellulosic material when compared to other leading pretreatment
technologies [98]. In the presence of water, ammonia leads to
ammonolysis (amide formation) and hydrolysis reactions (acid forma-
tion), which cleave LCC ester linkages [88]. Unlike other pretreatment
processes AFEX cause no sugar degradation and furthermore 97% of
the ammonia used can be recovered and reused for subsequent batches
of AFEX pretreatment [88,101]. An overview of different pretreatment
strategies was tabulated in Table 4.
3.4. Biological pretreatment
Lignin, a very crucial physical barrier that hinders the process of
efficient accessibility of cellulose and hemicellulose. The advantages
and challenges involved in biological delignification were given in
Table 3. In biological delignification, the organisms like white rot
basidiomycetes are usually employed on solid support that can
depolymerize lignin effectively [128,129]. Besides using fungus, certain
bacteria like Bacillus macerans, Cellulomonas cartae, Cellulomonas
uda and Zymomonas mobilis have also shown delignification abilities
with lignin degradation of up to 50% [130]. The ligninolytic enzymes
like laccase, lignin peroxidase (LiPs), manganese peroxidase (MnPs)
and versatile peroxidase play a prominent role in lignin biodegradation
[131]. The enzymes like lignin peroxidases and manganese peroxidases
were first described in P. chrysosporium [132]. The non-phenolic and
phenolic lignin units are oxidized directly by LiPs, while the Mn3+
generated by MnPs acts specially on phenolic units, as well also on non-
phenolic units via lipid peroxidase reactions [129].
Singh P et al. [130], reported that, using the fungus Trichoderma
reesei and Aspergillus terreus, resulted in the delignification of 60%
and 92% respectively. The use of genetically modified microorganisms
such as the cellobiose dehydrogenase deficient Tramates versicolor
strain [133], is found to be ideal for microbial pretreatment. The use of
enzymes to carry out delignification is the most feasible method next to
microbial delignification process. Laccase produced by Pleurotus
species was used to treat Ricinus communis (castor oil plant) that
resulted in the maximum delignification of 86% after 4 h. This further
increases the percentage of saccharification to a greater extent [134].
Suhara et al. [135] isolated white rot basidiomycete Punctularia sp.
(TUFC20057), showed high lignin degradation ( > 50%) and high
lignin/holocellulose loss ratios ( > 6) after 12 weeks of treatment using
bamboo culms as substrate. The combined strategy of microbial
delignification followed by chemical pretreatment was found to be
most promising. The details are given in Table 5. Biological pretreat-
 G. Jahnavi et al.

Table 4
Effect of various pretreatment methods on different lignocellulosic substrates and yields.

Substrate Pretreatment strategy Conditions Result Ref.

Oil palm frond fiber Ball milling Milling was carried out for a total time of 3–120 min (with a cycle of 10-min run recovered 87.0% and 81.6% glucose and xylose respectively [102]
and 10-min pause) at room temperature
Sugarcane bagasse Wet disk milling 3–17 min, depending on the type of biomass, slurry viscosity and disk clearance 68.0% glucose and 44.9 xylose was recovered [103]
and straw
Rice straw Wet disk milling Operation was repeated 2–20 times. Each operation time was approximately 3 min 78.5% of glucose and 41.5% of xylose was recovered [34]
Rape seed straw Gamma radiation 60Co g-irradiation (0 kGy–1200 kGy) Partially destroyed the intra- or intermolecular structure [104]
Wheat straw Microwaves An optimal condition of microwave power of 1000 W for 15 min ethanol yield of 148.9 (g/kg wheat straw) obtained when compared with untreated [105]
wheat straw which was 26.7 g/kg
Rice straw Electron beam The accelerating voltage was 2.5 MeV with a beam current of 25 mA. The absorbed The cellulose increased from 39.5% to 71.1%, and lignin decreased from 19.5% to [106]
dose ranged from 50 to 500 kGy. 3% NaOH is used at 121 °C for 5 h 6.4%. The yield of sugar increased with an increase in the dose of irradiation.
Corn stover Alkali pretreatment The optimum conditions were found to be 15 wt% ammonia, 60 °C, 12 h Removed 62% of lignin retaining 100% glucan and 85% xylan [107]
Corn stover Alkali pretreatment at 30 °C for four weeks using 55% delignification and 86.5% glucandigestibility by enzyme loading [108]
50 wt% of ammonia loading and 1:5 solid-to-liquid ratio
Prosopis juliflora Alkali pretreatment 2% Sodium dithionite Removed 80.4% lignin, with a minimum sugar loss of 2.5% [109]
Corn stover Alkali pretreatment 1% NaOH+8% NH4OH at 50 °C for 48 h Attained 65.8% delignification [110]
Corn Stover Alkali pretreatment 2% Alkaline H2O2 Resulted in the lignin removal of 82% [111]
Sorghum bicolor Alkali pretreatment 2% NaOH, 60 °C, 90 min 4.3-fold increase in total sugars [112]
Wheat straw Alkali pretreatment 2.15% H2O2 v/v, 35 °C, 24 h 8.6% w/v monomeric sugars [113]
Corn stover Alkali pretreatment 0.5 g Ca(OH)2, 55 °C for 4 weeks with aeration Recovered 93.2%, 79.5% glucose and xylose [114]
Corn stover Alkali pretreatment Aqueous ammonia, ammonia recycled percolation method Reduced lignin content by 70–80% [115]
Coastal Bermuda Alkaline pretreatment 0.75% NaOH, 121 °C, 15 min 86% removal of lignin with a recovery of 90.4% glucose and 65.1% xylose [116]

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grass
Switch grass Alkaline pretreatment 0.5% KOH, 24 h, 21 °C 91.8% sugar (582.4 mg) [117]
Rice straw Ionic liquids Imidazolium based ionic liquids, ethyl−3-methyl–imidazolium acetate, butyl−3- The CrI of the cellulose of 1-ethyl−3-methyl imidazolium acetate treated rice straw [118]
methyl-imidazolium tetra fluoroborate and methyl−3-octylimidazolium chloride was lowest 14% followed by 1-butyl−3-methyl-imidazolium tetrafluoro borate and 1-
methyl−3-octyl imidazolium chloride 29.8% and 31.4% respectively when compared
with untreated rice straw (35.8%)
Sugarcane bagasse Ionic liquids 1-ethyl−3-methylimidazolium acetate [Emim] [Ac] at 120 °C for 120 min 20.7% of the bagasse components remained dissolved and enzymatic saccharification [119]
resulted in 80% glucose within 6 h and 90% within 24 h.
Sugarcane bagasse Ozonolysis 3.44% (v/v) ozone concentration and 80% (w/w) moisture, 45 min Consumption of 0.12 g of ozone by dry bagasse, with 33% of delignification. [120]
Sugarcane bagasse Combination of Ozone+ NaOH Ozone 32 mg O3/minute for 60 min; NaOH 0.1 mol/L. Ultrasound irradiation (U) Glucose recovery of 391 mg/g (95%) was obtained after pretreatment [121]
+ Ultrasound for 5 min
Palm empty fruit Organosolv solid-liquid ratio (1:10) of aqueous ethanol. 55% vol (C2H5OH), at 120 oC for The optimum yield of total sugars of 98.89 mg/g [122]
bunch 60 min
Sugarcane bagasse Organosolv The optimum conditions were 30% (v/v) ethanol at 195 °C, for 60 min 29.1 g glucose/100 g bagasse [123]
Corn cobs Hydrothermal pretreatment Temperature 190 °C Cellulose digestibility’s of residues increased from 26.8% for the raw material to [124]
almost 100%
Wheat straw Liquid hot water pretreatment Different process conditions Recovery of 71.2% sugars at 184 °C for 24 min, whereas 214 °C for 2.7 min resulted [125]
184 °C for 24 min in 90.6%
Elephant grass Steam explosion 11.5 min, biomass impregnated with 0.5% H2SO4. 31% (w/w) lignin was removed with a recovery of 52.1% (w/w) cellulose [126]
Corn stalk Steam explosion combined treatments with alkali and steam explosion under 0.4–0.6 MPa 64.3–71.8% of lignin was removed with a recovery of 71.5–79.5% of hemicellulose [127]
Rice straw AFEX Two different Ammonia Fiber Expansion (AFEX) pretreatment conditions, AC1RS AC2RS−5 cm produced the highest sugar yield of 486.1 g/kg. AC1RS−5 cm gave the [101]
(low severity) and AC2RS (high severity) are used to pretreatment at different lowest sugar yield with only 107.6 g/kg
particle sizes
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G. Jahnavi et al. Renewable and Sustainable Energy Reviews 73 (2017) 798–820

Table 5
Consortium of microbial delignification with chemical pretreatment.

Substrate Concerted strategy Effect Ref.

Populus tomentosa Liquid hot water and microbial delignification Resulted in highest hemicellulose removal of 92.3%, which there by glucose yield [136]
(Lenzites betulina) increased to 2.66 fold than liquid hot water pretreatment
Corn stalks Mild alkaline pretreatment combined with microbial Increased lignin loss from 76% to 80% and this further improved the enzymatic [137]
delignification (Irpex lacteus) saccharification significantly
Wheat straw Steam explosion and microbial delignification (T. Initially T. versicolor alone could reduce the percentage of Lignin up to 31%, while [138]
versicolor) the combination decreased the lignin to 75%
Rice straw Treatment P. ostreatus followed by AFEX treatment Resulted in higher glucan and xylan conversion than treatment with the AFEX alone [139]
Wood chips of Acacia Organosolv and microbial delignification Increased the concentration of ethanol from 62% to 69% in SHF process. In SSF [140]
dealbata (Ganoderma austral) there was an increment from 77% to 82% than organosolv alone

ment is considered as an economical process when compared with
other alkaline pretreatment methods.
4. Hydrolysis
4.1. Acid hydrolysis
The hydrolysis process is currently based on thermochemical route
(acid-catalyzed hydrolysis) and biochemical route (enzyme-catalyzed
hydrolysis), a prior pretreatment step is required for both the methods
to utilize lignocellulosic biomass efficiently [141]. The differences
between acid and enzymatic hydrolysis is given in Table 6. The
lignocellulosic feedstock, only after hydrolysis will be able to generate
high quality and quantity sugars, and so it is referred as saccharifica-
tion. Acid hydrolysis is carried out in both dilute and concentrated
mode. Different kinds of catalysts like H2SO4, HCl, HNO3, H3PO4,
peracetic acid etc were usually employed to carry out acid hydrolysis.
States, Arkenol, Inc., a technology and project development company
stated that the concentrated acid hydrolysis process can be made
economically viable and are ready for commercial implementation. In
Northern America, the Masada Resource Group has also developed
full-scale cellulosics-to-ethanol project [32]. Due to the requirement of
large amounts of acid in concentrated acid hydrolysis, this method was
not previously regarded as economically viable. The improvement and
developments in acid recovery technologies has renewed interest in
adopting concentrated acid hydrolysis [142].
Several factors like solid to liquid ratio, temperature, reaction time,
type and concentration of acid strongly affects acid hydrolysis [143].
The release of reducing sugars during acid hydrolysis depends upon the
type and composition of lignocellulosic material and the reactors used
in this process [144]. To achieve maximum sugar recovery and to
minimize the concentration of inhibitors released during acid hydro-
lysis, optimization of the aforesaid factors is essential as this hydro-
lysate will be used further to carry out fermentation [145].

4.1.1. Dilute acid hydrolysis 4.1.3. Effect of acid pretreatments on cellulose, hemicellulose and
There are two types of dilute acid pretreatment processes: high lignin
temperature (e.g., 180 °C) during a short retention time (e.g. 5 min) During acid pretreatment under mild conditions, some part of
and lower temperature (e.g., 120 °C) for longer retention time (30– amorphous cellulose is solubilized and the porosity of the residue is
90 min). Dilute H2SO4 was found to be the most widely used acid [60]. increased [98]. The increase in crystallinity and recrystallization can
Dilute acid pretreatment (DAP) method is most feasible for industrial occur during acid treatment under severe conditions. However, the
purpose. The advantage of DAP over concentrated is that the concen- crystallinity of cellulose resists the attack of acid during dilute acid
tration of acid used is low and with short retention time. The hydrolysis, while it can reduce the degree of polymerization up to
disadvantage of this method is that it is operated at high temperature certain level, which is usually referred to as levelling-off DP [146]. One
[33]. of the major advantage of dilute acid pretreatment is that nearly 80–
90% of hemicellulosic sugars are recovered [147]. As deacetylation of
xylan occurs, the cellulosic fraction becomes more accessible and
4.1.2. Concentrated acid hydrolysis
digestible by enzymes [146]. During hemicellulose hydrolysis, repoly-
The concentrated acid hydrolysis is carried out at low temperature
merization of lignin occurs besides depolymerization [148].
(e.g. 40 °C) and high acid concentration (30–70%) and results in high
sugar yields, however concentrated acid hydrolysis makes the equip-
4.1.4. Phosphoric acid
ment corrosive with high acid consumption. The neutralization of the
Phosphoric acid can be more beneficial for hydrolysis when
acid hydrolysate produces large amounts of gypsum [33]. In the United
compared with other acids as it is less aggressive and the neutralization
of hydrolysate obtained after acid hydrolysis is done using NaOH, it
Table 6
Differences between acid and enzymatic hydrolysis [32]. results in the formation of sodium phosphate that need not be removed
from the hydrolysate as it acts as a nutrient to microorganism. In the
Comparing variable Dilute/concentrated acid Enzyme Hydrolysis work reported by Orozco et al. [149], the hydrolysis of municipal solid
Hydrolysis waste and hay crop was done by using dilute phosphoric acid in
Hydrolysis Temperature used is high Carried out at mild autoclave parr reactor. The reactor temperature was in the range of
(100–240 °C) conditions at 40–50 °C 135–200 °C and acid concentration was 2.5–10% (w/w). At the
Yields High sugar recovery is not High yields of sugars optimized conditions of 2.5 wt% H3PO4, temp of 175 °C and reaction
possible in dilute acid can be obtained time of 10 min, and at 5 wt% H3PO4, 150 °C, and 5 min reaction time,
hydrolysis
Inhibitors Results in the formation of No inhibitor formation
the production of xylose was 13.5 g/100 g dry mass corresponding to a
inhibitors yield of 67%. An average glucose yield of 25% was obtained at 5 wt%
Product inhibition No Yes H3PO4, 175 °C and 30 min. The degradation of glucose to HMF
during hydrolysis occurred at 10 wt% H3PO4 at 200 °C. The yield of arabinose was
Cost of catalyst Low High
100% that is 3 g/100 g dry mass. In the previous study reported by
Time of hydrolysis Occurs in shorter time Takes longer duration
periods Orozco et al. [150] the municipal bio-waste wood shavings were treated
with 2.5 wt% H3PO4 with a reaction temp and time of 175 °C and

806
G. Jahnavi et al.
10 min. The maximum concentration of xylose obtained was 17 g/
100 g dry mass, that corresponds to the yield of 100%. The yield of
glucose was 30% at 5 wt% H3PO4, at a reaction temp of 200 °C and
reaction time of 10 min. The production of xylose increased with an
increase in temperature and acid concentration, its conversion to
furfural was also catalyzed by those factors. The maximum yield of
furfural was 3 g/100 g dry mass, corresponding to the yield of 24%,
while total sugars of 60% was obtained using grass clippings treated
with 7.5% (w/v) dilute phosphoric acid at 160 °C with the reaction time
of 5 min [151].
4.1.5. Hydrochloric acid
The hydrolysis using hydrochloric acid was done by Hernandez-
Salas JM [152]. In this process the substrates depithed bagasse and
pith bagasse of sugarcane were treated with 1.2% (v/v) HCl at reaction
temp and time of 121 °C for 4 h. The maximum reducing sugar yield
was 37.21% for sugar cane depithed bagasse and 35.37% for sugar cane
pith bagasse. A similar quantum of reducing sugars that is of 30.29 g/L
was obtained when sugarcane bagasse was subjected to hydrolysis
using 2.5% (v/v) HCl along with furans (1.89 g/L), total phenolics
(2.75 g/L) and acetic acid (5.45 g/L) [10], whereas Y. Sun et al. [124]
used corn stover and reported only 19.93 g/L xylose, 1.2 g/L glucose,
1.5 g/L furfural and 1.3 g/L acetic acid with 5.8% hydrochloric acid
within 80 min.
4.1.6. Sulphuric acid
Pasha et al. [153] did a comparative study using phosphoric acid,
sulphuric acid and hydrochloric acid at different concentrations with
Lantana camara. The optimum concentration of 75% phosphoric and
sulphuric acid resulted in 120 and 210 mg/g sugars respectively
whereas 85% hydrochloric acid recovered 215 mg/g sugars. While in
dilute acid hydrolysis, the optimum concentration of 1% sulphuric acid,
phosphoric acid and hydrochloric acid resulted in the sugar recovery of
225 mg/g, 125 mg/g and 185 mg/g respectively. Recently Naseeruddin
et al. [154] also compared these three acids, where 1 & 2% (v/v)
combination of sulphuric acid hydrolyzed maximum holocellulose of
25.44% releasing 0.51 g/L of phenolics and 0.12 g/L of furans,
respectively. The selected acid was taken further to perform biphasic
dilute acid hydrolysis of delignified substrate P. juliflora using selected
acid by varying reaction time and temperature. At conditions of 110 °C
for 45 min in phase-I & 121 °C for 60 min in phase-II, hydrolyzed
55.58% of holocellulose releasing 2.11 g/L and 1.37 g/L of phenolics
and furans, respectively. The sulphuric acid treated hydrolysate was
fermented well and gave highest ethanol titer. Therefore, the study
concluded that dilute acid hydrolysis was found to be the best while the
concentrated hydrolysate showed left over sugars which were not
utilized. In a study conducted by Koti et al. [155] wheat straw was
subjected to two different stages of biphasic acid hydrolysis (first
phase- substrate was treated with 2% and 4% of sulphuric acid and
second phase was performed with 3% and 4% sulphuric acid) finally led
to the maximum sugar recovery of 52% and 41% respectively. Sachin
et al. [156] performed both dilute and concentrated sulphuric acid by
two-stage hydrolysis where sugarcane bagasse was hydrolyzed in a
high-pressure stain less steel autoclave to produce sugar monomers. In
the first-stage of hydrolysis, the dilute acid was in the range of 2–10%
(w/w), solid to liquid ratio was varied in the range of 1:8–1:4. At
optimum acid concentration of 8% and temperature of 100 °C and solid
to liquid ratio of 1:4 yielded 46.7 g/L a xylose, 3 g/L glucose and
0.58 g/L furfural/HMF. In the second-stage of hydrolysis, the residue
from first stage was soaked in concentrated acid ranging from 18% to
40%. At optimized acid concentration of 40%, with the same solid to
liquid ratio of 1:4 and at temperature of 80 °C, resulted in 65.2 g/L
xylose, 8.2 g/L glucose and 0.27 g/L furfural/HMF. Srilekha Yadav
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Renewable and Sustainable Energy Reviews 73 (2017) 798–820
et al. [157] also performed biphasic acid hydrolysis of rice straw that
resulted in the saccharification of 16.8 g/L, the hydrolysate when
subjected to vacuum distillation increased the concentration of sugars
to 31 g/L.
Therefore, biphasic acid hydrolysis can be preferred for maximum
sugar recovery where the concentration of inhibitors seems to be low
when compared with concentrated acid hydrolysis. The sugars in the
hydrolysate obtained after dilute acid hydrolysis can be increased by
concentrating the acid hydrolysate.
Solid loading and particle size also plays a vital role in dilute acid
pretreatment, usually it varies from 5% to 15% of dry lignocellulosic
biomass. Considering this, Sindhu et al. [158], optimized the particle
size of the sugarcane tops and showed that, mixed particle size was
found to be the most suitable with maximum reducing sugar yield of
0.64 g/g. This was followed by particle size > 1000 µm (0.57 g/g),
600–1000 µm (0.519 g/g) and < 600 µm (0.49 g/g). Therefore, the
particle size of feed stock plays an important role in determining the
efficiency of the process and suggested that, the use of mixed particle
size generated during milling prevents the wastage of lignocellulosic
materials. In a work conducted by Sindhu et al. [159], sugarcane tops
with mixed particle size was subjected to acid hydrolysis with 1–5%
acid concentration, biomass loading from 10% to 25% w/w and
incubation time of 30, 60 and 90 min. The optimized conditions of
3% w/w H2SO4, with 15% w/w solid loading and incubation time of
60 min, had resulted in 0.685 g/g of reducing sugars per gram of
pretreated biomass. Gupta et al. [160] reported the increase in
reducing sugars of 204.84 mg/g at optimized conditions of 3% acid
at 120 °C for 60 min. The hydrolysate was also found to contain
furfural (0.34 mg/ml), HMF (0.58 mg/ml) and caffeic acid (0.13 mg/
ml). Dilute acid hydrolysis using low acid concentrations with
Saccharum spontaneum was done by Chandel et al. [161] where
optimum conditions of 1.5% H2SO4 in 15 min at 160 °C led to the
maximum sugar recovery of 32.15 g/L, which is accompanied by furans
and phenolics concentrations of 1.54 and 2.0 g/L respectively. Redding
et al. [162] also used low concentration of sulphuric acid, where
bermuda grass was treated with 1.2% H2SO4 at 140 °C for 30 min
achieved a total reducing sugar yield of 97%. Noureddini and Byun
[163] reported sugar yield using low acid concentration. The optimized
treatment of corn fibers with 0.5% H2SO4 at 140 °C and biomass
weight of 5%, resulted in 56.8% yield of reducing sugars.
4.1.7. Dilute sulphuric acid in ionic liquid
The hydrolysis of microcrystalline cellulose (MCC) using dilute
sulphuric acid in ionic liquid in a batch reactor was studied. The
Imidazolium based ionic liquid (1-butyl-3-methylimdazolium chloride,
[BMIM] Cl) was synthesized by nucleophilic substitution reaction. The
hydrolysis of MCC was performed in two basic steps: In the first step,
initially the dissolution of MCC in [BMIM] Cl was done and in the
second step, dilute sulphuric acid was used to perform acid hydrolysis.
The process of dissolution process disrupts the structural network of
hydrogen bonding in MCC and reduces the recalcitrant nature of MCC,
thereby improving the accessibility of β-1, 4-glycosidic linkages to
hydrogen ions. The process of dissolution preceding the step of dilute
acid hydrolysis was found to be more efficient with higher yield of
reducing sugars. During the process of dissolution, the chlorine ions in
[BMIM] Cl forms hydrogen bonding with hydroxyl groups of cellulose,
there by disrupting the hydrogen bond. The authors performed three
experimental runs without treating with [BMIM] Cl (resulted in 12% of
total reducing sugars), with [BMIM] Cl but without prior dissolution
(yielded 25% total reducing sugars) and with prior dissolution using
[BMIM] Cl of MCC followed by acid hydrolysis (obtained a maximum
of 92% total reducing sugars). While all the three runs were done by
using sulphuric acid concentration of 5% v/v at temperature of 180 °C,
G. Jahnavi et al.
solid to liquid ratio of 1:100 at 500 rpm [164].
4.1.8. Use of Sulfonated carbons
Fraga et al. [165] reported that, depolymerization step is essential
to convert cellulose into glucose via hydrolysis of the β-1,4-glycosidic
bond, therefore a search for a strong solid acid catalyst is gaining
prominence for breakage of these bonds. Based on this, the solid acids
were derived from biomass which was used as effective hydrolysis
catalysts. The use of sulfonated carbons when compared with com-
mercial acid resins has shown higher activity and is found to be a very
good option for the generation of solid acid catalysts. The raw material
palm kernel shell was evaluated to produce sulfonated carbons. The
raw material was carbonized and sulfonated. The carbonization was
carried out at optimum temperature of 300–350 °C. The obtained solid
acids were tested for the hydrolysis of cellobiose. This reaction of
hydrolysis is the first step in converting renewable carbon sources into
chemical products and biofuels. Intriguing results utilizing sulfonated
carbons were reported in the literature, which might be effectively
reused and sulfonic acid groups has acid strength like that of sulphuric
acid [166].
4.1.9. Use of sulfated zirconia catalyst
Sulfated zirconia catalyst was found to be most promising and
potential in the hydrolysis reaction of lignocellulosic biomass to get
fermentable sugars as intermediate product to produce ethanol. Among
the sulfated zirconia catalysts tested, the highest rate of hydrolysis in
empty fruit bunches was 37.2%, while in another substrate (frond) it is
53.95% [167].
4.1.10. Release of inhibitors
The use of high concentration of acids and higher temperatures
results in the degradation of sugars, that further forms inhibitory
compounds which effects the rate of fermentation and at the same time
the recovery of acids in concentrated acid seems to be uneconomical.
Inhibitory compounds released during acid hydrolysis are categorized
into three major groups that include phenolics, furaldehydes and weak
acids. Furaldehydes in the hydrolysates generally contain furfural and
hydroxyl methyl furfural (HMF) released by sugar degradation i.e.
furfural from pentoses and HMF from hexoses. In aliphatic acids,
acetic acid is released from the acetyl groups of hemicellulose, when the
temperature and acid concentration are increased. Furfural and HMF
are further degraded to form weak acids like formic and levulinic acid.
Phenolic compounds like vanillin, syringaldehyde and coniferyl alde-
hyde are usually formed by degradation of lignin [168]. All these by
products will show adverse impact on fermentation and retard cell
growth.
4.1.11. Impact of inhibitors on fermentation
Acetic acid is not only released by the acetylation of hemicellulose
during mild acid hydrolysis but also a by-product of fermentation.
Acetic acid released during hydrolysis can be even higher than 10 g/L
[169]. The effect of undissociated part of acid is larger than the effect
caused by the dissociated part [170]. According to Verduyn et al. [171],
this undissociated aliphatic acid can easily enter into the membrane of
the cell, undergoes dissociation and thereby decreases the internal pH.
Baker’s yeast will be able to tolerate 5 g/L concentration of undisso-
ciated part of the carboxylic acid [169]. Both furfural and HMF,
together called furans have been found to have substantial inhibitory
effects on the yeast metabolism [168] and can inhibit protein and RNA
synthesis, reduces or weakens the yeast enzymatic and biological
activities, break down DNA [172]. According to Banerjee et al. [173]
furfural at a concentration of > 1 g/L is shown to inhibit different
glycolytic enzymes in the glycolysis such as hexokinase, phosphofruc-
tokinase, triose phosphate dehydrogenase, aldolase and alcohol dehy-
drogenase. Furfural strongly inhibits the metabolism in S. cerevisiae
but HMF is not toxic to S. cerevisiae as furfural [174]. Inhibition of
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Renewable and Sustainable Energy Reviews 73 (2017) 798–820
enzymes like pyruvate dehydrogenase and alcohol dehydrogenase by
HMF is less when compared to furfural. The rate of conversion of
furfural is many times faster than that of HMF, therefore HMF persists
longer than that of furfural in the medium and subsequently the effect
of HMF lasts longer than that of furfural [32]. The phenolics /aromatic
compounds are usually released after pretreatment are found in the
hydrolysate [168]. These aromatic compounds are the degradative
components of lignin formed during hydrolysis that includes vanillin,
syringaldehyde, 4-hydroxy benzoic acid, ferulic acid etc. Among
different phenolics, vanillin and syringaldehyde are found to be the
potent inhibitors [32]. Nevertheless, they can be assimilated by S.
cerevisiae during fermentation.
4.1.12. Strategies to overcome fermentation inhibitors
Till date there are many strategies developed for the removal of
fermentation inhibitors, yet it is also important to make the process
economical. These inhibitors may tend to involve and disrupt the yeast
metabolism. The elimination of inhibitors is carried out by chemical
and biological means. Detoxification is still necessary, though certain
yeast strains like S. cerevisiae are resistant to fermentation inhibitors
to attain the maximum productivity during fermentation [175].
4.1.12.1. Detoxification. The removal of inhibitors is usually done by
physical (evaporation, membrane mediated detoxification), chemical
(neutralization, calcium hydroxide over liming, activated charcoal
treatment, ion exchange resins, and extraction with ethyl acetate)
and biological detoxification (enzymatic mediated using laccase, lignin
peroxidase), in-situ detoxification, in-situ microbial detoxification etc.
The details of these methods were reviewed by Chandel et al. [161].
Among detoxification strategies used, the biological method was found
to be most promising to eliminate inhibitors. Hence, a brief discussion
on biological detoxification has been included in this review.
4.1.12.2. Biological detoxification. Use of microbes like fungi, bacteria,
yeast and enzymes to carry out detoxification for removal of inhibitors in
the acid hydrolysates seems to be more advantageous. The inhibitory
compounds are usually released into the hydrolysates upon hydrolysis of
lignocellulosic feedstocks. These compounds if left in the hydrolysate
may even affect the process of fermentation. Though the incubation time
is little longer, the process is environment friendly. The merits of
biological abatement over chemical detoxification is that the process
requires mild reaction conditions, doesn’t require toxic and corrosive
chemicals, fewer side-reaction toxic products and less energy demand
[176]. In the study conducted by Lopez et al. [177] the isolated fungus
Coniochaeta ligniaria NRRL30616 is capable of metabolizing furfural
and HMF as well as aromatic and aliphatic acids and aldehydes. Few
more fungi like Amorphotheca resinae (ZN1) [178], Aspergillus
nidulans (FLZ10) [179] have the ability to degrade inhibitors. A
thermophilic bacterium Ureibacillus thermophaercus has removed
furfural and HMF and phenolic compounds [180]. In a case reported
by Fonseca et al. [181], the yeast Issatchenkia occidentalis (CCTCC
M206097) degraded syringaldehyde, ferulic acid, furfural and HMF
from hemicellulosic hydrolysates prior to the start of fermentation
process. Immobilization of laccase was done onto the cellulose
nanofiber produced by the bacteria Gluconacetobacter xylinus. When
compared with that of free laccase, immobilized laccase showed a better
pH and thermal stability. The three lignocellulosic inhibitory derivatives
like furfural, acetosyringone, and coniferyl aldehyde were completely
degraded after 36 h of incubation at 40 °C by immobilized laccase. But
the ketone derivative acetosyringone required hydroxybenzotriazole
(HOBt) as mediator along with the immobilized laccase [182]. The
detoxification of furfural and HMF by newly isolated Enterobacter sp.
FDS8 was done that resulted in the degradation of furfural and HMF
rates of up to 0.54 g/L/h and 0.12 g/L/h respectively. As per the report
G. Jahnavi et al.
these are found to be the highest biodetoxification rates ever reported
with a total sugar loss of below 5% [183].
4.2. Enzymatic hydrolysis
Enzymes are biocatalysts gaining importance in the industries as
they have specific catalytic properties, easy to produce and environ-
mental friendly. The production of crude enzymes might not meet the
large-scale application, therefore use of high potential organisms that
are capable of producing enzymes at high grade can be used and will be
feasible to build up the cost-effective technology. Enzyme hydrolysis is
conducted during 24–150 h at 37–50 °C, pH 4.5–5. Once the hydro-
lysis is completed, the soluble monosaccharides are released in the
liquid form, while the unhydrolyzed portion of cellulose, lignin and
other components which are not soluble remain in the solid portion of
glucose molasses. They are extracted by filtering the suspensions, the
solid residue is washed thoroughly to increase the glucose yield [184].
4.2.1. Cellulases
Cellulose is a long linear polysaccharide consisting of hundreds to
over ten thousand glucose units linked by β-1,4 glycosidic bonds. The
cellulose chains aggregate into microfibrils via hydrogen bonding and
van der waals interactions [185]. The degradation of cellulose is slow as
it is tightly packed crystalline structure and cross links with hemi-
celluloses and lignin. The cellulose hydrolyzing enzymes (i.e. cellulases)
are divided into three major groups: endoglucanases, exoglucanases
(cellobiohydrolases) and β-glucosidases.
Endoglucanases(endo-1,4-β-glucanases): Catalyzes the random
cleavage of internal glycosidic bonds of the cellulose chain resulting
in the formation of new chain ends. The endoglucanases (GH5, GH6,
GH7, GH12, GH45) contain a shallow active site cleft and are not
processive [186].
Exoglucanases or cellobiohydrolases (exo-1,4-β-glucanases):
Attacks the free chain ends, releasing cellobiose. The tunnel-like active
site of these enzymes enables tight substrate binding and varying
degree of processivity. These enzymes are specific for the reducing end
(GH7; CBH-1) or non-reducing end (GH6; CBH-2) of cellulose [187].
β-glucosidases: Breaks the bonds of cellooligosaccharides and
cellobiose, there by releasing glucose subunits from cellobiose [188].
4.2.2. Hemicellulase
Xylan (β-1,4-linked D-xylose polymer), is the major component of
hemicelluloses which contains multiple side groups, such as arabino-
furanosyl, glucuronosyl, and acetylresidues. To attain complete biode-
gradation of xylan, several hydrolytic enzymes are required with
diverse specificity. Xylooligosaccharides are formed when xylan back
bone is depolymerized by endo-β-1,4-xylanase, these oligosaccharides
are further degraded by β-xylosidase, while different glycosidases like
β-glucouronidases, α-1- arabinofuranosidase, acetylxylanesterase,
ferulic and p-coumaric acid esterase and α-4-O-methylglucuronosi-
dases cleave the side groups [188].
4.2.3. Lignin
Lignin is an aromatic heteropolymer relatively hydrophobic with
three monolignols, coniferyl alcohol, sinapyl alcohol and p-coumaryl
alcohol, these are incorporated into lignin in the form of guaiacyl (G),
syringyl (S) and p-hydroxyphenyl (H) respectively [189]. Lignin in the
lignocellulosic biomass is crosslinked with carbohydrates by ether or
ester linkages via e.g. arabinose-ferulic acid or glucuronic acid [190].
4.2.4. Key parameters for pretreatment of lignocellulosic biomass
The pretreatment is an essential step to alter some structural
characteristics of lignocellulosic biomass increasing its accessibility to
the enzymatic attack. These modifications in the structure of lignocel-
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Renewable and Sustainable Energy Reviews 73 (2017) 798–820
luloses are highly dependent on the type of pretreatment employed and
shows impact on the enzymatic hydrolysis [98] and subsequent steps.
Some of the factors like substrate composition, type of pretreatment,
dosage and efficiency of the enzymes used for the hydrolysis have a
great influence on biomass digestibility; although the impact of these
factors on the enzymatic hydrolysis individually are still unclear [64].
a. Cellulose crystallinity
In enzymatic hydrolysis of lignocelluloses, the crystallinity of
cellulose plays a vital role in the biological conversion of cellulosic
materials and crystallinity is considered as one of the most im-
portant factors in determining the rate of hydrolysis of relatively
refined cellulosic substrates. The microfibrils in cellulose have both
crystalline and amorphous regions. Nearly 2/3 part of total cellulose
is in crystalline form [44]. Cellulases can degrade more accessible
amorphous portion of crystalline cellulose while it is not effective
enough in degrading the less accessible crystalline portion.
Therefore, the high crystalline nature of cellulose will be more
resistant to enzymatic hydrolysis, and it is obvious that the decrease
in the crystallinity of cellulose, increases the digestibility of ligno-
celluloses [98]. The amorphous region of cellulose will be able to
adsorb water easily and becomes soft and flexible [191] by which the
chemicals can penetrate easily and has higher accessibility to
enzyme binding when compared with the crystalline region of
cellulose [188].
b. Accessible surface area
The hydrolysis of cellulose depends upon the adsorption of
enzyme on to the substrate, this is usually determined by the
accessible surface area [192]. Lignocellulosic biomass shows two
types of surface areas both external and internal. The external
surface area is associated with particle size and shape; while the
internal surface area depends on the capillary structure of cellulosic
fibers [193]. The surface area of substrate can be measured by
Brunauer–Emmett–Teller method [194]. The differential scanning
colorimetry determines the variations in the pore size distribution.
The measure of porosity of the substrate gives the direct indication
of the accessible surface area [192].
c. Degree of polymerization
The recalcitrance of biomass is determined by the degree of
polymerization (DP) of cellulose. During enzymatic hydrolysis,
endoglucanases cleave the internal sites of the cellulose chains,
preferentially less ordered, which is majorly responsible for decreas-
ing the DP of cellulosic substrates [64]. According to Huang et al.
[195], the saccharification of cotton stalks is enhanced by steam
explosion treatment by reducing the DP of cellulose. The accessi-
bility of enzymes and bacteria to cellulose surfaces is limited by
higher DP that is associated with high number of hydrogen bonds.
The decrease in the DP results in high saccharification to a certain
extent. The DP of cellulose plays an important role in the hydrolysis
compared to its crystallinity. The shorter cellulose chains (with lower
DP) are more susceptible to hydrolysis than longer chains [196].
d. Acetylation of hemicellulose
The acetyl groups of the hemicellulose attached to lignin cause
hindrance to cellulose and hemicellulose hydrolysis. These acetyl
groups also inhibit the formation of hydrogen bonds between
cellulose and enzymes. Just like that of lignin even the acetyl groups
of hemicellulose are the impediments for enzymatic hydrolysis [44].
The degree of acylation of hemicellulose is the crucial factor in the
hydrolysis of cellulose [197]. It is finally said that the increase in
accessible surface area itself is not enough to make efficient
conversion of cellulose, even deacetylation is also necessary [198].
e. Effect of lignin
The presence of lignin in lignocellulose maintains the integrity,
structural rigidity and prevention of swelling of the substrate [98]. The
presence of lignin hampers the accessibility of enzymes to cellulose and
G. Jahnavi et al. Renewable and Sustainable Energy Reviews 73 (2017) 798–820

Table 7
The enzymatic saccharification of various pretreated feed stocks using commercial and in-house enzymes.

Enzyme/Organism employed Lignocellulosic biomass Result Ref.

Commercial cellulase (20 IU/ml), β-glucosidase (In-house, 10 IU/ Sodium hydroxide pre-treated rice straw The cocktail containing the three enzymes resulted a [203]
ml) and xylanase (In-house, 5000 IU/ml) by Aspergillus sp. maximum recovery of 574.8 mg/g of total reducing
sugars
In-house cellulase by T. reseei Acid treated sorghum straw 546.0 mg/g reducing sugars with 70% [204]
NCIM 992 (25FP U/g of cellulase) saccharification
Commercial enzyme 0.75% H2SO4 at 100 °C for 2 h treated Recovery of 45.6 g/L reducing sugars [205]
Accellerase 1500 (26 U/g) wheat Straw
Celluclast 1.5 L (15 FPU/g) Corn stover treated with AP and SP treated resulted in high recovery of 65% [206]
and Novozyme 188 (30 CBU/g) different pretreatment methods (LTMA, and 60% of enzymatic saccharification
HTDA, AP, SP)
Enzyme cocktail comprising Acremonium cellulase (40 FPU/ml) Ball milled treated oil palm empty fruit OPEFB and OPFF showed high recovery of glucose [102]
and Optimash BG (10%) bunch (OPEFB) and oil palm frond fiber (62.4% and 78.69%) and xylose (80.35 and (84.23%)
(OPFF) production
Enzyme cocktail of Acremonium cellulase (1–20 FPU/g substrate) Dry ball milled (60 min) and hot Glucose and xylose yields were 89.4% and 88.6% [34]
and Optimash BG (6.7 mg/g-substrate). compressed water treated (160 min) rice respectively
straw
Commercial cellulase (Celluclast 1.5 L, 15 FPU/g) supplemented Sugarcane bagasse pre-treated with dilute 29.1 g glucose/100 g sugarcane bagasse [207]
with β-glucosidase (Novozym 188, 15 IU/g) acid and organosolv
NS22146 enzymes (Novozymes) 1.67%, 3.33% and 6.66% (g Sulphuric acid pre-treated empty fruit 81.4% xylan and 74.8% of glucan [208]
enzyme/g glucan×100) bunches
Commercial cellulase (60 FPU/g) NaOH pre-treated cotton plant waste A total of 96% hydrolysis efficiency was found with [54]
pretreated residue
Trichoderma cellulase (Novozyme 50013) and β-glucosidase COSILF (95% ethanol) pre-treated bamboo The overall glucose and xylose yields were 86.0% and [209]
(Novozyme50010) 82.6% respectively
In-house enzyme production by A. flavus ITCC 7680, T. reesei Micro wave alkali (NaOH) pre-treated 82% enzymatic hydrolysis yield was obtained under [210]
MTCC 164 (Cellulase enzyme laoding−10FPU/g; β glucosidase wheat Straw optimum conditions
100 IU/g)
In-house enzyme by A. heteromophus (endoglucanase198.2 IU/g, Micro wave alkali pre-treated rice husk 16.2 g/L at 72 h after that a decline in the sugars was [211]
exo glucanase 8 IU/g, xylanase 134.3 IU/g, β glucosidase 147.8 observed
IU/g)

Low-temperature moderate acid (LTMA), high-temperature dilute acid (HTDA), alkali pretreatment (AP), and sulfite pretreatment (SP).

hemicelluloses there by reducing the efficiency of the hydrolysis. It is
recognized as an important factor for recalcitrance of lignocellulosic
materials. Therefore, an efficient process of delignification can improve
the rate and extent of enzymatic hydrolysis [199].
Cellulases are usually produced from Trichoderma reesei and
Trichoderma viridae which are widely studied. In general, cellulase
loading will be in the range of 5–35 FPU/g of substrate [32]. The
production of cellulase using a solitary organism may not be proficient
enough for the hydrolysis of the diversified feedstocks. For instance, T.
reesei is the most promising strain producing endoglucanases and
exoglucanases in substantial amounts, yet its β-glucosidase activity is
less to hydrolyse the biomass. Therefore, the objective of the commer-
cial cellulase producing industries are indeed manufacturing cellulase
cocktails showing multienzyme activity [200]. Considering this in
perspective, in 2010 Genencor has launched four new brands of
enzymes with different blends: Accellerase® 1500, Accellerase® XP,
Accellerase® XC and Accellerase® BG. Accellerase®1500 is a cellulase
complex, Accellerase® XP enhances both xylan and glucan conversion,
Accellerase® XC contains hemicellulase and cellulase activities;
Accellerase® BG is a β-glucosidase enzyme. Accellerase® Duet devel-
oped by Genencor in February 2010 has exoglucanase, endoglucanase,
β-glucosidase, and includes xylanase activity [201]. In addition to this,
Novozymes developed two enzymatic blends cellic Ctec, and cellic Htec
[202]. The enzymatic saccharification of various pretreated feed stocks
using commercial and in-house enzymes is given in Table 7.
Lignin seems to be the stumbling block, for the hydrolysis of
cellulose and hemicellulose, and this has prompted the researchers to
work for the progress in the advancements of pretreatment processes.
Although there is an extensive ongoing research in pretreatment and
saccharification technologies a completely reliable technology in the
conversion of lignocellulosic wastes to sugars is difficult. An efficient
pretreatment should be able to recover the fermentable sugars with low
impact on environment, with minimum release in inhibitors and with
less energy demand. Few pretreatment strategies like steam explosion
mentioned in the literature might seems to be promising and can be
implemented in large scale, while in enzymatic hydrolysis using
commercial enzymes includes high economics and may not be feasible
in large scale. Since the use of commercial enzymes in the industries
add to cost, focus ought to be on the production of enzymes using
lignocellulosic based feed stocks which requires an efficient organism.
The organisms capable of converting sugars, while tolerating the stress
levels will be a most promising approach. Every method used in the
conversion of agrowastes to bioethanol has got its own pros and cons.
The logistics should be taken care especially during scalability.
Therefore, a technology that minimizes these obstacles must be
developed or the existing pretreatment methods should be upgraded
for promising results.
5. Role of fungus in ethanol fermentation
Use of traditional microorganisms like S. cerevisiae and
Zymomonas mobilis for ethanol fermentation usually do not metabo-
lize pentoses. The use of pentose assimilating yeasts like P. stipitis,
Candida tropicalis and few fungal species could efficiently ferment
pentose sugars to ethanol. The ability of zygomycetes filamentous fungi
to ferment pentose sugars has been known since many years. This class
of fungi are filamentous and saprophytic organisms, which can produce
several metabolites including ethanol. Certain fungi like Aspergillus,
Rhizopus, Monilia, Neurospora, Fusarium, Trichoderma and Mucor,
have been studied which are capable of producing ethanol from
lignocellulosic biomass [212]. Mucor indicus has been found to be a
potential ethanol producing microorganism. The fungus could con-
sume xylose and produce ethanol in the aerobic cultivation, which is a
major advantage over S. cerevisiae and has got the ability to withstand
against the available inhibitors [213]. Among three genera of Rhizopus,
Mucor and Rhizomucor, M. indicus (formerly M. rouxii) and Rhizopus
oryzae showed good ethanol productivity from glucose and xylose
[214]. In the fermentation of sugars to ethanol, M. indicus follows the

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G. Jahnavi et al.
Embden–Meyerhof and the pentose phosphate pathways [215]. It can
convert xylose to ethanol only under aerobic and micro-aerobic
conditions [213,214]. The ethanol yield and productivity from xylose
by M. indicus are in the order of those obtained by P. stipitis, one of the
efficient strains used for ethanol production from xylose [216]. In a
study conducted by Molaverdi et al. [217], simultaneous saccharifica-
tion and fermentation of sweet sorghum stalk as substrate resulted in
the ethanol yield of 85.6% at 37 °C for 48 h. Bioethanol production
from spruce and birch wood hydrolysates using separate hydrolysis and
fermentation by M. indicus resulted in the ethanol yields of 0.44 g/g
and 0.46 g/g in laboratory scale, while in the pilot scale airlift
bioreactor the corresponding yields were 0.33 g/g and 0.40 g/g respec-
tively [218].
Millati et al. [219] reported that M. indicus could assimilate xylose
and produce ethanol, its concentration increased after 24 h and
reached its maximum value at 68 h, i.e., 2.3 g/L with an ethanol yield
of 0.45 g/g, using oil palm empty fruit bunch as substrate. Abedinfar
et al. [220] have investigated the fermentation of rice straw hydrolysate
with an ethanol yield of 0.43 g/g using M. indicus. Chatterjee et al.
[221] reported that, M. indicus has got the ability to consume glucose,
galactose, mannose, xylose, arabinose and produces ethanol with high
yield and productivity. One of the major advantages of using M. indicus
as an ethanol producing microorganism is its biomass which can be
used further to produce valuable products such as chitosan. M. indicus
has got few advantages over S. cerevisiae in terms of its ability to
metabolize several types of sugars including hexoses (glucose, mannose
and galactose) and pentoses (xylose and arabinose), grows at high
temperature than S. cerevisiae [214]. The fungus seems to be like S.
cerevisiae with respect to ethanol productivity and yield. The major
difficulty in using fungus for ethanol production is because of its
filamentous nature. However, it is possible to simply change and
control the morphology of M. Indicus [222]. It has been reported by
Sharifia et al. [222] and Lennartsson et al. [223] that M. indicus in
different morphologies, yeast-like and filamentous forms and their
mixture, can efficiently produce ethanol from different hexoses. M.
indicus under aerobic and anaerobic fermentation conditions and with
the difference in morphology produces maximum ethanol yields.
Filamentous (aerobic-0.40 g/g; anaerobic-0.42 g/g), mostly filamen-
tous (aerobic-0.40 g/g; anaerobic-0.41 g/g), mostly yeast like (aerobic-
0.41 g/g; anaerobic-0.42 g/g), and purely yeast like forms (aerobic-
0.39 g/g; anaerobic-0.40 g/g) [223].
6. Yeast: as a model organism in fermentation studies
S. cerevisiae classified as GRAS (generally regarded as safe) by the
U.S., Food and Drug Administration (FDA) is undoubtedly known for
its popularity in basic and applied research. This was the first
eukaryotic organism whose complete genome sequence was deter-
mined [224].
S. cerevisiae is relatively tolerant to low pH values and high sugar
and ethanol concentrations, which lowers the risk of contamination in
industrial fermentation. Yeast is resistant to inhibitors present in
lignocellulosic hydrolysates and can grow anaerobically. Thus S.
cerevisiae has a broader application in industrial white biotechnology,
focusing majorly on the fermentative production of industrially perti-
nent bio chemicals, e.g. glycerol, propanediol, organic acids, alcohol,
steroids, isoprenoids etc [225]. Yeast cells must stumble upon known
stresses such as osmotic, oxidative, thermo, ethanol tolerance, and/or
starvation. These stress conditions can radically affect the industrial
fermentation, including ethanol production [226].
7. Genetic engineering approach
Second-generation bioethanol production requires effective conver-
sion of all the sugars in lignocellulosic hydrolysate to ethanol. S.
cerevisiae, a well-known organism commonly being used in industrial
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Renewable and Sustainable Energy Reviews 73 (2017) 798–820
ethanol production cannot utilize xylose, the second most abundant
sugar in lignocellulosic substrates, although it effectively ferments
glucose [227]. Still there is a requirement in terms of xylose fermenta-
tion and inhibitor tolerance for effectual fermentation of lignocellulosic
hydrolysate, especially at high solid loading [228] as the fermentation
of xylose seems to be more sensitive to the inhibitors.
An efficient industrial yeast strain Ethanol red demonstrated and
proved its ability in carrying out fermentation with high yields. It is
robust and has got high tolerance to stress and has astounding
efficiency in fed-batch production on molasses. D-xylose fermenting
strain GS1.11–26 has been developed from Ethanol Red, i.e., it has the
genetic back ground of Ethanol red, but, it did not retain that tolerance
as Ethanol red strain due to various aspects [229].
Three xylose utilizing strains (GSF335, GSF767 and GSE16) were
developed from GS1.11–26. Though the utilization of xylose of these
three new strains was slower than that of GS1.11–26 strain, they
completely consumed 37 g/L D-xylose and 36 g/L glucose in about
32 h. In complex medium, the utilization rate of xylose by the three
strains ranged from 0.36 to 0.67 g/g DW/h, which was lower than that
of GS1.11–26 (1.10 g/g DW/h). These three strains produced up to
23% more ethanol compared to Ethanol Red and these are proficient
enough for direct application in industrial bioethanol production.
Recent literature suggests several genetic engineering strategies to
develop xylose-fermenting S. cerevisiae. Various research efforts have
been put forth in improving the ability of yeast to utilize xylose and
ferment this to ethanol, for this there should be a better understanding
of the metabolic pathways and regulatory points involved in the
conversion [229]. Genetic engineering usually termed as metabolic
engineering, as it involves the improved fermentation and product
formation [230]. Since S. cerevisiae is incapable of utilizing xylose the
strain is engineered to make it an efficient xylose fermenting organism.
The major enzymes involved in the metabolism of xylose are xylose
reductase (XOR or XYL1), xylitol dehydrogenase (XDH or XYL2),
xylose isomerase (XI) and xylulose kinase (XUK or XYL3). The
metabolism of xylose comprises of two pathways (Fig. 3).
The first pathway mainly includes the conversion of xylose to
xylulose by two step redox reactions, where the xylose to xylitol
conversion is catalyzed by NADPH dependent xylose reductase (XR)
and the xylitol is further converted to xylulose by the NAD+-dependent
xylitol dehydrogenase (XDH) [231]. S. cerevisiae cannot make use of
xylose due to lack of XR and XDH activity. Some yeast’s and
recombinant S. cerevisiae strains producing these enzymes are capable
of fermenting xylose to ethanol. But, during anaerobic conditions, there
exists different coenzyme specificities of XR and XDH which generates
a cofactor imbalance that results in substantial accumulation of xylitol
as a by-product and reduces the ethanol yield [232].
Genome-scale modelling of engineered S. cerevisiae metabolic
pathways envisaged that, if the cofactor system of XR/XDH is
balanced, that would increase the ethanol yield by 25% and xylose
consumption rate by 70% [233]. The expression of an NADH-prefer-
ring XR mutant together with the wild type XDH specific for NAD+
improved the ethanol yield and productivity with the decrease in xylitol
yield [234]. The ethanol production was improved with the reduced by
product formation by the metabolic engineering of recombinant S.
cerevisiae expressing both a mutant XR from Candida tenuis prefer-
ring NADH and a wild type XDH from Galactocandida mastotermitis
[235]. This suggests that cofactor imbalance is not a major factor
inhibiting the xylose fermentation [61].
The second pathway involves the direct isomerization of xylose to
xylulose, catalyzed by the enzyme xylose isomerase (XylA) that helps in
the excessive production of xylitol [236]. The xylose isomerase (XI)
pathway usually occurs in most prokaryotes like bacteria and in
eukaryotes like fungi. The expression of xylose isomerase genes into
S. cerevisiae to metabolize pentose’s were not much successful. The
xylose assimilating yeasts like P. stipitis, P. pastoris, C. tropicalis, C.
shehatae, Pachysolen tannophilus facilitates the conversion of xylose
G. Jahnavi et al.
Fig. 3. Illustration depicting the glucose and xylose pathways in yeast.
to ethanol. Some of the xylose fermenting yeasts may not be tolerant to
ethanol as S. cerevisiae. In bacteria, xylose isomerase catalyzes the
isomerization of xylose to xylulose, before entering the pentose
phosphate pathway.
There are many reports in cloning of the xylA gene of xylose
isomerase from several bacteria into S. cerevisiae [237]. xylA genes
from E. coli [238], Clostridium thermosulfurogenes [239] and from
few other bacteria, have been cloned in many yeasts. Walfridsson et al.
[237] has elucidated the cloning and expression of xylA gene of xylose
isomerase from Thermus thermophilus into S. cerevisiae. Kuyper et al.
[240] successfully introduced xylA gene with high enzymatic activity in
S. cerevisiae from the anaerobic fungus Piromyces sp. strain, on the
contrary, this enzyme is strongly inhibited by xylitol. Brat D et al. [241]
reported the most important finding from their work, that the XI gene
of Clostridium phytofermentans is considerably less sensitive to
inhibition by xylitol when compared with XI from anaerobic fungi
Piromyces. A soil metagenomic library in E. coli was constructed for
the first time by Parachin and Gorwa Grauslund [242], to isolate genes
coding xylose isomerase activity and could identify two novel XI
encoding genes xym1 and xym2, however, the incongruity between
the performance of xylose isomerase in E. coli and S. cerevisiae
proposes that, the further screening of XI activity should have been
followed directly using yeast as a host. The search for the gene should
be done in S. cerevisiae or the gene synthesis for optimizing their
codon usage is required, for the effective expression of bacterial xylose
isomerase genes in S. cerevisiae [61]. The expression of bacterial or in
a sense the prokaryotic genes in S. cerevisiae often require their codon-
optimization to attain the desired phenotypes [243].
xks1 gene coding for xylulokinase can either be over expressed
[244] or the insertion of xyl3 coding for P. stipitis xylulokinase [245]
improves the rate of xylose fermentation in S. cerevisiae. The yeast S.
cerevisiae is still the leading and the most prevailing organism meriting
its use in bio ethanol production with high tolerance to inhibitors,
ethanol, low pH, that usually inhibits the growth of spoilage microbes
and with efficiency to ferment hexose sugars, its genetic tractability etc.
[246]. There has been a considerable research and effort in developing
a xylose-fermenting recombinant S. cerevisiae by over-expressing the
XR and XDH enzymes from other xylose-fermenting yeasts and to
increase the expression of non-oxidative PPP enzymes. The develop-
ment of recombinant S. cerevisiae strains expressing XI represents a
promising area of research [247]. The genome of S. cerevisiae contains
the gene xks1 coding for XK, but in wild type S. cerevisiae, the XK
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Renewable and Sustainable Energy Reviews 73 (2017) 798–820
activity is too low to support ethanolic xylose fermentation in strains
engineered with a xylose metabolic pathway, nevertheless when addi-
tional copies of xks1 are expressed then the recombinant xylose
utilizing S. cerevisiae produces ethanol from xylose. It has been shown
experimentally that only fine-tuned expression of xks1 in S. cerevisiae
has improved fermentation of ethanol from xylose. Above all, there is
not just one rate-limiting step in metabolic flux from xylose to ethanol
by S. cerevisiae and therefore, strain engineering for enhanced capacity
for xylose fermentation remains a challenge [248].
According to Petersson et al. [249] the over expression of NADPH
dependent alcohol dehydrogenase enzyme (ADH6p) in S. cerevisiae
resulted in the higher uptake of HMF. Almeida et al. [250] fermented
spruce hydrolysate using the same strain which further improved the
ethanol productivity. ZWF1 gene (encoding pentose phosphate path-
way enzymes) of S. cerevisiae encoding Glucose-6-Phosphate dehy-
drogenase was over expressed which further improved the tolerance of
yeast to furfural [251]. Although, the genetic improvements made in
the microorganisms for ethanol fermentation seems to be noteworthy,
nevertheless in commercialization of ethanol, the conversion of pen-
toses to ethanol is still a challenge.
7.1. Role of laccase in ethanol production
Laccases [benzenediol: oxygen oxidoreductases (EC1.10.3.2)] are
considered as copper-containing enzymes capable of oxidizing a broad
spectrum of phenolic compounds and non-phenolic substrates using
molecular oxygen as the electron acceptor [252]. They find their
application in bioethanol production. Laccase genes were heterolo-
gously expressed in suitable hosts, especially in yeasts S. cerevisiae and
P. pastoris [253], or filamentous fungi, such as T. reesei [254]. This
kind of heterologous expression of laccase isoenzymes in yeasts would
make the ethanol fermentation and detoxification to occur simulta-
neously there by reducing the cost and time involved in performing
these steps separately. According to Simona Larsson et al. [255],
laccase from T. versicolor was expressed under control of the PGK1
promoter in S. cerevisiae to develop its resistance to inhibitors like
phenolics which is of a major setback for the yeast to carry out
fermentation of lignocellulosic hydrolysates. A novel laccase gene
pclac2 and its corresponding full-length cDNA were cloned and
characterized for the first time from the oomycete Phytophthora
capsici. This gene pclac2 was successfully expressed in P. pastoris. It
was also reported that pclac2, has biotechnological, industrial, and
G. Jahnavi et al. Renewable and Sustainable Energy Reviews 73 (2017) 798–820

Table 8
Advantages and disadvantages of various fermentation strategies.

Process Advantage Disadvantage Ref.

Simultaneous saccharification
and fermentation
Consolidated Bio processing
Separate Hydrolysis and
fermentation
Simultaneous saccharification
and co fermentation
Less equipment, reduces the investment cost, operation is also simplified, no prolonged reaction time, generates lower yield of [258]
end product inhibition by glucose and thereby increasing the rate of fermentable sugar resulting in lower ethanol yield
saccharification and ethanol yield.
Reduction of capital investment, utilities associated with enzyme production Lack of suitable thermophilic microbes. [258]
are eliminated, reduced usage of vessels for saccharification and
fermentation, simplified operation, reduced risk of contamination by
lessening glucose levels and producing ethanol, the product inhibition of
cellulase is alleviated for improvement of hydrolysis.
At optimum temperatures, both hydrolysis and fermentation are carried out Expensive and it is a time-consuming process as [259]
separately. hydrolysis and fermentation are carried out
separately
Short residence time, minimized in use of reactors, reduced capital cost, high High enzyme loading, difference of optimum [259]
productivity of bioethanol, the problem of feedback inhibition is resolved by temperature between hydrolysis and fermentation
continual removal of end products of saccharification micro-organisms

environmental applications, especially for the enzymatic degradation of
aromatic pollutants [256].
Larsson et al. [255] constructed S. cerevisiae strain with laccase
gene from white rot fungus T. versicolor overexpressing the homo-
logous t-SNARE Sso2p, a membrane protein involved in the protein
secretion machinery. This strain was more capable and showed high
laccase activity than S. cerevisiae carrying laccase gene alone and the
strain also has got the ability to coniferyl aldehyde. This transformant
was also able to ferment dilute acid hydrolysate obtained from spruce
giving higher ethanol productivity.
8. Fermentation technology
Fermentation of lignocellulosic hydrolysates involves the conver-
sion of sugars to ethanol which is mainly performed by bacteria or
yeast. The organism chosen should possess certain characters in terms
of tolerance i.e., towards inhibitors, sugars and ethanol concentrations
in the hydrolysates and should also withstand higher temperatures and
lower pH and with minimal by product formation [257]. Fermentation
is the key component where advancement in technology plays key role
and is required to be feasible. The most commonly employed technol-
ogies in fermentation include separate hydrolysis and fermentation
(SHF), simultaneous saccharification and fermentation (SSF), simulta-
neous saccharification and fermentation (SSCF) and consolidated bio
processing (CBP). The advantages and disadvantages of these processes
were given in (Table 8). This review mainly focuses upon SSF and CBP
technologies.
8.1. Thermophilic microbes and ethanologenic microorganisms
Thermophilic microorganisms like Clostridium thermocellum,
Clostridium thermoshydrosulfuricum, Thermoanaerobacterium sp
like T. saccharolyticum, T. acidotolerans, T. thermosulfurigenes, T.
ethanolicus, T. pseudoethanolicus, T. mathranii; Thermoanaerobacter
sp including T. pentosaceus, T. brockii, T. saccharolyticum;
Caloramator sp like C. viberbiensis, C. fervidus and Paenibacillus sp
etc are found to produce ethanol at high temperatures [260]. Orlygsson
[261] reported ethanol production at 45 °C using cellulose hydrolysate
with a yield of 0.34 g/g by Clostridium strain AK1.
Thermoanaerobacter BG1L1, is a novel candidate for lignocellulosic
bioconversion into ethanol, as the organism can ferment the undetox-
ified corn stover hydrolysate in a continuous immobilized reactor
system at 70 °C yielding ethanol of 0.39–0.42 g/g of sugars consumed
[262]. This species has been reported to be potential species as it
possesses many additional features like alcohol dehydrogenases, xylose
transporters, modifications to pentose metabolism suitable for CBP
strategies. Kluyveromyces marxianus appears to be most promising as
it grows well at high temperatures as high as 45–52 °C and can
efficiently produce ethanol at temperatures between 38 °C and 45 °C
[35,263]. Pichia sp. especially Pichia kudriavzevii and P. stipitis will be
able to withstand high temperatures and produce ethanol. According to
Kwon et al. [264], ethanol production with P. kudriavzevii IPE100 at
42 °C produced 85% and 93.8% of ethanol yield theoretically from
glucose and cornstalks hydrolysate.
8.2. Simultaneous saccharification and fermentation (SSF)
SHF and SSF are performed at 45–50 °C. The major down side with
these hydrolysis methods is the accumulation of end products. SSF is
usually carried out in a single step with the combination of enzymatic
hydrolysis and fermentation. SSF is more preferred when compared
with SHF because of low cost, reduced risk of contamination, sugars
are not much degraded to its inhibitory compounds [265] and can be
done in a single vessel. Enzyme hydrolysis is generally carried out at
45–50 °C, therefore to carry out fermentation simultaneously with
hydrolysis, there should be a yeast strain that is equally capable of
tolerating that temperature. In such cases use of thermotolerant yeast
strains would be more suitable and generally preferred. Saini et al. [35]
reported that some of the thermotolerant yeast strains like
Kluyveromyces marxianus, Fabospora fragilis, Saccharomyces uvar-
um, Candida brassicae, Candida lusitaniae are usually employed for
SSF. A thermotolerant yeast strain K. marxianus DBTIOC-35 was
isolated by which SSF was carried out at 42 and 45 °C using wheat
straw as substrate. Maximum ethanol concentration of 29.0 and 16.1g/
L were achieved, corresponding to the ethanol yields of 73% and 40.5%
at 42 and 45 °C respectively. At 42 °C, the ethanol productivity was
higher during SSF (0.92g/L/h) than SHF (0.49g/L/h). It was indicated
that, SSF without pre-saccharification led to higher ethanol production
(66.2g/L with 83.3% yield) faster than SSF with pre-saccharification
(PSSF) which produced ethanol titer of 61.8g/L that corresponds to the
77.7% ethanol yield and productivity of 0.86g/L/h. Miscanthus pre-
treated with NaOH was loaded with cellulase enzyme (Novozymes,
Cellic Ctec II). The mixture was fermented with thermotolerant S.
cerevisiae mbc 2 and incubated at 42 °C for 72 h with an agitation of
150 rpm that resulted in ethanol concentration and theoretical ethanol
yield of 15.3g/L and 90.1% respectively in 48 h [266]. Dahnum et al.
[267] conducted SSF by using NaOH pretreated empty fruit bunch and
was digested with Cellic® CTec2 and Cellic® HTec2. Upon fermentation
of this mixture using S. cerevisiae have resulted in ethanol concentra-
tion of 97% at 50 °C in 24 h. Chu et al. [268] performed a three stage
SSF using S. cerevisiae DQ1, a thermotolerant strain with high
quantum of substrate (corn stover) loading at a rate of 30% that finally
resulted in the ethanol yield of and 65.6%. The organism can tolerate
higher cellulase and solid loading and thus resulted in the increased
production of ethanol. SSF was performed using K. marxianus and
commercial cellulase on soybean cake and corn cobs. This produced

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G. Jahnavi et al.
maximum ethanol of 5.68g/L on corncob and 2.14g/L on soybean cake
after 48 h of incubation [269]. When steam exploded, duckweed was
subjected to SSF using an enzyme blend of Cellic CTec 2 (0.87 FPU/g
substrate) together with Novozyme 188 (2 Ug-1 substrate), has resulted
in the ethanol production of 80% at a substrate concentration of 1%.
The ethanol yield has reduced with an increase in substrate concentra-
tion. The concentration of ethanol can be improved by increasing the
yeast titer, that has enhanced the ethanol yield up to 70% at a substrate
concentration of 20% [270].
8.3. Consolidated bioprocessing
Consolidated bioprocessing (CBP) is a most promising and poten-
tial strategy which includes enzyme production, saccharification and
fermentation into a single process, for effective production of ethanol
from lignocellulosic materials. There is a requirement of highly
engineered microbial strain that is capable of hydrolyzing biomass
with enzymes produced on its own and producing high ethanol titer
[258]. CBP becomes feasible when an engineered CBP microorganism
or microbial consortium could be developed [271]. In a single
bioreactor ethanol, together with all the enzymes were produced by a
single microorganism's community [32]. CBP is expanding its recogni-
tion as a promising leap forward to produce bioethanol with low cost,
yet its feasibility extraordinarily relies upon whether a suitable micro-
organism can be found in nature or built by engineering strategies in
the laboratory [212]. As S. cerevisiae in not capable of utilizing
pentoses and due to the lack of suitable enzymes for exploiting the
lignocellulosic feed stocks, is making the organisms unsuitable for CBP
applications [272]. Genetic engineering approaches in this area like
integration of genes from a cellulase producing strain into the ethanol
producing strains like S. cerevisiae was found to be inappropriate since
transfer of very high number of these genes might influence the
execution of cell, their co-expression at the transcriptional level is
often lop-sided causing ER-stress to the host cell [212]. Although the
fungi like T. reesei are found be efficient producers of cellulase, they are
not broadly being proposed as possibility for CBP applications because
of low ethanol yields obtained, as well as the slow fermentation rates
[273]. However, few fungi like Mucor [222] seems to be fermenting
lignocellulosic components to ethanol. Fusarium oxysporum is the best
contemplated filamentous organism for CBP applications with cellulo-
lytic and hemicellulolytic properties [274] to the enhancement of its
CBP execution through Genetic engineering methodologies [275]. The
ascomycete Paecilomyces variotii (ATHUM 8891) was evaluated as a
candidate species in CBP applications. The fungus is capable of
fermenting glucose and xylose to ethanol, closer to the maximum
theoretical yields, edifying an unusually powerful pentose metabolic
pathway and the fungus possesses the necessary enzyme factory for the
exploitation of lignocellulosic biomass, as it can grow and produce
ethanol on common agro-industrial derivatives [276]. White rot fungus
Lentinula edodes (Shiitake mushroom) is one of the most important
edible mushrooms in Asia. The chemical composition of the spent
mushroom waste produced by Lentinula edodes cultivation for CBP
fermentation using ethanol producing white rot fungus Phlebia sp.
(MG-60) was evaluated. When this fungus Phlebia sp. was cultured
with 20 g/L of unbleached hardwood kraft pulp, 8.4 g/L of ethanol was
obtained after 168 h incubation, with an ethanol yield of 0.42 g/g
corresponding to the ethanol efficiency of 71.8% [277]. A semi
consolidated process was designed by combining two microorganisms
for the fermentation. In this process Fusarium oxysporum culture was
added at the SSF stage along with S. cerevisiae. The use of F.
oxysporum culture resulted in the rise in ethanol production by 19%,
leading to a final ethanol concentration of 58 g/L. This semi-consoli-
dated process has not only increased the ethanol yields predominantly,
but also lowered the overall cost of the process by incorporating in-situ
enzyme production [278]. One of the major disadvantages of CBP is
that the saccharification and fermentation, usually carried out at
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Renewable and Sustainable Energy Reviews 73 (2017) 798–820
ambient temperatures but the hydrolysis by cellulases is usually higher
at higher temperature. This drawback could be dodged if thermophilic
microbes could be utilized as a host for consolidated bioprocessing
since it allows saccharification and fermentation to be carried out at
higher temperature [279]. The most demanding step in developing a
cellulolytic organism is the stimulation of cellulase expression and
secretion system. For the efficient hydrolysis of lignocellulosic biomass,
cellulase of different types should either be secreted extracellular (free
cellulases) or bound to the surface (cellulosomes). Most of these
enzymes ought to be brought into a CBP microbe that seems to be
ideal for the proficient depolymerization of lignocellulosic residues and
their consequent conversion to simple sugars. For developing a
potential CBP microbe, cellulase secretion has become a long-standing
limitation. The most viable initial move towards CBP is to attain an
effective cellobiose metabolism in industrial microbes. To keep away
from extracellular accumulation of cellobiose during CBP, the rate of
cellobiose metabolism in the recombinant strain should be higher than
the rate of glucose metabolism [271].
9. Third generation biofuels: Algae to Ethanol
Algae are a diverse group of eukaryotic organisms of which most of
them are aquatic and autotrophic. Algae are attaining wide considera-
tion as an alternative biomass which is also renewable to produce
bioethanol and is considered as the ‘‘Third generation biofuels” [280].
In correlation to different feed stocks, algae can give a high return
wellspring of bio fuels without trading off sustenance supplies, rain-
forests or arable area [281]. There are many reports archiving the
capability of algal biomass to biofuels [282]. Microalgae can endure
and use considerably larger amounts of CO2 and consequently can use
CO2 transmitted from petroleum-based power stations or other in-
dustrial sources which thus can lessen emanation of greenhouse gases
[280]. Microalgae is usually used as feed stock for bio ethanol
production. Few microalgae like Chlorella, Dunaliella,
Chlamydomonas, Scenedesmus, Spirulina contains a large amount (
> 50% of the dry weight) of starch and glycogen which are often used as
raw materials for ethanol production [283]. Ueda et al. [283] elabo-
rated the system microalgae fermentation. In the first stage, fermenta-
tion of micro algal cells was carried out in anaerobic and dark
environment to produce ethanol. The ethanol obtained from fermenta-
tion was used as fuel. The CO2 generated during fermentation was
recycled for cultivation of microalgae. Second stage involves the use of
remaining microalgal biomass in an anaerobic digestion process, this
process resulted in the production of methane, from which electricity
can be produced. They provide carbohydrates and proteins which are
used as sources of carbon in fermentation [284]. Micro algae also can
absorb CO2 and other greenhouse gases for photosynthesis, there by
diminishing the release of emissions into the atmosphere [285]. Dry
algal biomass of 1 kg can utilize about 1.83 kg of CO2 [286]. The macro
algae Gelidium amansii (red algae) usually found in the shallow coast
has high content of carbohydrates typically galactose and glucose
serves as a promising feedstock for bioconversion to ethanol [287].
In the production of bioethanol, during screening of algal strains high
biomass with high starch/cellulose substance ought to be additionally
considered as an alluring trademark since starch/cellulose can serve as
substrate for ethanol fermentation [288]. Harun et al. [284], has
reported a maximum ethanol concentration of 3.83 g/L obtained from
10 g/L of lipid-extracted microalgae (Chlorococcum) debris. In the
future, there will be a huge dependence on algal biomass for biofuels.
10. Conclusion
Bioethanol, not only reduces the release of harmful pollutants and
GHG emissions, it also additionally builds the energy security, employ-
ment and the country need not rely much on the oil imports. The
Government is already into ethanol blending program; however, there
G. Jahnavi et al.
is also a need to focus more on the technology where the cost
economics play a vital role. If a policy is taken up and analyzed in
terms of inadequacy and constraints involved in the production and
focussed on certain parameters involved in commercial level, that will
be of a greater help to the country. The use of petroleum based
resources has already shattered the eco system; therefore, there is a
tremendous need to switch on to lignocellulosic feed stocks. A
substitute for sugarcane crops by other cost effective lignocellulosic
feed stocks is essential and it is also recommended to make use of
multiple or mixed feed stocks to produce ethanol on large scale, as few
crops seems to be seasonal and the residues may not be available
throughout the year. It is evident that there are technical problems like
cost economics involved during processing, procurement, transport,
strategies involved in feed stock conversion to ethanol and distillation.
Nevertheless, the planet is waiting for the alternative and sustainable
energy sources, the bio ethanol research must be strengthened from lab
to industrial level probing into the difficulties and making the process
commercially feasible. A new and innovative technologies in the
automobile industries can also be brought about to reduce these
emissions. Efforts should be made to create awareness among people
besides the use of fossil fuels. It is also possible to adopt the procedures
from the countries successful in the production of bioethanol and such
strategies can also be implemented. Research itself has got many folds
which are to be crossed to reach the desired level. Henceforth the step
headed by R & D should be towards the operational costs, logistics
involved and other parameters which has become more challenging
these days and without which the production of cost effective bioetha-
nol shall become arduous. As explained earlier, pollution will cause a
very serious and disastrous impact on the environment in the coming
years. The population is increasing day by day and the usage of vehicles
never seems to cease. There should be a greater concern about the
pollution and its impact on environment. An initiative is to be taken as
to how to utilize the agro wastes and weeds generated in the country
and the impediments involved in its usage are to be considered.
Production of bioethanol seems to be inevitable for the country and
should be escalated for a brighter future.
Acknowledgements
The authors wish to acknowledge the financial support from
University Grants Commission (UGC) (F.4-1/2006(BSR) 7-369/
2012(BSR)/2013-2014/03) for BSR-RFSMS and BSR-faculty fellow-
ship.
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