Sonali Mohapatra, Chinmaya Mishra, Sudhansu S. Behera, Hrudayanath Thatoi, 2017, Vol. 78, PP 1007-1032

Renewable and Sustainable Energy Reviews 78 (2017) 1007–1032
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Application of pretreatment, fermentation and molecular techniques MARK

for enhancing bioethanol production from grass biomass – A review
⁎
Sonali Mohapatraa, Chinmaya Mishraa, Sudhansu S. Beherab, Hrudayanath Thatoic,
a
Department of Biotechnology, College of Engineering and Technology, Biju Pattnaik University of Technology, Bhubaneswar 751003, India
b
Officer at Odisha Public Service Commission, Government of Odisha, India
c
Department of Biotechnology, North Orissa University, Sriram Chandra Vihar, Takatpur, Baripada 757003, Odisha, India
A R T I C L E I N F O A BS T RAC T
Keywords: Grasses as lignocellulose biomass are promising feed stocks for renewable bioethanol production, since these
Grass biomass raw materials have high productivity, require low agricultural inputs, have positive environmental impacts, are
Pre-treatment easy to process and do not compete with the food crops. However, bioethanol production from grass biomass
Fermentation requires efficient pre-treatment, enzymatic hydrolysis and microbial fermentation processes which varies with
Molecular tools
types of grass species and the microorganisms used. Pretreatment is an important process for delignification of
lignocellulose biomass and is dependent on the type of lignin present in the biomass and the degradation
pathway employed for removal of the specific type of lignin. Further, enzymatic hydrolysis converts the cellulose
and hemicellulose into monomers, making it feasible for the fermenting microorganisms to convert it into
bioethanol where use of improved strain and biomass can yield higher ethanol on industrial scale. This review
paper presents an overview of the types of grass species, their composition and cultivation practices,
fermentation process used for bioethanol production and genetic tools used for improvement in bioethanol
production from grass biomass on a sustainable basis. The current knowledge and future prospect for industrial
bioethanol production from grass biomass along with its economic aspects have also been discussed in this
review.
1. Introduction consumes, marks its potential as a sustainable biofuel, that can be

utilised as an alternative to fossil fuel in commercial scale [2].
The high dependence on fossil fuels has led to uncertainty of Bioethanol can be produced from different biomass such as sugar or
petroleum resources and concern about climatic changes, which starchy materials as well as lignocellulosic biomass which are rich in
mandates for search of an alternative and eco-friendly energy source hexoses and pentosans [2,3]. While, in one hand the use of sugary and
[1]. It is expected that the global fossil fuel reserves will get depleted by starchy biomass as first generation biofuel production leads to high cost
next 40–50 years due to rapid increase in the consumption rate of these of bioethanol production due to the high price of raw materials [4,5],
non-renewable fuels. Thus, biofuels derived from biomass sources can the use of food stocks like sugarcane, corn and cereal grains on the
be an alternate source of energy in future. Among the different biofuels, other hand might possibly lead to the food crisis. Biofuels generated
bioethanol produced from biological sources represents one of the from lignocellulosic biomass (second generation biofuel) represent one
potential renewable resources of energy that can replace fossil fuels and of the potential renewable sources of energy that are non-polluting and
gasoline that are particularly utilised in the transport sector [2]. The are sustainable [3]. Lignocellulosic biomass are plentily available and
fact that bioethanol produces nearly twice as much energy as it can often be locally produced at low cost feedstock for bioethanol
Abbreviations: ABTS, 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid); AFEX, Ammonia fibre explosion; ARP, Ammonia recycles percolation; CBP, Consolidated
bioprocess schemes; CF, Co-fermentation; CMC, Carboxymethylcellulose; CMCase, Carboxymethylcellulase; Crl, Crystallinity index; DBU, 1, 8-diazabicyclo [5.4.0] undec-7-ene;
DMC, Direct microbial conversion; DP, Degree of polymerization; FPase, Filter paper activity assay for cellulases; GAXs, Glucuararonoxylans; GCE, Guaiacylglycerol-β-guaiacyl ether;
GlcA, Glucuronic acid; HBT, Hydroxybenzotriazole; ILs, Ionic liquids; LHW, Liquid hot water pretreatment; LMS, Laccase mediator system; Mg, Megagallons; MEA,
Monoethanolamine; MLG, Mixed-linkage glucan; NREL, National renewable Energy Laboratories; PDC, 2-pyrone-4,6-dicarboxylate; PEF, Pulsed-electric- field; QTL, Quantitative
trait loci; SHF, Separate hydrolysis and fermentation; SILs, Switchable ILs like; SmF, Submerged fermentation; SOC, Soil organic carbon; SPORL, Sulfite pretreatment to overcome
recalcitrance of lignocelluloses; SSCF, Simultaneous saccharification and co-fermentation; SSF, Simultaneous saccharification and fermentation; SsF, Solid state fermentation; TCA,
Tricarboxylic acid; Xyl, Xylose
⁎
Corresponding author.
E-mail address: [email protected] (H. Thatoi).
http://dx.doi.org/10.1016/j.rser.2017.05.026
Received 14 November 2016; Received in revised form 9 March 2017; Accepted 5 May 2017
Available online 13 May 2017
1364-0321/ © 2017 Elsevier Ltd. All rights reserved.
S. Mohapatra et al. Renewable and Sustainable Energy Reviews 78 (2017) 1007–1032
production. Biofuels from lignocellulosic biomass have been shown to quantity of cell wall (cellulose, hemicellulose and lignin) and other cell
offer most environmentally attractive and technologically feasible near- components (protein, lipid and sugars) in the grass. It has been
term alternative bioethanol with 94% lower greenhouse gas emissions reported that while the structural carbohydrates in cell wall increase
than that of gasoline [6,7,8]. In a study by Ryan et al., [9] it has been with the maturity of the grasses, the reverse phenomena is observed for
stated that for every 1000 L of bioethanol from lignocellulosic biomass, the cell components [16]. Whenever the rate of photosynthesis super-
approximately 2.6Mg carbon dioxide emission is saved. sedes the rate of plant growth or the plant gets into a stress,
There are different types of lignocellulosic biomasses such as wood carbohydrate accumulation starts in the plants. In these conditions
based, non-wood biomasses that include agricultural residues, sugar high concentrations of carbohydrates like starch, sugar and fructans
cane bagasse, switch grass, cotton fibre etc. Among the different can be seen in dry hay of cool season grasses. The carbohydrates are
lignocellulosic biomasses short rotation crops like grasses have high mostly accumulated in the lower regions of the grass stem like stem
yields of upto 40 Mg/ha/year as compared to corn feedstocks which bases, stolons, corms, and rhizomes [17,18]. More particularly, sub-
have yields of 7 Mg/ha/year [10]. In future, these fast growing plants stantial amounts of soluble carbohydrates are stored in the parench-
can be targeted as potential energy crops not only because of their high yma cells that surround the vascular bundles located within internode
productivity per hectare but also due to their abundancy, availability tissues [19,20]. Hence, these cells can be good targets for enhanced
and utilisation of the whole plants, high percentage of total cellulose carbohydrate storage in grass biomass, which would result in increased
and hemicellulose content and comparatively less lignin content [11]. carbohydrate yields successively, not interfering in the plant growth
Further, grasses can grow globally in a wide range of geographies, pattern.
climate and soil types [12]. However, the main bottle neck in large Further, depending on the temperature or climatic conditions,
scale ethanol production from grass as lignocellulosic biomass is the grasses are divided into tropical and temperate region grasses.
technological impediments of breaking down plant biomass (lignin in Tropical grasses or C4 grasses can withstand optimum temperatures
the cell walls) and releasing carbohydrate polymers (cellulose and and are high yielding varieties as compared to temperate or C3 grasses
hemicellulose) that can be fermented into fermentable sugars and due to their highly efficient mode of photosynthesis. Sucrose and
further refined fuels [11]. fructose are the predominant reserve constituents of temperate-origin
In order to obtain bioethanol, lignocellulose biomasses have to be grasses while sucrose and starch, are the major constituents of tropical-
pre-treated followed by enzymatic hydrolysis and fermentation. Pre- origin grasses [21,22].
treatment of grass biomass prior to enzymatic hydrolysis is necessary The type of grass variety and the environmental conditions in which
to break the recalcitrant lignin. But grasses have the advantage of low it is cultivated play an important role in determining the chemical
lignin content, which eventually leads to milder pre-treatment condi- composition of the grasses and thereby it's potential to be considered as
tions [13]. Further, enzymatic hydrolysis converts the cellulose and a bioethanol crop. Therefore, attentive considerations of the ecological
hemicellulose into monomers, making it feasible for the fermenting aspect serve as an integral part for bioethanol production from grasses.
microorganisms to convert it into bioethanol. However, improvement The consideration of the above mentioned factors will be crucial in
in grass biomass and microorganisms (specifically pentose fermenting) utilising many underutilised grass varieties in future for sustainable
by application of molecular techniques in genetic modification of plant bioethanol production.
biomass and microbes would play significant role for commercializa-
tion of bioethanol production from grasses. Further, biofuel production 2.2. Composition and potential of grass biomass for bioethanol
depends upon the availability of the feedstock, production and supply production
pathways, availability of the technologies and the cost-effectiveness,
which varies from region to region. The most favourable biomass resource for biofuel production
should be readily available, should have high yielding biomass per
2. Grass biomass around the world for bioethanol dry weight, unwavering desirable chemical concentrations and should
production be economical [23]. Other features like elevated carbon and hydrogen
concentrations and minimum concentrations of oxygen, nitrogen and
2.1. Types of grass biomass other organic components are also crucial for a biomass to be
considered for bioethanol [24]. In addition to the above mentioned
There are around 11,369 accepted grass species that have been features the importance of a biomass also lies on the fact that the
known worldwide till now [14]. The habitat of these grasses ranges industries using the biomass should produce less effluent and offer low
from infertile land mass to well drained fertile soil in varied climatic CO2 emission ability.
conditions. Grasses are composed primarily of carbohydrate polymers Grasses have the advantage of possessing possibly all these features.
(cellulose and hemicellulose) and phenolic polymers (lignin) along with The general composition of grasses along with the process of conver-
other compounds, such as proteins, acids, salts, and minerals. The sion of grasses to bioethanol is represented in Fig. 1. Grasses grow
accumulation of carbohydrate can be attributed to the photosynthetic naturally and do not require any special requirements for cultivation,
cycle in plants. The carbohydrate content is not similar for all the types which makes the biomass growth cost effective, as application of
of grasses, and significantly varies due to a lot of factors such as 1) fertilizers and pesticides is not a necessity [25]. It also has good
variety of the grass 2) developmental stage of the grasses and 3) the above-ground foliage and much denser growth which maximizes the
environmental conditions in which it is grown. amount of biomass that an acre of land can produce. Additionally,
Among the different varieties of wild and cultivated grasses, blue- grasses are composed primarily of carbohydrate polymers (cellulose
stems, Indian grass, and switchgrass are some of the most common and hemicellulose) and phenolic polymers (lignin) and lower concen-
examples of wild grasses. The cultivated grasses such as smooth broom trations of various other compounds, such as proteins, acids, salts, and
grass, timothy, meadow foxtail are some of the species that are derived minerals. These carbohydrate polymers, which typically make up two-
from wild species of grasses. They are developed through different thirds of cell wall dry matter, are polysaccharides that can be hydro-
breeding methods viz., pure line selections, mutants, polyploids and lysed to sugars and then fermented to ethanol. Further the carbohy-
inter-generic/interspecific hybrids. Both wild and cultivated grasses are drate concentration in grasses is directly related to the bioethanol yield
considered as suitable plant biomass because of their high carbohy- from biomass and the maturity of the grass is the key factor that
drate content, their longevity, redevelopment after the cut off, and determines its quantity in the grass. Another feature that makes grass
effective capability to tolerate the drought [15]. an attractive energy crop is its potential to increase carbon storage by
The maturity of the grass is the key factor that determines the increasing above and below ground biomass, specifically in C4 grasses.
1008
Fig. 1. The representation of lignocellulose content in grasses. (a) Grass;(b) Plant Cell;(c) Lignocellulose structure in cell wall;(d) Lignin polymer structure;(e) Hemicellulose structure;
(f) Cellulose structure. Source:http://www.nature.com/scitable/content/ne0000/ne0000/ne0000/ne0000/14464273/rubin_nature07190-f2.2_1_2.jpg.
This carbon storage competency helps grasses to eliminate carbon grass annual yields of 20–36 Mg/h have been reported [33]. The
dioxide emission nearly to zero during the fermentation processes for cellulose, hemicellulose and lignin contents generally vary from 37%
bioethanol production [12]. This have been exemplified in a study by to 40%, 25% to 29%, 18% to 25% respectively. Moreover, switch grass
Tan et al., 2008, which shows the carbon sequestering capacity of requires low maintenance with little or no fertilizer application.
Miscanthus to be 7–9 Mg C ha−1 in the soil in the first four years of Morrow et al. [34] has reported that a mature bioenergy switchgrass
cultivation [26]. crop production system would yield 330–380 l of ethanol per Mg of dry
Accounting the numerous advantages of different grass varieties switchgrass. These assessments are consistent with those from the
many research groups have widely studied the biomass for bioethanol studies of Forde et al. [35] and national renewable Energy laboratory's
production. Among the different varieties the most commonly used [36] theoretical ethanol yield that assumes conversion of both hexoses
herbaceous biomass are Miscanthus sp. followed by switch grass, and pentoses.
Napier grass and costal Bermuda grass. Giant miscanthus Napier grass (Pennisetum purpureum) is a native to eastern and
(Miscanthus x giganteus) which grows fifteen feet tall and is a central Africa and has been introduced to most tropical and sub-
sustainable feedstock for cellulosic ethanol production and is mostly tropical countries. It has high cellulosic fibre content, zero utilisation of
considered an Asian grass. It is a new leading biomass crop in the nitrogenous fertilizers and fast growing capability is an excellent cheap
United States and is commercially grown in the European Union as a feedstock for ethanol production. The ability of Napier grass to produce
combustible energy source [27]. It has an annual ethanol production adequate biomass under limited nitrogen levels is linked to the
yield of 1198 gal per 0.404 ha [28]. Miscanthus x giganteus is a variety occurrence of diazotrophic nitrogen fixing bacteria with the grass.
of sawgrass that is capable of producing 5–8 times as much ethanol per Presence of these bacteria in soil augments the nitrogen requirement of
acre as corn. The main feature distinguishing giant miscanthus from the plant by fixing atmospheric nitrogen [37]. Due to its highly efficient
other biomass crops is its high lignocellulose yields with cellulose (40– CO2 fixation, it is capable of producing 60 t/ha/yr. of dry biomass
60%), hemicellulose (20–40%) and lignin (10–30%) contents [27]. under optimal condition and 30 t/ha/yr. of dry biomass under sub-
While harvestable miscanthus yields (dry matter) have been estimated optimal condition. Other features that make this grass suitable for
to be in the range of 2–44 t/ha, yields of 27–44 t/ha has been reported bioenergy purposes include the cellulose content of 40–50% by weight
in Europe and US Midwestern locations and 10–11 t/ha of small-scale followed by hemicelluloses and lignin which is about 20–40% and 10–
trials at spring harvest in Montreal Canada [29,30]. 25% respectively [38].
Switchgrass (Panicum virgatum) is a primary biomass crop in the Bermuda grass (Conodont dactyl on) is a warm-season perennial
United States. Switchgrass is a native warm-season grass that has been grass grown in tropical and subtropical regions [39] especially in the
promoted as a model bioenergy crop because of its high-yield potential, Southern US [40]. The Bermuda grass also has the advantage of good
low input requirements on marginal soils, and potential for soil carbon biomass yield of 14.1–24.2 t/ha [41]. Increase in the nitrogenous
sequestration [31]. It has been reported that switchgrass stores large content of the soil has been seen to enhance the biomass yield from 1.2
quantities of carbon, with four farms in Nebraska storing an average of to 16.6 t/ha/yr [42]. Bermuda grass has high carbohydrate content
2590 pounds of soil organic carbon (SOC)/acre/year [32]. Typical (cellulose and hemicellulose) of 40–55% and low lignin content of 20–
yields range between 11–24 t/ha, but with newer varieties of switch 25% [43].
1009
Table 1
Composition and potential of herbaceous biomass for bioethanol production.
Grasses Adaptation Place Of Origin Composition (~) Production/ha Theoretical Advantage as an energy crop References
ethanol yield
C H L
S. Mohapatra et al.
Alfalfa (Medicago Good fertile well drained soil and Area around the 27 12 6.5–12 5–7 t/ac. 300 gal/acre Mostly the stem fraction is used as a [269,270]
sativa.) near neutral pH. Mediterranean Sea source of fermentable sugars to produce
cellulosic ethanol. Potentially increasing
profitability per acre.
Bigblue stem Soils with low moisture holding Central and eastern 37 28 18–20 80–150 t/hac 400 gal/acre More drought tolerant than other warm- [271]
(Andropogon capacity, pH, and phosphorus. United States. season grasses
gerardi)
Carrot grass sandy to heavy clay soil, but better India 35–37 21 17–19 12.14g/L(Actual excessive growth rate and wider [3]
(Parthenium growth is observed in moist type yield) adaptability
hysterophorus) of soil.
Chrysopogon Although occurring on neutral Asia, Polynesia, and 40 35 6 100–120 t/ha – Can grow on abandoned cultivations on [272]
aciculatus soils, it favours sandy acidic loams Australia poor sandy soils.
(Andropogon with pH 5.1–6.1. It prefers moist
aciculatus) soil
Cocksfoot grass grows well in a variety of soils with Europe, Asia and North 36 24 19–21 80–100 t/hac 2171 L/ha Tolerant of shade, high temperatures [273,274,44]
(Dactalis pH greater than 4.0. Africa and drought.
glomeruta) High tolerance of aluminium
Costal Bermuda grass Prefers deep soils but produces Southern USA 30–32 25–27 20–22 60–127 t/ha 208 L/ton drought tolerant [275,43,41,77,40]
(Cynodon dactylon) well on moderately shallow sites.
It withstands pH ranges from
about 5.0–8.5 and is boron
tolerant.
Cyper grass (Cyperus Grows best in rich,fertile soil. Afghanistan 33 ~36 ~6 4–19 t/hac/a 498 L/ton But can grow in poor sandy or clay soils [276,277]
1010
compactus) of unused lands or in fallow rice fields.
Deenanath grass Grows well in both dry and wet India and Africa 32 23 2.9 13–14 t /ha – It spreads quickly and is tolerant of [61,278]
(Pennisetum lands (ASL) saline and infertile soil.
pedicellatum)
Eastern gama grass Soils which are moist, little Eastern US 57.2 −64.1(total – 22.9–26.4 24,965 kg/ha 386.1 L/ton High biomass yield, longevity of [279,280,281]
(Tripsacum drained fertile soil that has an cellulose and established fields for decades,
dactyloides) annual precipitation of 900– hemicellulose) adaptation to different soil and climate
1500 mm (35–59 in) and a pH of conditions, non-invasiveness, carbon
5.5–7.5. sequestration capacity, soil
phytoremediation ability, and easy
integration into existing farming
operations
Energy cane (hybrids of soils ranging from highly fertile South Asia and Melanesia 43 23 21 17–19 t/ha 75 L/ton High density of energy, favourable cost [282,283,284]
Saccharum sp) well drained mollisols, through of production and delivery, produced
heavy cracking vertisols, infertile under stress conditions and so not to
acidoxisols, peaty histosols to compete with land used for food
rocky andisols. production
Giant cane (Arundo Dense stands, particularly in Mediterranean Basin and 31 35 18 8–10 t/ha 75 L/ton high growth rate and productivity even [285]
donax) riparian areas – boundary areas middle east Asia on marginal croplands
between land and streams.
Indian grass It grows well in deep, well-drained North America 39 29 4 t/acres 400 gal/acre It is tolerant of poor and welldrained [286,287]
(Sorghastrum floodplain soils and in well- soils, acid to alkaline conditions, and
nutans) drained upland sandy loam soils. textures from sand to clay.
Indian rice grass It prefers sandy, coarse textured plains, foothills, 32–35 NM NM NM NM drought tolerant [288,289]
(Achnatherum soils and can also be found on mountains, and
hymenoides) sands, fine sandy loams, silt intermountain basins of
loams, clay loams, gravelly, rocky the western United States
and shale soil textures
Intermediate wheat well drained loamy to clayey Europe and Western Asia 35–38 29 – 5.9–8.3 Mg/hac 0.41–0.47g/g tolerates slightly acidic to mildly saline [290,291]
(continued on next page)
Table 1 (continued)
Grasses Adaptation Place Of Origin Composition (~) Production/ha Theoretical Advantage as an energy crop References
ethanol yield
C H L
S. Mohapatra et al.
grass ( Thinopyrum textured soils conditions, is cold tolerant, can

intermedium) withstand moderate periodic flooding in
the spring, and is very tolerant of fire
Kallar grass Dry soil, drought area Pakistan and cholistan 45–50 20–22 20–40 t/hac/a 0.39–0.42g/g salt-tolerant grass [292,293,294]
(Leptochloa fusca) desert of India
Kans grass (Saccharum silt plains created each year by the South Asia and occurs 43 24 23 – 0.44–0.46g/g grow quickly without requiring any [140,189,172]
spontaneum) retreating monsoon floods throughout India along economic input;
the sides of the river tolerates many soil types and moisture
levels. Resistant to many diseases and
pests, and can produce high yields with
low applications of fertilizer and other
chemicals.
It is also tolerant to poor soils, flooding,
and drought; It uses less water per gram
of biomass produced than other plants
Little bluestem soils ranging from sandy to clay- U.S. and Canada 35 31 – 200–300 lb/acre – broad adaptation to diverse sites [227]
(Schizachyrium loam in texture
scoparium)
Meadow foxtail Moist, fertile soil, avoiding water- England and Wales 28–31 15–18 11–15 6–13 t/hac/a – Early growth and potential productivity [295]
(Alopecurus logged or light, dry ground
pratensis)
Miscanthus sp. Rich, moist, well-drained soil to Asia 28–49 24–32 15–28 9–54 t/hac 1198 gal/acre Cold and drought tolerance, little water [296,297]
attain maximum growth potential. or fertilizer inputs. Can tolerate a variety
of poor conditions, including soils of
1011
various pH, compacted soils, nutrient
poor soils.
Napier grass Grows best in deep, fertile soils Africa 40–50 20–40 12–19 54–60 t/ha 2621.06 L/ha/ Perennial, it can be vegetatively [298,237,15]
(Pennisetum through which its roots can forage. year propagated and it can withstand
purpureum schum) Deep, friable loams are preferable. repeated cutting/harvesting and
regenerates, lower content of sulphur in
biomass
Prairie cordgrass ( found on lower, poorly drained Northern Canada 39–41 31–33 19 6–9 t/ha 1748–4368 L/ha Grows well on seasonally dry sites, [299,300]
Spartina pectinata) soils along roadsides, ditches, tolerates alkaline conditions and high
streams, marshes and potholes. It water tables, chilling tolerance.
also occurs in floodplains, wet Tolerates alkaline conditions and high
meadows and back dune areas water tables
Reed canary grass ( Wet and humus rich soil Europe,Asia and north 38–45 20–25 18–21 7–13 t/hac/ 20g/l Superior drought and water logging [301,302]
Phalaris america tolerance, confer adaptation to a wide
arundinacea) range of soil types,
Highly productive yields
Saw grass ( Cladium forming dense pure stands, California, Arizona, New 25–29 NM grows in extremely infertile conditions, [303,304]
mariscus) usually on neutral or alkaline soils. Mexico, Nevada carbon neutral, absorbing about the
It can grow in semi-shade (light same amount of greenhouse gases while
woodland) or no shade. It prefers it's growing as it emits when burned as
moist or wet soil fuel
Smooth broom grass grows best on deep, well-drained Hungary 32 36 – 4–7 t/ha resistant to temperature extremes and [305,306]
(Bromus inermis) silt or clay loam but may also drought
establish itself in sandier soils.
Sudangrass (Sorghum Does best on fairly fertilie soil, Tropical and subtropical 33 27 – 3–4 t/ha Tolerates very high pH and salinity, [307]
vulgare) tolerates very high pH 8.0–9.0) regions of Eastern Africa most heat and drought-tolerant
Switch grass (Panicum Swamplands, plains, and streams, Continental United States 28–37 25–34 9–13 5–23 t/hac 2000–4000 L/ha High cellulose content,Self-seeding and [308,309]
virgatum) and along the shores and except California and the resistant to many diseases and pests,
interstate highways Pacific Northwest high productivity,Low N,P and K
Other grasses like cocksfoot grass, reed canary grass, big blue stem
grass, alfalfa etc. which are also documented to be potential feedstocks
References
for bioethanol production are listed along with the major bioethanol
[311,312]
[78,310]
producing grasses in Table 1. The wide scale adaptability of these
[313]
grasses, the major structural composition of the cell wall of these
grasses, their production on dry mass basis and the their theoretical
requirements,high moisture efficiency
ethanol yields are presented in Table 2. This reported information from

Produces good yields of high quality
forage, high yield and relatively low
Tolerates submersion, low pH, and

several research findings give us information on many grass varieties
Advantage as an energy crop
that remain unexploited for commercial bioethanol production inspite

of good amounts of cellulose and low levels of lignin present in them.
heat and drought tolerant.
Further, some grass varieties have also good amounts of hemicellulose

present in them. These grasses can be utilised by extraction of the
hemicellulose fraction for a pentose fermentation leading to bioethanol
production cost
production. An example can be cited from the study done by Njouk

anoxic soils
et al. [44] who had used the hemicellulose fraction of the cocksfoot
grass for bioethanol production with an ethanol yield in the range of
89–158 ml/kg of dry biomass. Similarly other features like good
productivity of dry biomass per hectare and adaptability to a variety
2211 L/ha l/ha
ethanol yield
of soil and climatic conditions can be considered for biomass like big
255.27 L/dry
Theoretical
metric ton
blue stem, deenanath grass, energy cane, giant cane, kallar grass,
8270g/kg
meadow foxtail, reed canary grass and tall fescue grass. Though their
annual production per hectare on dry mass basis is not as attractive as
the leading grass varieties, still undoubtedly these grasses with
Production/ha
qualities like good carbohydrate content, salt, stress and drought

tolerant have good potential as biomass for bioethanol production
8–14 t/hac
10.5 t /ha
even in arid lands. With minimum or nearly zero maintenance costs

these grasses can be cheap sources of biomass for bioethanol produc-
–
tion. However, more laboratory scale and pilot scale studies have to be
implemented for technological improvements for better ethanol pro-
duction in industrial levels.
11.5
14
L
3. Bioethanol production from grasses

25
30
10
Lignocellulosic biomass has a complex and recalcitarant structure

H
which requires a pretreatment step prior to enzymatic hydrolysis and

Composition (~)
subsequent fermentation for bioethanol production. This prior step is

mainly aimed for the disruption of the recalcitrant material of the plant
biomass. This increases substrate porosity with lignin redistribution in
the cell wall and enables maximal exposure of cellulose surface area for
the enzymes to reach an effective hydrolysis with minimal energy
25
28
28
C
consumption and a maximal sugar recovery. Herbaceous biomass like

Europe, western Asia and
US, Canada and Europe
grasses do not require as much as energy as recalcitrant woody

Large parts of Europe,
Asia and North Africa
material for removal of lignin since grasses have the lowest contents
Place Of Origin
of lignin. However, pretreatment seems to be a prerequisite for efficient

northwest Africa
enzymatic hydrolysis of grasses [45]. This is because of the fact that

although the content of the lignin is low but the main bond types of
grass lignin with the cellulose and hemicellulose are the same as those
of lignin in wood [46]. Moreover, it is not possible to determine the
best pretreatment method for lignocellulose biomass which however
It is an invasive species and grows
to temperate, moist environments

Cool-season forage grass, adapted
Very wet conditions and is often
depends on factors such as type of biomass and desired products.

woodlands and other habitats
as weed in native grasslands,
Keeping this in view many research groups have developed various

kinds of pretreatment techniques which are discussed in the subse-
common in wetlands.
quent sections. The flowchart representation which briefly summarises

the pretreatment category is given in Fig. 2.
Adaptation
3.1. Pretreatment technologies
Pretreatment is aimed mainly for delignification of plant biomass.

There are different types of pretreatment techniques such as physical
Tall fesecue (Festuca
(mechanical comminution) [47], chemical (dilute acid or alkali),

Table 1 (continued)
Timothy (Phleum
ammonia percolation, physio-chemical (steam explosion, ammonia

arundinacea)
pseudacorus)
Yellow flag (Iris
fibre expansion (AFEX) [48]), ultrasonication and microwave pretreat-

pratense)
ment [49], biological pretreatments (e.g. using white rot fungi), sulfite
Grasses
pretreatment to overcome recalcitrance of lignocelluloses(SPORL)

[50], pulsed-electric- field (PEF) pretreatment [45], organosolvic
pretreatment [51], ozone and liquid hot water (LHW) pretreatments
1012
Table 2
Total sugar/Reducing sugar yields of different types of grasses with different types of pretreatment.
Pretreatment technology Parametres maintained Grass variety Release of sugars after Delignification (%) Reference
pretreatment
S. Mohapatra et al.
Alkaline NaOH-7%,Incubation time-4 h Napier grass Hollocellulose-88.46% 9.77 [111]

Cellulose-82.21%
NaOH-0.5%,Temperature-120 °C,Incubation-120 min Kans grass Reducing sugar-350 mg/g < 79% Delignification [172]
NaOH-1%,Incubation time−15 min,Temperature- Ryegrass, Tall fescue, NMa NM [78]
180 °C Bentgrass
NaOH-7.29%,Incubation time- Napier grass Reducing sugar- 113.43 mg/ml 56.27% maximum lignin [314]
75.48 mins,Temperature- 43.45 °C solubilisation
(NaOH-1.7%,Incubation time- 30 mins), +Extursion Prairie cord grass NM NM [64]
pretreatment
(screw speed −122 rpm Temperature- 114 °C)
0.75% NaOH, incubation time 30 min, temperature Bermuda grass Glucose 257.1 mg/g raw biomass NM [315]
121 °C Xylose 117.5 mg/g raw biomass
NaOH 1%, 30 mins incubation time, temperature Switchgrass Glucose 253.8 mg/g raw biomass NM [316]
121 °C Xylose 114.7 mg/g raw biomass
NaOH-1%,Incubation time- 127.5 min, Guinea grass Reducing sugar- 57.42% NM [317]
Temperature- 110 °C
Alkaline peroxide Napier grass, Dwarf napier grass, King napier grass, Average cellulose of 18 grass varities Average lignin of 18 grass [15,318]
Bana grass, Purple guinea grass, Ruzi grass, Pangola −31.85–38.51%, varities−3.10–5.64
grass, Atratum grass, Vetiver grasses Average hemicellulose- 31.13–42.61%
Aquoeus ammonia Switch grass 72% reducing sugar NM [319]

NH3−1.1 g,Temperature−80 °C, Incubation time−86 h Napier grass-dwarf type Glucan−39.7%,Xylan−21.4%, NM [186]
Ca(OH)2−0.1%, Temperature−50 °C,Incubation Switch grass Glucose−239.6 mg/g 13–21 [320,321]
1013
time−24 h Xylose−127.2 mg/g, and total reducing
sugars 433.4 mg/g
Acid H2SO4−1.2%,180 °C, Switch grass 91.4% cellulose recovery(in form of NM [193]
glucose and cellobiose)
1% H2SO4,Temperature 150 °C,Incubation time Switch grass 337.8 mg/g Glucose, 157.6 mg/g Xylose NM [322]
10 min
H2SO4−0.6% v/v, Incubation time−20 min, Cogon grass Total sugar−39.98 mg/ml NM [323]
Temperature−127 °C
1.2% H2SO4, Bermuda grass 269.9 mg/g Glucose NM [77]
Temperature 140 °C 168.9 mg/g Xylose
Incubation time 30 min
Biological Innoculum size−20 ml,Incubation time−3 weeks, Napier grass Hollocellulose−76.4%, Cellulose−58.3% NM [111]
microganism-Phanerochaete chrysosporium `
Ozonolysis 1% NaOH at 121 °C for 60 min Miscanthus ×giganteus, Glucan−34.1% 20.7–59.9% reduction [324]
Xylan−11.3%
M. sinensis ‘Gracillimus’, Glucan−30.0%
Xylan−13.6%
Saccharum arundinaceum Glucan−38.0%
Xylan−18.2%
Saccharum ravennae Glucan−32.7%
Xylan−15.1%
Low-moisture anhydrous NH3−1.1g,Temperature−80 °C, Incubation time−86 h Napier grass-dwarf type Glucan−39.7%,Xylan−21.4%, 7.1 [186]
ammonia (LMAA)
Combination pretreatments
Alkali+Extursion NaOH-1.7%,Incubation time- 30 min, +screw Prairie cord grass Total sugar 67.2% NM [64]
speed −122 rpm Temperature- 114 °C
[52]. The flowchart representation which briefly summarises the
[325]
[326]
[297]
[327]
Reference
classification of these pretreatment techniques is given in Fig. 2.
Physical pretreatments like milling and grinding mostly aim for the
size reduction of the substrate of up to 0.2 mm. But, when physical
pretreatment processes are amalgamated with other pretreatments the
effectiveness of the process is improved to many folds leading to better
delignification of the biomass [53]. Chemical pretreatments are more
effective in solubilising the lignin and hemicelluloses content thus
Delignification (%)
making the biomass more available for the enzymatic hydrolysis [54].
However, physicochemical pretreatments are highly effective either
individually or when combined with chemical pretreatments. But the
most effective physicochemical pretreatments are ultrasonication and
85.1%
thermal pretreatment processes where the cell disruption accompanied

NM
NM
6.9
by degradation of the crystalline complex of the cellulose is achieved.

This leads to swelling of biomass with higher availability of the biomass
Glucose-61% xylose-95%
Glucose-95% xylose-73%
Total sugar 58.7g/100 g
to the cellulose and hemicellulose hydrolysing enzymes during the

process of enzymatic hydrolysis [55]. Biological and enzymatic pre-
Glucose- 0.4 g/g
treatments are moreover aimed at decomposition of lignin in milder

Release of sugars after
conditions.
biomass
3.1.1. Physical pretreatment for size reduction of grasses

pretreatment
Physical pretreatments are mainly aimed at reduction of particle

size of grasses, which in turn decreases the degree of polymerization
and crystallinity of cellulose. This size reduction also allows in
reduction of the heat and mass transfer limitations that occurs due to
large particle size of the biomass and increases the bulk density, thus
allowing the pretreatment of more concentrated feedstock [53,56].
Physical pretreatment mainly includes chipping, milling, grinding,
extrusion and pyrolysis.
Chipping processes are mainly required for woody biomasses. The
findings of Zhu and Pan [57] emphasizes on the fact that the
pretreatment method of woody biomass differs substantially from the
herbaceous biomass. This is mainly attributed to the differences in
their chemical composition and the low lignin content in herbaceous
biomass [57]. Milling and grinding are techniques which can be utilised
for size reduction of the biomasses. Though grinding and milling have
not been an integral part for grass delignification, but substantial
Grass variety
differences have been observed during the enzymatic hydrolysis step if

Switch grass
Miscanthus
Miscanthus
Miscanthus
these two techniques are included in the pre-treatment scheme. A

significant difference in the hydrolysis yield was observed in switch-
grass and alfalfa grass chops when they were subjected to size
reduction through grinding process [58,59]. Further, different types
H2SO4- 0.73 wt%,Temp-150 °C, Incubation time-
of milling processes can be used for size reductions in grasses like ball
milling, knife milling and hammer milling [25]. Among all of these,
6 min+Ca(OH)2- 0.024 g/g,Temp-202 °C
particularly ball milling with different sieve sizes have been seen to be
an efficient technique in size reduction of grasses [60]. Ball milled
Dennanath and Hybyrid Napier grasses have been shown to improve
the alkaline pretreatment for enhanced delignification and cellulose
exposure [61]. Menegol et al. [62] also demonstrated enhanced
Alkali 0.1 g/g of biomass
Treatment time: 30 min
Parametres maintained
delignification of elephant grass when exposed to physical pretreat-

Temperature: 190 °C
58 g/L solid content
ment, leading to higher bioethanol production.

Extrusion pretreatment is another commonly used pretreatment
technique in lignocellulosic biomass which generally includes heating,
mixing and shearing of the biomass thereby leading to cell disruption.
NM
NM
It is found that single-screw and twin-screw extruders are widely used

extrusion techniques in pretreatment process. Among the two, the later
is more preferred due to extended control of residence time distribu-
tion and mixing as well as superior heat and mass transfer capabilities
Pretreatment technology
Dilute-acid presoaking+Wet
[63]. In grasses, extrusion pretreatments have been seen to be effective

NM-Not mentioned.
Dilute-acid presoaking
in exposing the cellulose to enzymatic attacks. Extrusion pre-treatment

Table 2 (continued)
of switchgraas and prairie cord grass resulted in a total sugar recovery

Alkali+microwave
+Organosolv
of 45.2% and 65.8% respectively [64]. The same authors in 2012,

explosion
Acid+alkali
attempted for optimisation of extrusion pretreatment of other varieties

of grass biomasses like switchgrass, bluestem and prairie cord grass.
The study resulted in significant recovery of fermentable sugars with
a
the highest values of 28.2%, 66.2% and 49.2% for switchgrass, big
1014
Fig. 2. Classification of Pretreatment techniques. Based on: http://pubs.rsc.org/en/content/articlelanding/2016/ra/c6ra09851g/unauth.
bluestem and prairie cord grass respectively [65]. The difference in the experimented in extruders for long-term usage. Further, low energy
results for switchgraas and prairie cord grass was probably due to the requirements will also be a requirement for economical pretreatment of
different parameters, specifically the high screw speed and the higher the biomass. Hence, a multidisciplinary approach will be required to
residence time that were observed to be the most effective parameters. achieve an economical output in this type of pretreatment technique.
Lamas [66], also had similar conclusions for switchgrass treated with
extrusion pretreatment. Brudecki et al. [67], also investigated on
ground prairie cordgrass and found more promising results of 87% 3.1.2. Chemical and physico-chemical pretreatments for
delignification and 92% glucose yield. Though twin-screw methods delignification of grasses
have been seen to be more effective for many lignocellulosic biomasses In order to increase the elimination of lignin and/or hemicelluloses
but studies on grasses have been limited using this technique. As and decrease the crystallinity index (Crl) and degree of polymerization
saccharification yields of 80% [68], has been achieved with twin-screw (DP) of cellulose, chemical and physico-chemical pretreatment meth-
extrusion pretreatment of rice straw, equivalent results can also be ods have evolved as attractive methods [69]. Chemical pretreatments
achieved in grasses. which include the use of acids, alkali, ionic liquids and organic acids
Although physical pretreatments processes have the advantage of such as oxalic acid, acetyl-salicylic acid and salicylic acid as catalysts
producing zero toxic or inhibitory by-products in the substrate, but the are mainly used for internal degradation of lignin and hemicelluloses
high energy requirement makes it a cost intensive process. Secondly, by breaking the internal lignin and hemicellulose bonds [69]. The
the metal surfaces in extruder's needs constant replacements due to the hemicellulose and lignin bonds mainly refer to the chemical bonds
aberrations observed after two to three cycles of pretreatment. These between the lignin, galactose and arabinose residues on the side chains
factors should be taken into account and more durable surfaces can be of hemicellulose molecules [70]. In general lignin can be divided into
three types of monolignons i.e hydroxycinnamyl aldehyde (H), guaiacyl
1015
(G) and syringyl (S) lignin. Lignin of grasses is particularly high in p- bottlenecks of the process. For example, in a recent study, Merino et al.
hydroxycinnamates or H-monolignon [71]. The H-monolignols are [84] used a series of ionic liquids for pretreatment of Taiwan grass and
primarily connected via carbon-carbon and carbon-oxygen bonds achieved thirty-five times higher reducing sugar yields after enzymatic
among its phenylpropanoid building blocks with aryl ether bonds (β- hydrolysis in pretreated biomass in only one hour of pretreatment time.
O-4) which are important interunit linkage [72]. This was achieved due to the use of Q-tube which was very efficient as
It has been demonstrated by Samuel et al. [73] that dilute acid compared to the conventional round bottom flask.
pretreatment led to a thirty-six percent decrease of β-O-4 linkages in Physicochemical pretreatments, which are a combination of both
lignin of pretreated switchgrass. Focusing mainly on the lignin the chemical and physical processes, are mostly useful for dissolving
aliphatic linkages Teramura et al. [74], revealed that dilute sulphuric the hemicellulose and altering the lignin structure [85]. In this regard
acid predominantly reduces the lignin aliphatic linkages or ferulate in AFEX pretreatment has been effective for higher delignification in
rice straw. Savy et al. [75] also found similar results for Miscanthus alfalfa [86], and for over 90% hydrolysis of hemicellulose in Bermuda
and giant reed grass. Apart from the breakage of lignin bonds, the effect grass [87]. Further, combination of pretreatments has been shown to
of dilute acids has also been studied for decrease in Crl and DP of decrease the formation of by-products, thereby decreasing inhibition in
grasses. Foston and Ragauskas. [76] during their study on pretreat- the final fermentation step [88]. Ammonia recycle percolation (ARP)
ment, indicated that it is not only the debonding of lignin which has been paired with the AFEX pretreatment process for grasses. Iyer
determines the effectiveness of pretreatment but the DC and DP of the et al. [89] applied the pretreatment process on switchgrass and
biomass are equally responsible. The authors concluded that cellulose achieved 85% delignification without the formation of inhibitors.
CrI and DP are altered during dilute acid (H2SO4) pretreatment and Chan et al. [90] performed a CO2 added AFEX pretreatment on rice
can affect biomass recalcitrance in switchgrass. Another important straw and achieved a glucose conversion of 93.6% after the pretreat-
feature in the pretreatment technique is it is greatly affected by the ment. CO2 explosion is another physicochemical method which has
parameters maintained during the process. It has been observed that been experimented on switch grass by Luterbacher and coworkers [91]
though high temperature yields higher delignification, but substantial and has proven to give good glucose yields of up to 81%. Similarly
loss of biomass also occurs in the substrates. Redding et al. [77] found LHW, which are much alike as the steam explosion pretreatment has
similar conclusions after pretreatment of costal Bermuda grass at high also been an interesting physicochemical method. Recently, Yu et al.
temperatures with dilute H2SO4. In analogous studies it has been seen [92] pretreated two varieties of Pennisetum hybrid (I and II) and a
that high acid concentrations and high temperatures during dilute acid switchgrass variety with LHW to enhance the release of sugars and has
process can cause degradation of sugar monomers to furans, which are achieved encouraging results for delignification of the biomasses.
inhibitory to yeast during fermentation [78]. SPORL pretreatments which involves the treatment of biomass with
The importance of alkali pretreatment in grasses is due to the fact calcium or magnesium sulfite followed by significant reduction of size
that, alkali pretreatment not only offers solubilisation of more than of the pretreated biomass using mechanical disk miller has been
50% of grass lignins by destruction of alkali-labile ester linkages but effective in removing hemicellulose and lignin [93]. Zhang et al. [94]
also solubilizes high free phenolic content, thus improving the lignin used sulphuric acid dosage (0.8–4.2%) and sodium sulfite dosage (0.6–
solubility [79]. Alkaline pretreatment also causes less sugar degrada- 7.4%) followed by size reduction pretreatment on switchgrass biomass.
tion, and are economical as many of the caustic salts can be recovered Improved elimination of hemicellulose and partial removal of lignin
and/or regenerated [45]. Li et al. [80] in a very interesting study along with decrease in hydrophobicity of lignin due to sulfonation was
concluded that alkaline hydrogen peroxide effectively reduced total observed.
lignin content and the content of p-hydroxycinnamates, which had the Apart from the conventional process of pretreatment, advanced
maximum effect on enzymatic hydrolysis. The authors also found that studies have been carried out in the recent past using microwave,
the amount of S/G (syringyl to guanicyl) ratios did not appear to ultrasonication pretreatments because of their unique heating mechan-
contribute much to the enzymatic digestibility or delignification of four ism, which selectively heats the more polar parts of the biomass [95].
different grass cultivars, and the predominant effect of this phenom- This unique factor creates a ‘hot spot’ within heterogeneous biomass
enon was specifically observed in case of switchgrass cultivar. In a [96] enhancing the disruption of the recalcitrant structures of ligno-
similar study by Savy et al. [75], Miscanthus and giant reed grass were cellulose. Zhu et al. [97] conducted chemical assisted microwave
seen to have breakage of C–C bonds between lignin monomers and the treatment on Miscanthus and led to the conclusion that the selective
degradation of phenolic species using alkaline hydrogen peroxide. heating and electromagnetic field created by the microwave are the
Ionic liquids (ILs) have recently attracted substantial attention due prominent factors in efficient pretreatment. Sharma et al. [98] and
to their ability to dissolve a wide range of lignocellulosic materials and Hong and Mai [99], attributed higher delignification of switchgrass and
have been seen for associated environmental benefits [81]. Soudham Bermuda grass to the similar unique factor of ultrasonication pre-
et al. [82] conducted IL pretreatment on reed canary grass using new treatment.
acidic switchable ILs (SILs) like DBU–MEA–SO2 (DBU: 1, 8-diazabi- While many researchers have used chemical, physio-chemical and
cyclo [5.4.0] undec-7-ene; MEA: monoethanolamine) and DBU–MEA– other pretreatment techniques for delignification and higher glucose
CO2. The authors obtained significant delignification with excellent yields of different grass varieties, the high energy costs and liquid
glucan-to-glucose conversion levels (between 75% and 97%) after the loadings required specifically in physicochemical techniques are still
enzymatic hydrolysis of IL-treated substrates. In order to evaluate the the major draw backs owing to its less preference. Further, though
efficiency of IL pretreatment on grasses comparative studies have also SPORL pretreatment has been popular in the recent times because of
been performed using acid/alkali and ILs. In one such research, Li its very high conversion rate of cellulose to glucose and maximum
et al. [83] compared the efficiency of two pretreatment techniques i.e. removal and recovery of hemicellulose and lignin, but post-pretreat-
dilute acid hydrolysis and dissolution in an ionic liquid. Significance of ment washing and high cost of recovering pretreatment chemicals need
the pretreatment was seen for delignification and saccharification to be addressed. The scalability of the techniques in the commercial
yields for switchgrass. The authors observed that switchgrass exhibited production of biofuels is still a challenge that has to be considered.
reduced cellulose crystallinity and lignin content and higher sacchar-
ification yields in IL treatment as compared to acid treatment. 3.1.3. Biological delignification of grass
Even if IL pretreatment have presented promising results as Chemical and physicochemical delignification generally leads to
compared to acids and alkalis but the expensive chemicals, with high energy demand, corrosions, and by-product formations and
retention time of several days has made the process inconvenient. adversely effects the environment. Thus, biological delignification
But, the recent advances in the technique have decreased some of the which does not produce aforesaid conditions can be a promising
1016
alternative. Both, bacteria and fungi can be involved in biological converted to (1Z, 3Z)-4-hydroxybuta-1, 3-diene-1, 2, 4-tricarboxylate
delignification, but the effective depolymerisation of lignin is generally in a route that hasn't been characterized yet. (1Z, 3Z)-4-hydroxybuta-
achieved by using white-rot fungi or basidiomycete [100]. A represen- 1,3-diene-1,2,4-tricarboxylate spontaneously converts to its tautomeric
tation of the biological delignification process that has been proposed form (1E,3E)-4-hydroxybuta-1,3-diene-1,2,4-tricarboxylate, which is
by different researchers using fungi and bacteria have been amalga- degraded into TCA cycle intermediates by the action of two additional
mated and presented in a single figure as given in Fig. 3. As mentioned enzymes-4-oxalomesaconate hydratase (lig J) and oxaloacetate β-
earlier grass lignins have three types of monolignons and the H lignin is decarboxylase/4-oxalocitramalate aldolase (lig K) [108].
the most abundant form of lignin found in grasses. The pathway shows With the use of these defined pathways, Kuhar et al. [109] used a
the formation of the specific monolignons from the three major basidiomycete fungus RCK-1 and pretreated wheat straw under solid
sections of lignin i.e. H, G and S that are formed from coumaryl state fermentation. The results indicated an increase in the ethanol
alcohol, conniferyl alcohol and sinapyl alcohol respectively. The path- yield by 33%. However, the residence time observed was more in this
way can be divided into three parts which are a) degradation of H into study. But, a similar study on wheat straw by Salvachu et al. [110]
protocatechuate, b) degradation of guaiacyl into vanillin which further using fungi Panus tigrinus and Trametes versicolor gave 47% increase
degrades into the TCA Cylcle, c) degradation of syringate into the TCA in delignification with lowest residence time of 7 days. Liong et al.
Cycle. [111] did a comparative study of pretreatment of Napier grass with a
The pathway of degradation of H into protocatechuate by fungus Phanerochaete chrysosporium for three weeks and another one
Aspergillus flavus (a basidiomycete) was proposed by Gold and Alic with NaOH. The authors reported that though delignification was
[101]. Fungi generally use extracellular and unspecific oxidative higher in biological delignification but 1.5 fold higher ethanol yields
enzymatic system for delignification. The process not only involves was obtained from NaOH pretreated Napier grass. Suhara et al. [112]
various enzymatic activities like that of oxidases, reductases and detailed the study of lignin degrading basidiomycetes on bamboo clums
peroxidases but compounds (low molecular weight) that mediate the and stated that it was effective in removing 50% of the lignin content.
processing of these enzymes. As given in the Fig. 2, H is first converted Wan and Li et al. [113] evaluated the biological pretreatment of various
back to p-coumarate which is then degraded into p-Hydroxybenzoic feedstocks like corn stover, wood and grass using Cereporiopsis
acid. The conversion of p-coumarate to p-hydroxybenzoic acid involves subvemispora. A two to three fold increase in reducing sugar produc-
a β-oxidation type of reaction. After formation of protocatechuic acid, tion was observed. Similarly, Cianchetta et al. [114] also reported
intradiol cleavage takes place by the enzyme protocatechuate 3,4- efficient delignification of wheat straw using Cereporiopsis subvemis-
dioxygenase which gives rise to β-ketoadipic acid and leads to the TCA pora. The addition of external carbon sources, inducers and chemicals
cycle [102]. like alkalis, acids and AFEX has also been reported to enhance
Similarly, Guaiacylglycerol-β-guaiacyl ether (GCE) degradation biological degradation of lignin [115]. Recently, Torreiro et al. [116]
pathway was proposed by using Sphingomonas sp. GCE consists of found that delignification of fungal (Irpex lacteus) pretreated wheat
two diastereomers which needs to be cleaved. But, prior to the cleavage straw increased with addition of a mild alkali pretreatment. Balan et al.
of the ether bond the compounds have to be oxidized by dedicated [117] also reported a high glucan and xylan conversion rate using
alcohol dehydrogenases. The organism has multiple such enzymes biological pretreatment of rice straw (Pleurotus ostreatus) followed by
(ligD, ligL and ligN) with differing selectivity [103,104]. Following AFEX pretreatment. Many other studies have revealed the high
oxidation, the compounds are activated and cleaved by the enantiose- conversion effects of glucans and xylans using combination of biologi-
lective glutathione-S-transferase/β-etherase enzymes encoded by ligE cal pretreatments with liquid hot water and acids (H2SO4) in various
and ligF. The glutathione moiety is cleaved by glutathione lyases such substrates like poplus, rice hull and water hyacinth respectively [118–
as the enzyme encoded by ligG, generating β-hydroxypropiovanillone, a 120].
non-stereoactive molecule [105]. β-hydroxypropiovanillone is de- An alternative biological pretreatment method is the method of
graded into vanillin or vanillate in a process that hasn't been defined ensiling or biomass preservation. Ensiling holds potential as an
yet, and those are degraded to central metabolism intermediates as integrated storage and pretreatment method with low cost and low
described in super pathway of vanillin and vanillate degradation. energy requirements plus brings about multiple advantages with
Sphingomonas sp. converts vanillin to vanillate by vanillin dehydro- regards to agricultural management [121]. Jensen et al. [122] explored
genase (encoded by ligV) [103]. Vanillate is converted in the ensiling pretreatment for temperate grasses like Festulolium
Sphingomonas sp. to protocatechuate by a tetrahydrofolate-dependent Hykor. However, the pretreatment effect of ensiling, and the overall
demethylase, vanillate/3-O-methylgallate O-demethylase, encoded by effects for further conversion are limited.
ligM [106]. Protocatechuate is degraded by this organism into central With a limited number of studies on the mechanism of the
metabolism intermediates using the Meta cleavage pathway, as de- delignification processes carried out by micro-organisms, further
scribed in protocatechuate degradation I (meta-cleavage pathway) research is required to completely understand the intermediates
[102]. Protocatechuate is then converted to pyruvate which is then obtained during the delignification process. This will be particularly
directed into the TCA cycle. helpful in enhancing of our insight into the conversion pathways, which
The final conversion of S lignin begins with the degradation of would further help us in structuring of genes for genetically improved
syringate with its conversion to 3-O-methylgallate by the tetrahydro- microorganisms. Further organisms can be designed for targeted
folate-dependent syringate O-demethylase (encoded by desA) [105]. 3- delignification in grasses with low S/G ratio and higher H lignins.
O-methylgallate can be degraded further via three different routes that Combination of biological pretreatment with other pretreatments and
converge back at (1Z, 3Z)-4-hydroxybuta-1, 3-diene-1, 2, 4-tricarbox- ensiling pretreatment also seem to be attractive technologies that can
ylate. The main route, via gallate, is catalysed by two enzymes – a be utilised for herbaceous biomass for enhanced delignification, never-
second tetrahydrofolate-dependent demethylase, vanillate/3-O- theless more research is required for cost-effective combination
methylgallate O-demethylase (ligM), and gallatedioxygenase (des B) biological pretreatments.
[107]. The other two routes are the result of direct dioxygenation of 3-
O-methylgallate. This reaction is carried out by two dioxygenases – 3.2. Enzymatic delignification of grasses
protocatechuate 4,5-dioxygenase (ligA and ligB) and 3-O-methylgallate
3,4-dioxygenase (des Z), both of which produce a mixture of two Enzymatic delignification of grasses is an alternative to biological
different products -2-pyrone-4,6-dicarboxylate (PDC) and 5-carboxy- pretreatment where the objective is to increase the rate of reaction and
vanillate (CHMOD) [108]. While the first product is hydrolysed by 2- delignification efficiency. This strategy not only offers the possibility of
pyrone-4, 6-dicarboxylate hydrolase (ligI), the second product is substrate specificity but also reduces the processing time from weeks to
1017
Fig. 3. Pathways of conversion of the lignins present in grass biomass.
1018
hours without consumption of carbohydrate [123]. Enzymes for et al. [140]. 2% NaOH was used as an extractant which was followed by
delignification can either be collected from culture supernatant of acidification, precipitation and washing by 70% ethanol. A 26.2% yield
lignolytic microorganisms or commercial enzymes which contains of pentosans was observed. Another study by Singh et al. [141] used
purified and concentrated enzymes [124]. Mostly, lignin degrading dilute acid for hemicellulose extraction from Kans grass and found a
enzymes like laccase, mannase and peroxidases are produced by higher yield of xylan sugars in the liquid hydrolysate, which was further
P.chyrosporium, Ceriporiala cerata, Cyathus stercolerus, used for ethanol production. Njoku et al. [44] applied a different
C.subvermispora, Pyconoporus cinnarbarinus, Trametes villosa and approach by applying wet explosion treatment on cocksfoot grass for
P.ostreaus [125]. extraction of the hemicellulose fraction. The authors concluded that the
From various types of lignolytic enzymes, laccase (copper containing release of hemicellulose sugars is maximal in the liquid hydrolysate,
oxidase enzymes) in the form of laccase mediator system (LMS) have been with application of less severe pretreatment condition.
extensively used in industrial delignification processes, as without these Extraction of the hemicellulose fraction will not only improve the
mediators laccases have limited usage [126]. In LMS, a redox material fermentation of pentosans but will also increase the subsequent
which is stable in its oxidized and reduced state and does not inhibit the enzymatic hydrolysis of cellulose. This effect was shown by Spindler
catalytic activity of laccase is used [127]. In this context, Guitierrez et al. and co-workers [141] who observed an increase in cellulose digest-
[128] observed the synergistic ability of laccase enzyme from Trametes ibility by five to seven times in wheat straw. Similar results were also
villosa and I-hydroxybenzotriazole (HBT) which is a synthetic mediator, on given by Kong et al. [142] who observed a major effect on cellulose
milled elephant grass. Following the treatment, the delignification increased digestibility by the removal of the hemicellulose content. But, contra-
by 50%, enzymatic hydrolysis by 12% and the ethanol concentration dictory reports have also been seen. For example, Weimer et al., 2007
reached 2 g/L. In a similar study by Sidhu. [129], switchgrass was subjected [281] advocated that cellulose and xylan association does not restrict
to LMS with different mediators like HBT, ABTS (2, 2′-azino-bis (3- the hydrolysis of the polysaccharides. Conversely, the removal of
ethylbenzothiazoline-6-sulphonic acid) and violuric acid. Reduction of 28% hemicellulose removes acetyl groups which alter the structural form
lignin was observed with violuric acid. In another study [130], acid pre- of left lignin, thereby making the isolation of the most influential
treated wheat straw with laccase-HBT in combination with alkaline factors responsible for improving hydrolysis, difficult and time con-
peroxide extraction was studied. It was observed that LMS increased the suming [143]. In a more recent research, xylan and mannan have been
saccharification yield by 35% which was attributed to enhanced lignin reported to produce inhibitory effects on the proceeding cellulose
extraction due to LMS-induced formation of Cα oxidized groups in lignin. hydrolysis step [125]. With divergent reports on extraction of hemi-
The most significant effect of LMS was observed in wheat straw where high cellulose, it is imperative to focus on the structural characterization of
delignification yield (up to 97%) was achieved using laccase from the biomass before and after the extraction of hemicellulose from
Pyconoporus sanguineus and violuric acid as a mediator [131]. biomass for better understanding of its chemistry with cellulose with
LMS is advantageous in terms of significant reduction in pretreat- respect to bioethanol production.
ment time, elucidation of sugar consumption and thereby improving
the saccharification and fermentation. But, from an economical pro- 3.4. By-products of pretreatment
spect the main disadvantage is the production cost of the enzyme and
the synthetic mediators [127]. Alternatives can be replacement of Formation of by-products during pretreatment is mostly observed
natural mediators like acetosyringone, syringaldehyde and p-coumeric with the solubilisation and degradation of pentosans and lignin,
acid extracted from lignin in place of synthetic mediators [132] and although other extractives and hexosans also contribute minutely for
growth of laccase producing microorganisms in lignocellulosic feed- the formation of inhibitors [144]. In sufficiently high concentrations
stocks, rather than synthetic medium [133,134]. An example of laccase these inhibitors inhibit fermenting microorganisms, such as yeast and
producing microorganism with the capability of enzymatic hydrolysis bacteria [145]. Some important inhibitors produced during different
have been seen in Pycnoporus sp using switchgrass as a substrate pretreatment methods from different type of grasses are exemplified in
[135]. Thus, screening of such microorganisms can not only be Table 3.
beneficial for consolidated bioprocessing approaches leading to re- In this context, an intensive study on lignin chemistry is required
duced processing stages, but will also help in reducing the usage of for the elimination of lignin from the biomass that could potentially
chemicals which further needs downstream treatment processes in affect the subsequent hydrolysis process. Though previously, much
industrial sectors. importance was not given on hydrolysis process with respect to the
lignin components, but recent works are mainly focused on these
3.3. Extraction of hemicellulose fraction in grasses aspects. This holds true due to the fact that the structure and
concentration of the lignin components affects their hydrolysis [146].
The secondary walls of grasses predominantly consist of xylans, More recently it has been studied that the presence of lignin in the
more specifically Glucuararonoxylans (GAXs) and mixed-linkage glu- biomass leads to higher requirements of cellulases in the subsequent
can (MLG). GAXs has a β-(1,4)-linked xylose (Xyl) backbone and ara saccharification step. This is because the enzyme binding generates a
and glucuronic acid (GlcA) side chains, while MLGs are unbranched non-productive attachment with certain components of lignin and
chains of ~30% β-(1,3) and ~70% β-(1,4)-linked glucopyranosyl limits the accessibility of cellulose to cellulase [147]. Furthermore,
residues [135–137]. The GAXs are immensely underutilised sources phenolic groups are formed from the degradation of lignin. These
for bioethanol [138] and this is mainly due to the economic challenges components substantially create hindrance for cellulolytic enzymes to
that limit the extraction and utilisation of these valuable products. reach cellulose and hence influence enzymatic hydrolysis. Phenolic
Mostly the GAXs are either degraded in the pretreatment step or are compounds that are typically formed in grass processing are p-
present in minimal quantities along with the celluloses. coumaric, ferulic and diferulic acids. Though these are not representa-
Extraction of the GAXs generally involves the alkali dissolution tive of lignin components, but contribute to crosslinking with hemi-
followed by precipitation using alcohols such as 2-propanol, methanol, celluloses and esterified to arabinoxylans form ether- or ester-linkages
or ethanol. Following this an oxidative bleaching is done to remove the with lignin [148]. To overcome these, grasses with relatively low
contaminating lignin in the precipitate [139]. In one such study, recalcitrance, which in turn can be pretreated under mild conditions
Stoklosa and Hodge. [138] used increasing levels of NaOH followed can be employed. To exemplify the positive effects of mild pretreatment
by precipitation using ethanol on switchgrass biomass. This resulted in Chiaramonti et al. [149] and Larsen et al. [150] carried out hydro-
high yields of the hemicellulose from the grass. The same approach was thermal pretreatment without addition of acid catalysts for bioconver-
also tested in bamboo (generally considered as a woody grass) by Luo sion of Miscanthus grass and wheat straw respectively. They concluded
1019
Table 3
Inhibitors produced during different pretreatment methods from different type of grasses.
Pretreatment method Type of grass used Chemicals involved By-products formed Reference
Mild acid treatment combined with Cocksfoot grass H2SO4 High concentrations of acetic acid and low [44]
wet-explosion treatment amounts of Furfural, Hydroxymethy furfural
Mild acid with high temperature Bermuda grass H2SO4 Furfural, Hydroxymethy furfural [77]
treatment
Mild acid with higher incubation time Bermuda grass H2SO4 Acetic acid, Furfural, Hydroxymethy furfural [328]
Mild acid with higher incubation time Bermuda grass HCl
Mild acid with high temperature Rice straw H2SO4 5- Hydroxymethy furfural and Formic acid [74]
treatment
Mild acid and alkali aasisted with Miscanthus H2SO4/NaOH microwave Furfural, Hydroxymethy furfural [97]
microwave pretreatment assisted pretreatment
Hydrothermal pretreatment Wheat sraw High temperature Formic acid, Acetic acid, Furfural, [329]
Hydroxymethy furfural
Hydrothermal pretreatment Prairie cord grass High temperature and low Acetic acid, Furfural, Hydroxymethy furfural [330]
incubation time
Ozonolysis Miscanthus ×giganteus, M. sinensis Low temperature and low alkali Gluconic acid [324]
‘Gracillimus’, Saccharum concentration
that mild pretreatment produced low concentrations of furan alde- not have much role in conversion of these complex carbohydrates to
hydes and phenols, but the concentrations of acetic acid were reported simple sugars which can be utilised by the microorganisms. The
to reach 17 g/kg/1 for Miscanthus and 5.1 g kg/1 for wheat straw. performance of enzymatic saccharification of lignocellulose strongly
Lower temperatures have also been reported to yield less inhibitor in depends on many factors such as the diverse species, complex chemical
grass biomass. In one such study, Redding et al. [77], pretreated composition, efficiency of the pretreatment technique, the mode of
coastal Bermuda grass at with acid at temperatures lower than 180 °C enzyme action and structural characteristics of the feedstock used
and reported much lower formations of formic and levulinic acids. [155]. Rivers and Emert. [156] in their study on lignocellulosic
Other effective techniques could be adding of catalysts during the biomass for bioethanol production, showed that, a specific pretreat-
pretreatment process. Jacquet et al., 2012 [151], experimented the ment is required for every individual lignocellulosic substrate depend-
use of catalysts like CO2 or SO2 during steam pretreatment of rice ing on its composition, if maximum enzymatic hydrolysis is to be
straw, bagasse and giant Miscanthus which resulted in significant achieved. Since the compositions of grasses are significantly different
decrease in inhibitory compounds in the subsequent hydrolysis and from other lignocellulosic biomasses, the main factors mostly affecting
fermentation steps. Advanced approaches could be integrated pretreat- the enzymatic hydrolysis are the cellulose crystallinity and the presence
ment processes combining two or more pretreatment techniques. of hemicellulose. In this context, Yoshida et al. [157] studied the effects
Recently, effect of microwave treatment on NaOH- and H2SO4- of cellulose crystallinity, hemicellulose and lignin on enzymatic hydro-
pretreated Miscanthus was studied by Zhu et al. [97]. An increased lysis in Miscanthus sinensis. The authors concluded that yield of
(twelve times higher) sugar yield with low concentrations of inhibitors monosaccharides increased with decrease in cellulose crystallinity,
was obtained in half the time compared to single pretreatments. To add delignification and complete hydrolysis of hemicellulose by xylanases.
advantage to the combined pretreatment techniques, “in-situ detox- This can be attributed to the increase in enzyme absorption capacity of
ification” method wherein organisms like some species of S. cerevisiae, the amorphous cellulose [158]. Hence, during the biochemical conver-
having capacity to transform furfural and HMF into less toxic sion of lignocellulosic biomass to fuels, cell wall-deconstructing en-
compounds of furfural alcohol and 2, 5-bishydroxymethylfuran, re- zymes are used to convert plant cell wall polysaccharides into fermen-
spectively can be utilised [69]. Further, some species of Pichia stipitis table sugars. Cellulases and hemicellulase are the cell wall deconstruct-
which do not get affected by furfural in low concentrations up to 0.5 g/ ing enzymes which are mostly produced in higher quantities by
L are viable options to overcome the toxic effects of inhibitory bacteria and fungi. Commercially, cellulases have been commercially
compounds in the substrate that are produced during pretreatment available for more than thirty years, and have been used both in
[152]. Genetically improved strains for better inhibitor tolerance academic and industrial researches (Singh, 2007) [159]. But, techno-
capacity or adaptability would also be a preferable option. logical developments have also prompted most of the academicians and
Conversely, lowering the lignin content in grasses by molecular tools research institutes for isolating cellulolytic and xylolytic enzyme
has also been experimented. Chen et al. [153] demonstrated that lignin producing organisms and further extraction and application of these
modification via genetically engineering practices targeting its biosyn- enzymes for various purposes (Kuhad et al., 2011) [160]. Application of
thetic pathways could considerably reduce lignin formation and the enzymes on the substrate of interest can be made taking two
improve ethanol yield. However, this could be quite challenging as important things into consideration. The foremost thing is the compo-
lignin components serve as the major plant defence system to pathogen sition of the biomass i.e. presence of higher crystalline or amorphous
and insects and its modification could disrupt the plants’ natural cellulose and the amount of hemicellulose present. Other than the
protection [154]. composition, the proficiency of the enzyme (i.e. its activity), to
hydrolyse the carbohydrate of interest in the biomass is also a vital
parameter that has to be considered. With these factors in considera-
4. Enzymatic hydrolysis tion, this section of the review aims to highlight the work conceded by
the research groups on application of both commercial and isolated
4.1. Enzymatic saccharification enzymes for higher saccharification yields of grass biomass.
The foremost objective of enzymatic saccharification is to induce

structural changes of cellulose and other carbohydrate polymers in the 4.1.1. Commercial enzymes for enzymatic hydrolysis
pretreated biomass into fermentable sugars by using biological or The major source of commercial cellulase enzymes are filamentous
chemical approaches [143]. Though pretreatment is essential in break- fungi and mutant strains of Trichoderma (T. viride, T. reesei, T.
ing of the bonds that holds lignin with the carbohydrates, but it does longibrachiatum) [161,162]. But, production of enzymes from a single
1020
organism leads to variable problems like lower thermostability and lack ml),from T.ressei and achieved total reducing sugars of 350 mg/g.
of specific activities [163]. An example is cellulases from Thrichoderma Belal, [173] taking rice straw as her lignocellulosic substrate also
reesei which produces endoglucanases and exoglucanases in bulky concluded that T.ressei is an efficient strain for cellulase production
quantities, but has low βglucosidase activity which results in a lower and cellulose hydrolysis. In a recent study, Shrestha et al. [174] isolated
biomass saccharification. To avoid such circumstances, some enzymes thirty fungal sp. from decaying leaves of Miscanthus and sugarcane and
producers have marked new cellulase mixtures or multienzyme mix- also produced a variety of cellwall degrading enzymes showing
tures which can sustain stability at higher temperatures. A commercial activities for xylanases, exocellulases, endocellulases, and beta-gluco-
enzyme Accellerase 1500 which is a mixture of cellulases complex (i.e. sidases. This approach will not only produce value added products but
exoglucanase, endoglucanase, hemi-cellulase and β-glucosidase) pro- will also utilise the decaying lignocellulosic biomasses and in turn help
duced from a genetically modified strain of T. reesei. was utilised on in restricting environmental pollution. The study also suggested that a
four perennial grasses ryegrass, tall fescue and bentgrass and high majority of fungi tested equalled or exceeded the bioconversion
hydrolysis yields (~80%) were observed for all three grasses [78]. abilities of native T. reesei.
Meineke et al. [164] also reported enhanced hydrolysis of switch grass Apart, from fungus many bacteria have also been investigated in
using Accellerase 1500. But, similar results were not obtained by Tut production of cellulase that has been seen to efficiently saccharify
and Olt. [165] who also used Accellerase 1500 on energy grasses like cellulose to reducing sugars. Another advantage of using bacteria as a
Miscanthus saccharifloris and achieved a low glucose yield of 59.80% cellulase source is, mostly the enzymes are thermostable. Zambare
after saccharification. The glucose and xylose yields can be enhanced by et al. [175] isolated cellulase and xylanase from thermophilic con-
using accessory enzymes in combination with the above. Examples of sortium of bacteria with maximum activities of 367 U/L and 489 U/L
such enzymes are Accelerase®XP which enhances both xylan and at 60 °C and 70 °C taking prairie cord grass and corn stover as
glucan conversion; Accelerase®XC which contains hemicellulose and substrates respectively. It was further demonstrated that enzyme-
cellulase activities and Accelerase® BG which is a β-glucosidase generated using prairie cord grass as a substrate, was a good source
enzyme. Singh et al. [166] studied on the effect of particle size on for bioethanol production. Sharma et al. [176] used cellulase from
enzymatic hydrolysis of pretreated Mischanthus sp. Singh and his co- isolated bacillus (S3B8) species, with activity of 0.12 U/ml on switch
workers performed the enzymatic hydrolysis using Accellerase 1500 in grass for bioethanol production. Higher enzyme activities and sacchar-
combination with all the accessory enzymes as stated above and found ification of cellulases from bacterial strains have also been reported by
an increased polysaccharide conversion. Badhan et al. [167] experi- researchers. An example can be cited by the work done by Singh et al.
mented Accellerase 1500 with Accelerase®XC in combination with [177], who used CMCase (1.7 mg/ml) from isolated Bacillus amyloli-
isolated rumen enzymes on alpha alpha hay and barley straw. The quefaciens for enzymatic hydrolysis of pretreated Parthenium hyster-
authors got some interesting findings and concluded that while a 1.6 ophorus and achieved 187.4 mg/g of fermentable sugars.
fold increase in glucose yield was observed with the addition of With the reported results it can be inferred that the isolated
complementary rumen enzymes, the xylose yield enhanced to seven enzymes from fungus and bacteria, in the labrotary scale can be a
fold higher in alfalfa and fivefold higher in barley straw. A variant of the good alternative for hydrolysing herbaceous substrates. Though redu-
commercial enzyme i.e. Accellerase 1000 has also shown satisfactory cing sugars yields post saccharification is not as promising as com-
results on alkali pretreated cogon grass [168]. Other, commercially mercial enzymes, but application of molecular and genetic tools can
important enzymes are Celluclast 1.5 L (Sigma), cellic Ctec and cellic further enhance the saccharification yields by many folds. The strate-
Htec (Novozymes). The latter is used in mixtures and works well on a gies for using economical substrates like agro-wastes are a good
variety of pretreated feedstocks for converson of the polymeric sugars alternative to combat the high cost of the commercial enzymes.
to monomers [169]. With the advent of the enzymes, Xu et al. [40]
performed the enzymatic hydrolysis of pretreated switch grass and 5. Fermentation of grasses for bioethanol
costal Bermuda grass using cellic Ctec and cellic Htec. More reducing
sugars were obtained from enzyme hydrolysed costal Bermuda grass Pretreatment and saccharification processes are designed to opti-
than switchgrass. Recently, Thomsen et al. [170] used CellicCTec2 and mize the fermentation process. In general many fermentation techni-
HTec2 in ratio of 90:10 on pretreated wheat straw and achieved higher ques are employed for bioethanol production depending on the
enzymatic convertibility of both glucan and xylan. substrate and microorganism used. The general fermentation techni-
Though commercial enzymes, in small quantities, have accelerated ques can be broadly classified into submerged fermentation (SmF) and
the enzymatic hydrolysis processes by many folds, but the economical solid state fermentation (SsF) either in batch, fed-batch or continuous
availability of the same is not yet achieved. Hence, innovative culture systems that can be utilised for different type of substrates. But
bioprocesses for the production of new generation of enzymes are in order to improve the economic viability of lignocellulose-to-ethanol
needed. An alternative, for small scale labrotary preparations can be conversion, both the cellulose and hemicellulose hydrolysates should
the use of isolated enzymes from microorganisms like bacteria and be utilised for ethanol production. In that regard many improved
fungi. techniques of fermentation have been developed and been implemen-
ted in fermentation of grasses. A number of studies are carried out in
4.1.2. Isolated enzymes for enzymatic hydrolysis switch grass [178], reed canary grass [179], Bermuda grass [180],
The high cost of the commercially available cellulolytic enzymes has silver grass [181], kans grass [182], sea grass [183], cocksfoot grass
led to many reports using isolated cellulase and xylanase enzymes [44], elephant grass [62] and mostly Napier grass [184–186] using the
either from native or from recombinant strains [171]. In that regard improved fermentation techniques. These outlines are summarized in
many researchers have come up with promising results for enzymatic Table 4.
hydrolysis of grasses using isolated enzymes. Wongwatanapaiboon Fermentation of grasses has mostly been carried out by different
et al. [15] used T.ressei for production of cellulase and xylanase with processes such as separate hydrolysis and fermentation (SHF), simul-
activity of 0.948 ± 0.05 and 92.13 ± 6.86 U/ml. The enzymes were taneous saccharification and fermentation (SSF) and simultaneous
used for saccharification of eighteen varieties of grass cultivars, which saccharification and co-fermentation (SSCF). In SHF enzymatic hydro-
showed total reducing sugars of 500–600 mg/g of substrate. The lysis is performed separately from the fermentation while in SSF/SSCF
production of reducing sugars by isolated enzymes was quite significant cellulose hydrolysis is carried out in the presence of the fermentative
to that of the commercial enzymes. In a similar study, Katarina and microbes [187] like yeasts, bacteria, and fungi which can ferment
Ghosh, [172] achieved a crude enzyme mixture of cellulase and ethanol in lignocellulosic hydrolysate. SHF is a process where separate
xylanase enzyme (CMCase-1.41, FPase-1.12, and Xylanase-6.23 U/ hydrolysis and fermentation are carried out in separate units. In a first
1021
Table 4
Global vision of fermentation process for bioethanol production using divergent grass varieties.
Sl no Substrate Microorganisms Process of Parameters Ethanol yield Reference

fermentation
1 Dwarf Napier grass Saccharomyces cerevisiae NBRC SSCF 36 °C, pH 5.0 74.1% [186]
(Schumach) 2044+Escherichia coli KO11
2 Dwarf Napier grass S. cerevisiae NBRC 2044 SSF 35 °C, pH 5.0 121 mg/g [185]
(Schumach)
3 Dwarf Napier grass Saccharomyces cerevisiae NBRC SSF 34 °C, pH 6.6 68.9% [331]
(Schumach) 2044+Escherichia coli KO11
4 Dwarf Napier grass E.coli KO11 SSF 34 °C, pH 5.0 144 mg/g [184]
(Schumach) (44.2%)
5 Kans grass S. cerevisiae SA 28 °C, pH 5 0.46 g/g [182]
(Saccharum spontaneum) 200 rpm
6 Napier grass E. coli LY01 SSCF 35 °C, pH 5.5 224.5 mg/g [209]
(Merkeron) (73%)
7 Napier grass S. cerevisiae TV2 SA-HF – SA: 91.8% [332]
(Pennisetum purpureum) HF: 76.9%
8 Napier grass Klebsiellaoxytoca THLC0409 SSCF 31 °C, pH 7.04 82 mg/g [333]
(Pennisetum purpureum) 472 rpm (76.9%)
9 Napier grass Clostridium strain TCW1, Bacillus sp. SSCF 60 °C, pH 7.0–7.2 40 mg/g [334]
(Pennisetum purpureum) THLA0409, 150 rpm
Klebsiellapneumoniae THLB0409,
Klebsiellaoxytoca
THLC0409, Brevibacillus strain AHPC8120
10 Napier grass Bacillus sp. THLA0409, Klebsiella oxytoca SSCF 30 °C, 276 g/kg [335]
(Pennisetum purpureum) THLC0409 100 rpm (59%)
11 Napier grass S. cerevisiae (KY3 and KY-NpaBGS) SSF 40 °C, pH 5–6 3.32 mg/ml [153]
(Pennisetum purpureum)
12 Napier grass S. cerevisiae SSF 37 °C, pH 4.8 162 L/t DM [336]
(Pennisetum purpureum) 100 rpm (52%)
13 Switch grass S. cerevisiae 424 A (LNH-ST) SSCF 30–35 °C, pH 4.8–5.5 32.1g/L [337]
(Panicum virgatum) 180 rpm (72.7%)
14 Italian ryegrass E. coli KO11, S. cerevisiae SSCF 36 °C, pH 5.6 333 mg/g [338]
(Lolium multiflorum Lam) (84.6%)
15 11 types of grasses* S. cerevisiae+Pichia stipitis SSCF 35 °C, 150 rpm 0.02–0.14 g/g [15]
(Max. 32.72%)
16 Purple guinea grass (P. maximum S. cerevisiae TISTR 5596 SHF 30 °C, pH 5.0 5.92 g/L [194]
cv. TD53)
17 Grass silage (fibre fraction) Lactobacillus sp. SSF – 14.6 g/L [203]
18 Elephant grass S. cerevisiae CAT-1 SSF 6 min at 200 °C 107.72 µL/g [339]
(Pennisetum purpureum, Schum.)
19 Elephant grass S. cerevisiae CAT-1 SSF 28 °C, 12 h 6.1 g/L [62]
20 Switchgrass (Panicum virgatum) Kluyveromyces marxianus IMB3 SSF 45 °C, 168 h 22.5 g/L [340]
(86%)
21 Mission grass S. cerevisiae TISTR 5596 SHF pH 10, 24 h 16 g/L [189]
(Pennisetum polystachion)
22 Elephant grass Aspergillus niger and S. cerevisiae SSF pH 5.5, 35 °C, 72 h, 23.4 g/L [341]
(Pennisetun purpureum) 300 rpm (78%)
23 Reed canary grass S. cerevisiae D5A and SSF 35 °C, 100 rpm,72 h (81–84%) [179]
(Phalaris arundinacea L.) S. cerevisiae YRH400
24 Thatch grass Zymomonas mobilis SSF pH 4.3, 30 °C, 8.8 g/L [264]
(Hyparrhenia rufa) 120 rpm, 60 h
25 Napier grass E. coli KO11 SSF 35 °C, 24 h 18.5 g/g [111]
26 Sea grass S. cerevisiae SSF pH 4.5, 28 °C, 24 h 0.047 ml/g [183]
(Cymodocea serrulata)
27 Elephant grass (Pennisetum S. cerevisiae SSF 37 °C, 36 h, 150 rpm 26.05 g/L [342]
purpureum)
28 Cocksfoot grass Pichia stipitis CBS 6054 SSF 160 °C, 15 min, 87 psi 158 ml/kg DM [44]
(Dactylis glomerata) oxygen
29 Giant miscanthus Scheffersomyces (Pichia) stipitis SSF pH 5.5, 30 °C, 96 h 12.1 g/L [343]
(Miscanthus x giganteus) CBS 6054
30 Giant reed (C3 grass sp.) S. cerevisiae SSF 37 °C, 200 rpm, 48 h 8.2 g/L [344]
(Arundo donax L.)
31 Mission grass (Pennisetum Saccharomyces cerevisiae SHF pH 10, 30 °C, 24 h 16 g/L [189]
polystachion) TISTR 5596
32 Purple elephant grass S. cerevisiae SSF pH 4.5, 30 °C, 1.8g/L [345]
(Pennisetum purpureum Schum.) 4h (95%)
33 Rice grass Trichoderma reesei SEMCC 3.217 and S. SSF pH 4.5, 30 °C, 28.1 g/L [346]
(Spartina spp.) cerevisiae SEMCC 2.157 4 h, 150 rpm
34 Elephant grass Aspergillus niger and S. cerevisiae SSF pH 5, 35 °C, 300 rpm 16.8 g/L [347]
35 Cogon grass S. cerevisiae F1BY3 SSF 37 °C, 120 h 9.11 g/L [323]
(Imperata cylindrical L.)
36 Bamboo Zymomonas mobilis SHF 30 °C, 24 h 4.72 g/L [190]
1022
Table 4 (continued)
Sl no Substrate Microorganisms Process of Parameters Ethanol yield Reference

fermentation
(55.8%)
37 Silver grass Candida shehatae SHF pH 5.5, 24 °C, 150 rpm (64.3%) [181]
(Miscanthus floridulus)
38 Cogon grass S. cerevisiae SSF 37 °C, 72 h 19.08 g/L [202]
(Imperata cylindrical L.) (76.2%)
Abbreviation:
[SA: Saccharification of cellulosic components; SSF: Simultaneous saccharification and fermentation; SHF: Separate hydrolysis and fermentation; SA-HF: Saccharification of cellulosic
components/hexose fermentation; SSCF: Simultaneous saccharification and co-fermentation; DM: Dry matter; *11 types of grasses: Napier grass (Pennisetum purpureum), Dwarf
napier grass (P. purpureum cv. Mott), King napier grass (P. purpureum cv. King Grass), Bana grass (P. purpureum × P. americanum), Purple guinea grass (Panicum maximum TD 58),
Ruzi grass (Brachiaria ruziziensis), Pangola grass (Digitaria decumbens), Atratum grass (Paspalum atratum) and Vetiver grasses]
unit pretreated grasses are degraded to monomeric sugars by cellulases have been experimented by Yasuda et al. [184], which resulted in an
and xylanases and thereafter fermented to ethanol in a separate unit. ethanol concentration of 144 mg/g of untreated Napier grass. This
The main advantage is that the two processes (hydrolysis and technique is also beneficial for the diauxic behaviour (inability to use
fermentation) can be performed at their own individually optimal xylose in presence of hexose) of the pentose fermenting microorganism
conditions. This is quite essential because cellulases are efficient at in the fermentation medium [188]. Further to enhance the ethanol
temperature between 45 and 50 °C while commonly used fermenting yields amalgamations of SHF and SSF with co-cultures and immobi-
organism perform at an optimum temperature of 30–37 °C [188]. lized cultures have also been proposed by Chen. [70]. The major
Recently Prasertwasu et al. [189] conducted SHF on Mission grass advantage that a co-culture offers is the flexibility of combining a
(Pennisetum polystachion) with maximum ethanol yield of 16 g/L. But pentose fermenting microorganism with a hexose fermenting microbe
many researchers have also reported low ethanol yields using SHF [47]. [208]. Since the xylan or pentose sugar is relatively low in grasses and
He et al. [190] used SHF for bamboo residues and achieved an ethanol are mostly removed in the pretreatment step, using of a relatively not
concentration of 4.72 g/L. This was attributed to the unutilised so efficient pentose strain in combination with a highly efficient hexose
reducing sugar (25.0–40.0%) that was produced during the enzymatic fermenting microorganism can also be advantageous in achieving high
hydrolysis and was not fermented to ethanol. Other possible reasons of ethanol yields. These techniques have been reported to produce high
obtaining low ethanol concentration are the end product inhibition that titters of ethanol production from Napier grass [209], Bermuda grass
occurs, when glucose and cellobiose released in cellulose hydrolysis and bunch grasses [210] through a SHF process involving the enzy-
strongly inhibits the cellulase efficiency. Further, microbial contamina- matic saccharification and co-fermentation (CF) of hexose and pentose
tions in the medium may also lead to low ethanol yields [191]. To sugars by a recombinant E. coli LY01 (a derivative of E. coli KO11).
achieve a reasonable ethanol yield, lower solid loadings and higher Fermentation efficiency has also been seen to have increased by almost
enzyme additions could be needed [192]. SHF studies have been 20% using immobilized yeast and broth supplemented with Mg, Zn, Cu
conducted on switch grass and purple guinea grass by Chung et al. or capantothenate [211]. Since a fermenting organism shares the same
[193] and Ratsamee et al. [194] with an ethanol yield of 0.44 g/g and pathway for both glucose and xylose utilisation, and the affinity for
5.92 g/L respectively. xylose is 200-fold less than that of glucose, the transport of xylose into
An alternative to overcome the end product inhibition in SHF is the cell is inhibited by high amounts of glucose [207]. To overcome
simultaneous saccharification and fermentation (SSF) in which sugar this, low glucose concentrations should be maintained in the fermenta-
concentrations are kept low [195–198]. SSF can be carried out in two tion slurry. Lee and his co-workers suggested fed-batch and immobi-
different routes viz. either by processing the liquid pentose stream lization techniques can be utilised to overcome the aforesaid problem.
produced after pretreatment separately or by a co-fermentation step Another modified form of immobilization is co-immobilization of both
[199]. Considering SSF as an efficient technique as compared to SHF, hexose and pentose fermenting organisms in a single immobilized
Yasuda et al., [186] performed fermentation of Napier grass using E. bead. This allows hexose fermenting organisms to be fixed outside the
coli KO11 in SSF conditions and obtained an ethanol yield of 74.1%. beads and the xylose fermenting microbes inside the bead allowing
Similarly, Das et al. [200] reported seven fold increases in ethanol early utilisation of hexose with simultaneous consumption of xylose
concentration from thatch grass in SSF as compared to SHF using [212]. New techniques have led to immobilization of fermenting
Zymomonas mobilis as the fermenting organism. SSF has also been microorganisms like yeasts with natural lignocellulosic materials like
highly effective for high solid loadings or in scale-up processes. This sugarcane bagasse [213], alginate-chitosan beads [214], sweet sor-
has been demonstrated by Santos et al. [201] using sugarcane bagasse ghum pith [215], corncob pieces [216], cashew apple bagasse [217],,
as a substrate where an ethanol concentration of 60 g/L was obtained dried spongy fruit of luffa (Luffa cylindrica) [218], carboxymethylcel-
using 30% (w/w) substrate loading. Lin and Lee. [202] conducted SSF lulose (CMC) grafted with N-vinyl-2-pyrrolidone [219], sodium algi-
experiments on cogon grass in both shake flask (7 g) and 5 L rotary nate grafted with N-vinyl-2-pyrrolidone [220], lentikat discs [221] and
drum reactor (1 kg) and obtained an ethanol yield of 80.3% and 76.2% rice flour and white glutinous rice flour [222]. Sugarcane bagasse and
respectively. Similar results of obtaining higher ethanol yields have also agar-agar cubes have been successfully recycled with ethanol yields of
been observed by Suryawati et al. [204] and Isci et al. [205] from 0.44 g/g and 0.33 g/g in sugarcane bagasse [223]. Saccharum sponta-
switchgrass using SSF. Sieker et al. [203] used SSF for grass silage and neum pith, which are not toxic to microorganisms have also been used
obtained an ethanol concentration of 14.6 g/L and also stated that as immobilization matrixes for lignocelluloses [224].
enzyme hydrolysis is an essential requirement before fermentation. The In recent years focus has been made on emerging approaches in
finding of Sieker et al. [203] was well evidenced, when low ethanol yield bioethanol production for reduction of production costs. This approach
from SSF of switchgrass was reported by Wymann [206]. The low yield aims at developing consolidated bioprocess schemes (CBP) wherein the
of ethanol was reported because of the use of strategies like direct same microorganism is accomplished for cellulase production, sub-
microbial conversion (DMC), with evasion of enzymes during sacchar- strate hydrolysis, and fermentation in a single step [225]. CBP is
ification process. advantageous as it employs microbes to perform all the four biologi-
Recently, techniques like SHF followed by pentose fermentation cally-mediated transformations viz. the production of saccharolytic
1023
enzymes, the hydrolysis of carbohydrate components present in genetic tools or markers have also been developed in which the initial
biomass to simple sugars, the fermentation of hexose sugars and finally stages of plant growth can predict its yield. Recently, this robust
the fermentation of pentose sugars in a single step [226,227]. Thus, technique has been applied on Miscanthus. This technique was used to
this process can substantially lower the production cost and enhances identify links between early establishment traits and biomass yield in
the efficiency of bioethanol production, as compared to independent Miscanthus. This marker-assisted selection technique could save years
hydrolysis and fermentation steps. Fusarium oxysporum is one such of waiting for Miscanthus plants to establish and become mature [240].
fungus that is capable of degrading and fermenting a wide variety of The technique can be used to reliably select higher-yielding plants
different lignocellulosic substrates including grasses under CBP [228]. earlier in the grass life cycle.
Little has been studied on co-immobilization and CBP techniques A new approach of gaining control over the lignocellulosic recalci-
on grasses. Hence, more research with these modern techniques in trance is gaining insight into the molecular dynamics simulations of the
grasses could lead to better substrate utilisation and higher ethanol lignocellulosic structure. Molecular dynamics simulations have been
yields. Further, the interaction mechanism of the aforesaid biological used to investigate decrystallization of cellulose, the interactions
matrices with the fermenting organisms can be studied intensively to between cellulose and noncellulosic components of plant cell walls,
examine any toxicity effects of these matrices on the organisms. Since, and the structure and dynamics of lignin. Molecular dynamics simula-
pore size is also an important aspect in immobilization studies, the tions are mostly used for addressing both solvation structure and
matrix chosen for immobilizing a particular microbial species should thermodynamics of effective interactions in cell walls based on
also be considered. Undoubtedly, CBP processes are the next genera- molecular forces [241]. Silveira et al. [241] employed the statistical-
tion technologies in commercial ethanol production from herbaceous mechanical, 3D reference interaction site model with the Kovalenko–
biomasses. Therefore, identification of microorganisms capable of Hirata closure approximation (or 3D-RISM-KH molecular theory of
carrying out the CBP process in grasses remains an important area of solvation) to reveal the supramolecular interactions in this network and
research that is yet to be exploited. provide molecular-level insight into the effective lignin–lignin and
lignin−hemicellulose thermodynamic interactions. The research group
6. Improvements in bioethanol from grasses found that such interactions are hydrophobic and entropy-driven, and
arise from the expelling of water from the mutual interaction surfaces.
The success of grass biomass as an energy crop for bioethanol The molecular origin of these interactions was carbohydrate–π and
production in large scale will depend mainly on two factors i.e. the π–π stacking forces, whose strengths are dependent on the lignin
biomass yield and the level of biomass recalcitrance including other chemical composition and the methoxy substituents in the phenyl
properties of the cell wall. In the following section, we discuss about the groups of lignin, promote substantial entropic stabilization of the ligno-
improvements that have been carried out till date on either the crop or hemicellulosic matrix. A study on glucuronoarabinoxylan (mainly
the microorganism involved in bioethanol production, by means of consists of glucuronic acid and arabinose) an abundant hemicellulose
genetic and molecular processes. This will not only update the findings in grasses has been carried out using similar techniques by Mortimer
of the researchers in this area but will also improve our insights to the et al. [242,243], where genetic manipulation of glucuronic acid
modern technologies that have been implemented for developing grass branching has been shown to significantly improve xylan extractability
biomass as a potential candidate for bioethanol production. from cell walls without impairing plant growth.
6.1. Genetic improvements in grasses 6.2. Molecular Improvements
With thousands of grass genotypes being deposited in seed banks, Molecular techniques can be employed to either minimize the
only few varieties have been accessed and screened for identification of recalcitrance of the grasses or in increasing the efficiency of micro-
bioenergy traits [229]. Transgenic manipulation or plant breeding organisms in increasing the enzymatic digestibility of lignin, cellulose
programs can be employed to achieve genetic development in grasses. and hemicelluloses.
In order to design the biomass for higher biomass yields ploidy level
and genome architecture are important factors that are to be con- 6.2.1. Molecular Improvements in grasses
sidered [230]. In this regard, leaf yield, stem yield and total plant Molecular improvements in grasses have been mostly targeted to
height in Miscanthus, have been identified [231] leading to potential eliminate the use of the pretreatment step or the enzymes, both of
increases in total biomass. Other studies on genetic development of which contribute to the high processing cost of bioethanol from
Miscanthus spa with identification of ploidy levels have also been grasses. In this context, enzymes from the plants itself are an attractive
reported [232,233]. Similarly genome sizes of various bioenergy alternative which can be utilised in grasses for converting the poly-
grasses like switchgrass (1.88 pg), maize (2.73 pg), sorghum (1.21 pg) saccharides into monosaccharides.
[234] and various species of Miscanthus [235] have been identified Approaches like promoter modifications for enhancing the accu-
which can be utilised towards the genetic development. mulation of enzymes, accumulation of enzymes in specific tissues or
Another approach of crop development is QTL mapping. QTL organs and production of induced expressible enzymes have been
mapping has been employed by Liu et al. [236] to develop a high widely used in case of monocots like rice and maize [244], for
density genetic map for switchgrass. Other research groups like Ma enhancing the hydrolysis of the carbohydrates in the cell wall.
et al. [237] and Swaminathan et al. [238] have also developed high Promoters that are used are generally derived from either monocot
resolution linkage maps for Miscanthus sinensis. The development of or dicot or from viral vector that can target a monocot or dicot species.
these maps has made QTL mapping studies easier. This can be well But a promoter is much effective if it is expressed in the same type of
illustrated from the intense studies done by Feltus and Vandenbrink. plant from where it has been derived [245]. Another molecular
[229] on Miscanthus. Their studies revealed that sorghum (a variety of approach is apoplast targeting using pathogenesis-related protein
grass) has the closest syntenous relationship to Miscanthus. These signal peptides which has been successfully accomplished in maize
genetic maps which are made using QTL studies will allow scientists to [246–248], rice [249,250] and alfalfa [251] without disturbing cell wall
sequence genome via synteny relationships from sorghum to integrity and with reasonable enzyme yields (1.2–6.1%). One recent
Miscanthus. On the other hand undetected QTLs also form a valuable approach which has gained much attention is the auto hydrolysis of the
method to identify DNA markers, often in multiple genome positions plant biomass using internally generated enzymes [252–255]. In this
and can be used to select improved feedstock varieties before a crop case, no further pretreatment of the substrate is required and the
development cycle is complete [239]. Apart from QTL studies novel biomass is auto hydrolysed using either heterologous enzymes pro-
1024
duced within the plant or in combination with some other additional for first generation biofuel production while switchgrass and organic
enzymes [256]. residues are used for second/next generation biofuel production.
However, variation in the conversion efficiency of herbaceous Unlike first generation feedstocks, the market value of second genera-
biomasses does not solely rest on the lignin content and composition. tion feedstock is not known since residue markets are not yet developed
Numerous studies on biomass recalcitrance have investigated the in many countries. Also, little commercial production has been
impact of differences in the composition and structure of cell wall experienced in developing countries of energy crops such as switch-
polysaccharides, and the interactions between polysaccharides and grass and thus their market value is not known. So, the cost analysis of
other cell wall components. These demonstrated how cell wall char- second generation biofuel can be made only based on production cost.
acteristics other than lignin— including the degree of cell wall porosity, The cost that are taken into account include land preparation to
cellulose crystallinity, polysaccharide accessible surface area and the harvesting biomass incorporating labour, material expenses, land rent,
protective sheathing of cellulose by hemicellulose—can also contribute fertilisers and other supplies. Another important aspect that will affect
to the natural resistance of plant biomass to enzymatic degradation the cost of biofuel production is cost development of biofuel conversion
[257–259]. Consequently, strategies to develop novel genotypes, with technology for second generation biofuels. Grasses, as discussed
reduced recalcitrance, through targeted modifications of cell wall previously in this review, have high levels of cellulose and hemicellu-
biosynthesis genes are beginning to gain momentum. For instance, lose with the total sugar content between 55% and 70% of the total cell
alterations in the cellulose synthesis machinery—or its accessory wall content on an average. It has been documented that grasses have
complexes— may lead to modifications in the structure of cellulose very high production rate leading to around 80–150 t of grass biomass
micro fibrils, with, for example, reduced crystallinity, a lower degree of production per hectare (Zhao et. al. 2015) [266]. Various studies have
polymerization and/or a higher degree of porosity. Vandenbrink et al. been conducted over the world to optimize and reduce the cost of the
[260] demonstrated a large variation in cellulose crystallinity within a end product from grasses. One such example is that of switchgrass that
diverse association mapping panel in sorghum, and reaffirmed that is grown in Argentina and the process economics has been studied by
genotypes with lower cellulose crystallinity exhibit higher enzymatic Eijck et al., 2014 [267]. The production costs of switchgrass are
hydrolysis rates, as has been reported for pure microcrystalline estimated to be 3.2 $/GJ in 2020 and is proposed to decrease to 3.0
cellulose samples [261] and ground Miscanthus powder [155]. $/GJ by 2030. These prices include the land rent in Argentina (100–
Thus, the robust genetic and/or molecular tools that have been 300 $/ha/year), labour wages (2.18–3.18 $/h), export of switchgrass
widely studied in other monocots for improving the biomass yield and seeds from US (20$/kg) and fertiliser costs (0.13–0.48 $/kg). Mostly,
reducing lignin recalcitrance and cellulose crystallinity with successful the decrease in the costs of production can be obtained by local
results, can also be implemented in grass biomass. Though some production of the seeds of switch grass which would can be available at
research have been conducted on some varieties of Miscanthus and 10 $/kg. Further, the ethanol cost of switchgrass in 2014 was 18.3$/GJ
switchgrass, but the other potential grass varieties which can be as compared to Eucalyptus that has production costs of 16.8 $/GJ in
sustainable source for bioethanol production requires substantial Brazil and 19.8 $/GJ in Mozambique. Compared to the first generation
research. feedstock like cassava, where the production costs of bioethanol are
around 15 $/GJ, the second generation ethanol production still
6.2.2. Molecular Improvements in microorganisms requires more improvements (Chum et al., 2011) [268]. A comparative
Engineered microorganisms can be utilised for improved biomass analysis of the feedstock cost and their ethanol yield are given in
processing, which would allow the use of bioethanol from grasses Table 5. Although, ethanol production from first generation feedstocks
commercially. Many researchers have approached for recombinant have been commercialized in many countries but the unavailability of
microorganisms with either higher delignification rates or enzyme feedstocks restricts its use in the future generations. In this context,
production capacity and high fermenting capacities. In this regard, bioethanol from grasses, though in the present scenario are still in the
Ogawa et al., 1998 [262] introduced cDNA of Postreatus in Coprinus process of optimisation for cost-effective ethanol production in many
cineresus which resulted in high MnP production of the later followed developing countries, but the present production costs are at par with
by increased delignification after 16 days. Similarly, increased sacchar- the first generation feedstocks. Further, the technological develop-
ification have been reported by Mutreja et al. [263] who used ments for production of enhanced grass biomass and efficient micro-
recombinant cellulase for bioethanol from wild grass using E. coli organisms will be helpful in achieving lower ethanol production costs
BL21 cells which were transformed with CtLic26A-Cel5-CBM11 full- in the near future.
length gene from Clostridium thermocellum. In a similar study, Das
et al. [264] developed a recombinant strain of Escherichia coli strain by 8. Research gaps in bioethanol production
expressing GH5 cellulase and GH43 hemicellulase genes from
Clostridium thermocellum for increasing the cell biomass production The conversion of lignocellulosic biomass into bioethanol with
and enzyme activity from wild grass. In a more recent approach, utmost productivity/yields is a crucial biochemical process in bior-
researchers from the University of Georgia and Tennessee's Oak Ridge efinery. However, the lack of industrially robust strain for converting
National Laboratory engineered the thermophilic bacterium biomass into bioethanol has been a major issue for the success of
Caldicellulosiruptor bescii to directly convert switchgrass into ethanol bioethanol industries. The major bottlenecks in bioethanol productions
[265]. The engineered microbe was successfully experimented for are lack of consistency in the composition of lignocellulosic biomass in
ethanol production from switchgrass without the use of any pretreat- divergent grasses varieties, non-uniformity in environmental condi-
ment or enzymatic hydrolysis step. Though ethanol production was on tions, instability of the performance of the microorganisms involved in
the lower side but this has elucidated the potential of the next steps biological fermentation processes, optimizing process parameters and
that has to be taken in this field. above all, constraints in modelling/designing the appropriate bioreac-
tors.
7. Economic analysis
9. Critical comments and future prospective
Economic evaluation of biofuel production include a detail analysis
of the viability of feedstock production and total production cost (in The outlook for global bioethanol will depend on number of
terms of net present value-NPV) and total production cost (TPC) which interrelated factors such as, (i) continuous hike of oil price; (ii)
includes conversion, transport and distribution. The most commonly availability of feed stocks; (iii) technological breakthroughs and (iv)
used feedstocks in developing countries include sugarcane and palm oil sustainable policies by government. Keeping in mind the above
1025
Table 5
Minimal ethanol selling price (MESP) of grasses as compared to various feedstocks with their production(on dry ton basis) and their ethanol yields.
Sl No. Type of Feedstock Production of feedstock (dry ton/ Feed stock cost ($/dry Ethanol yield (gal/dry MESP ($/gal) References
day) ton) ton)
1. Corn stover 2200 51 90 1.49 [348]

2. Corn stover 2200 59 79 2.15 [36]
3. Eucalyptus 4.5 −35 tdm/ha 78 130–142 1.35 [349]
4. Hardwood 2200 65 75 3.43–4.03 [350]
5. Rice straw 1000–1200 35–68 60–80 – [267]
6. Straw, Eucalypatus and 1760–2200 57–127 70–84 2.12–2.91 [351]
poplar
7. Sugarcane 100 (t/ha/yr) 102–108 116 0.88–1.16 [267]
8. Switchgrass 5000 44 97–105 0.63–0.83 [352]
9. Switchgrass 4600 30.6 80–85 1.26–1.28 [353]
10. Wheatstraw 1200–1700 38–45 50–70 2.36 [353]
discussed outlooks and constraints, genome mining of compassionate our understanding in enhancement of bioethanol production from
microorganisms can be an important step in suitable conversion of grasses which are at present considered as the most important
lignocellulosic biomass into bioethanol for improving the productivity. lignocellulosic substrate.
For example, suitable gene from remote sources are traced and cloned
into host organism to obtain optimum bioethanol production. The Acknowledgement
future of cellulosic fuel will continue to emphasize on group of C4
grasses for sustainable large scale production of biomass to support Authors are grateful to the authorities of college of engineering and
cellulosic fuel industries. The C4 plants mainly grasses which use C4 technology, BPUT, Bhubaneswar for providing necessary facilities to
photosynthesis are important sources of bioethanol due to their high carry out this research.
yield potential, wide adaptability across diverse environments, low
water requirement, high carbohydrate content and low fertiliser References
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Contents lists available at ScienceDirect

Renewable and Sustainable Energy Reviews

Application of pretreatment, fermentation and molecular techniques MARK

1. Introduction consumes, marks its potential as a sustainable biofuel, that can be

grass ( Thinopyrum textured soils conditions, is cold tolerant, can

ethanol yields are presented in Table 2. This reported information from

Tolerates submersion, low pH, and

that remain unexploited for commercial bioethanol production inspite

Further, some grass varieties have also good amounts of hemicellulose

production. An example can be cited from the study done by Njouk

qualities like good carbohydrate content, salt, stress and drought

even in arid lands. With minimum or nearly zero maintenance costs

3. Bioethanol production from grasses

Lignocellulosic biomass has a complex and recalcitarant structure

which requires a pretreatment step prior to enzymatic hydrolysis and

subsequent fermentation for bioethanol production. This prior step is

consumption and a maximal sugar recovery. Herbaceous biomass like

grasses do not require as much as energy as recalcitrant woody

of lignin. However, pretreatment seems to be a prerequisite for eﬃcient

enzymatic hydrolysis of grasses [45]. This is because of the fact that

to temperate, moist environments

Very wet conditions and is often

depends on factors such as type of biomass and desired products.

Keeping this in view many research groups have developed various

quent sections. The ﬂowchart representation which brieﬂy summarises

3.1. Pretreatment technologies

Pretreatment is aimed mainly for deligniﬁcation of plant biomass.

(mechanical comminution) [47], chemical (dilute acid or alkali),

ammonia percolation, physio-chemical (steam explosion, ammonia

ﬁbre expansion (AFEX) [48]), ultrasonication and microwave pretreat-

pretreatment to overcome recalcitrance of lignocelluloses(SPORL)

Alkaline NaOH-7%,Incubation time-4 h Napier grass Hollocellulose-88.46% 9.77 [111]

Aquoeus ammonia Switch grass 72% reducing sugar NM [319]

[52]. The ﬂowchart representation which brieﬂy summarises the

thermal pretreatment processes where the cell disruption accompanied

by degradation of the crystalline complex of the cellulose is achieved.

to the cellulose and hemicellulose hydrolysing enzymes during the

treatments are moreover aimed at decomposition of lignin in milder

3.1.1. Physical pretreatment for size reduction of grasses

Physical pretreatments are mainly aimed at reduction of particle

diﬀerences have been observed during the enzymatic hydrolysis step if

these two techniques are included in the pre-treatment scheme. A

deligniﬁcation of elephant grass when exposed to physical pretreat-

ment, leading to higher bioethanol production.

It is found that single-screw and twin-screw extruders are widely used

[63]. In grasses, extrusion pretreatments have been seen to be eﬀective

in exposing the cellulose to enzymatic attacks. Extrusion pre-treatment

of switchgraas and prairie cord grass resulted in a total sugar recovery

of 45.2% and 65.8% respectively [64]. The same authors in 2012,

attempted for optimisation of extrusion pretreatment of other varieties

Fig. 2. Classiﬁcation of Pretreatment techniques. Based on: http://pubs.rsc.org/en/content/articlelanding/2016/ra/c6ra09851g/unauth.

Fig. 3. Pathways of conversion of the lignins present in grass biomass.

The foremost objective of enzymatic sacchariﬁcation is to induce

Sl no Substrate Microorganisms Process of Parameters Ethanol yield Reference

Sl no Substrate Microorganisms Process of Parameters Ethanol yield Reference

6.1. Genetic improvements in grasses 6.2. Molecular Improvements

1. Corn stover 2200 51 90 1.49 [348]