Renewable and Sustainable Energy Reviews 80 (2017) 330–340

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Renewable and Sustainable Energy Reviews

journal homepage: www.elsevier.com/locate/rser

Recent advances in second generation bioethanol production: An insight MARK

to pretreatment, saccharification and fermentation processes
⁎
Meenal Rastogi, Smriti Shrivastava
Amity Institute of Biotechnology, Amity University Uttar Pradesh, Sector 125, Noida 201313, Uttar Pradesh, India

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

Keywords: In response to the scarcity of non-renewable energy sources, sustainable and renewable biofuels from biomass
Bioethanol have gained utmost attention. Utilization of lignocellulosic biomass for production of varied energy forms
Consolidated Bioprocessing (CBP) (second generation fuels) like biogas, biodiesel, bioethanol, etc has increased in the past decade. Their
Second Generation biofuel properties of being naturally abundant and easily accessible throughout the year, makes them an attractive
Lignocelluloses
energy alternative. Efficient pretreatment techniques for effective transformation of lignocelluloses to varied
Sustainable energy
products, by increasing digestibility of celluloses and hemicelluloses can be achieved through acid, alkali
treatment, enzymatic hydrolysis, and steam explosion. Idea behind optimizing pretreatment protocol is to
maximize release of monosaccharide sugars for conversion to value added products. Tailoring of hydrolytic
enzymes through various approaches is well accepted for increasing specific activity of particular enzymatic
reaction and can also be clubbed with other pretreatment processes minimizing chemical usage. We at our
laboratory are working on optimization of process parameters for enhancing efficiency of saccharification
process to obtain maximal monosaccharide sugars that can be converted to bioethanol.
Present review compiles various approaches made by prominent scientists for efficient utilization of
celluloses and hemicelluloses for ethanol production and also describes recent advanced techniques utilized
for the same. Greater emphasis has been led on comparative study on utilization of simple sugars by bacteria
and fungi and effect of consolidated bioprocess system on ethanol production from varied agro-industrial
wastes.

1. Introduction used as fuel source upon blending with the primary fuels and these can
be produced through varied ways by processing suitable biomasses [2].
Till date, fossil fuels have been the only source to address the As per current details, ethanol is one of the bio-based products, which
increasing energy demand of the mankind. Limited availability of fossil dominates global production of fossil based substitutes. Bioethanol
fuels, economic concerns, national security and accelerated global dominates the market with a sale of 58 billion dollars per year. Globally
warming has shifted the focus on new alternative renewable energy ~86,000 kton/year is produced primarily for applications in biofuels.
fuels [1]. Fossil fuels are depleting continuously leading to its crisis and Almost 50% of global sugar is used for ethanol production [4]. Brazil
their subsequent price hikes are a matter of great concern, especially in and US are leading biofuel producers, with the current bio-fuel
underdeveloped countries. On the other hand, alternative renewable mandate in Brazil being 27.5%. Renewable energy grew by 15.5% in
fuels provide an environmental friendly, cheap, pollution-free option terms of power generation by the year 2010 and could account for
and also increase energy security. Thus, renewable energy sources such almost 1.3% of primary global energy consumption [5]. Europe and
as solar, hydro, wind and biomass are being sought as solution to the Eurasia have highest share in global power generation [6]. Second
pollution caused by using fossil fuels [1,2]. Biomass from different generation biofuel has great potential and it has been predicted
sources such as agricultural, aquatic, and forestry can be taken into through various surveys that only 10% of global residues can suffi-
consideration for production of biofuels (solid, liquid or gaseous fuels) ciently satisfy about 50% of entire biofuel demand tree by 2030.
[3]. Since decades primary biofuels like fuelwood are used in their Expected major development in this field is from countries with
natural form primarily for heating, cooking or electricity production. emerging economies such as India, China etc. Depending upon the
Conversely, secondary biofuels such as bioethanol and biodiesel can be source and production technology, biofuels are broadly categorized into

⁎
Corresponding author.
E-mail addresses: [email protected] (M. Rastogi), [email protected] (S. Shrivastava).

http://dx.doi.org/10.1016/j.rser.2017.05.225
Received 5 January 2017; Received in revised form 16 May 2017; Accepted 24 May 2017
1364-0321/ © 2017 Elsevier Ltd. All rights reserved.
M. Rastogi, S. Shrivastava Renewable and Sustainable Energy Reviews 80 (2017) 330–340

Nomenclature PK Pyruvate kinase

PDC Pyruvate decarboxylase
CBP Consolidated Bioprocessing ADH Alcohol dehydrogenase
MSW Municipal solid waste EDP Entner-Doudoroff pathway
XK Xylulose kinase GPDH Glucose-6-phosphate dehydrogenase
PPP Pentose phosphate pathway Lac Lactonase
XI Xylose isomerase PGD 6-phosphogluconate dehydratase
XR Xylose reductase KDPG 2-keto-3-deoxy-6-phosphogluconate
XDH Xylitol dehydrogenase KDPGA 2-keto-3-deoxy-6-phosphogluconate aldolase
PPE Phosphopentose epimerase ILs Ionic liquids
TAL Transaldolase DESs Deep eutectic solvents
TK Transketolase NADES Natural deep eutectic solvents
EMP Embden-Meyerhof-Parnas Pathway AFEX Ammonia fiber explosion
PKL Phosphoketolase ARP Ammonia recycle percolation
AR Arabinose reductase SAA Soaking aqueous ammonia
LAD L-arabitol dehydrogenase SHF Separate hydrolysis and fermentation
AI Arabinose isomerase SSF Simultaneous saccharification and fermentation
RK Ribulokinase SSCF Simultaneous Saccharification and Co-Fermentation
R5PE Ribulose-5-phosphate-4-epimerase DMC Direct microbial conversion
TCA Ttricarboxylic acid cycle ABPP Activity-based protein profiling
HK Hexokinases GHs Glycoside hydrolases
PFK Phosphofructokinase TPSSF Two-phase simultaneous saccharification and fermenta-
FA Fructose bisphosphate aldolase tion
TPI Triosephosphate isomerase SVSCF Same Vessel Saccharification and Co-fermentation
GDH Phosphate dehydrogenase HMF Hydroxymethylfurfural
PGK Phosphoglycerate kinase CMC Carboxymethylcellulose
PGM Phosphoglycerate mutase PASC Phosphoric acid swollen cellulose
Eno Enolase

first, second, third and fourth generations. Biofuels derived from food applied recently for pretreatment processes for delignification (redu-
crops like sugar, starch, vegetable oil and even animal fats consolidate cing lignin content) and detoxification (removing chemical compounds
as first generation biofuels. The process is easy but utilizing food crops from sugar hydrolysates) of lignocellulosic biomass [14]. These fungi
as feedstocks for fuel directly competes with food consumption and include Trametes versicolor [15], Pleurotus ostreatus IBL-02 [16],
poses a negative impact on agricultural areas [7]. Second generation Trametes villosa [17], Ceriporiopsis subvermispora [18], Trametes
biofuels comprise those made from non-food crops such as lignocellu- reesei [19], Cyathus stercoreus [20], and many more; however, the
losic biomass and biowastes while third generation biofuels utilize process is of long duration, cost is high and cannot efficiently modify
algae as feedstock. Engineering algae to increase sustainable biofuel cellulose [14]. Various biomass processing and new genome-based
production and studying algal biodiversity in an attempt to reveal breeding techniques are under investigation to improve the production
highly competent fuel producing species are still under extensive of second generation bioethanol by reducing the costs of manufacturing
research [3]. The fourth generation biofuels (presently on paper) and enhancing the yields of bioethanol.
includes photobiological solar fuels and electrofuels. Using novel Broadly, conversion of lignocellulosic feedstocks to ethanol is
synthetic biology technologies, it will be possible to generate fuels carried out using two technologies i.e., biochemical conversion (com-
from solar energy and inexhaustible, economical and readily available monly used) and thermochemical conversion [21]. In biochemical
raw materials through biological systems. Though at an infant stage, conversion, enzymes are utilized for conversion of biomass to ethanol
these 4 G fuels seem to provide a fundamental breakthrough in biofuel and comprises of four major steps: physicochemical pretreatment,
production [8]. So far, ethanol accounts for upto 75% of the total enzymatic hydrolysis of the sugar polymers fermentation of the derived
biofuel usage [9]. Bioethanol production has increased from 13.1 sugars into ethanol and distillation [7]; whereas in thermochemical
billion gallons in 2007 to 25.7 billion gallons in 2015 [10]; with conversion, the feedstock is gasified under high heat levels to produce
United States and Brazil being the leading producers, dominantly using syngas (carbon monoxide, hydrogen and carbon dioxide) which is then
simple substrates such as corn and sugarcane, respectively [11]. converted into ethanol through chemical catalysis using Molybdenum
Although India is the second largest producer of sugarcane in the disulphide [22] or biologically using microorganisms like
world, its bioethanol production accounts for only 2% of global Saccharomyces cerevisiae, Clostridium ljungdahlii, or Zymomonas
production. mobilis [23–26]. The biochemical route is accomplished by using
On the contrary, non-food crops overcome the limitations of 1 G vigorous glycosyl hydrolases- and ethanol-producing microbes, both
fuels. Despite abundant supply of agricultural residues for fuel ethanol native and recombinant strains. Microorganisms such as C. thermo-
production, challenges still exist for their commercial conversions, cellum and T. reesei have been used widely for their native ability to
owing to their recalcitrance (due to lignin sheath) to degradation and produce ethanol [27]. Potential saccharifying microbes such as C.
unique chemical composition [12]. Highly crystalline structure of thermocellum, T. mathranii, K. oxytoca, F. oxysporum have been
cellulose and heterogenous composition of hemicelluloses in the modified to be ethanologenic while potential fermenting organisms
lignocellulosic biomass requires chemical or physical pretreatment such as S. cerevisiae, E. coli, Z. mobilis, P. stipitis, H. polymorpha, K.
besides enzymatic treatment processes which increases the cost of marxianus have been modified to be cellulolytic [27].
the entire process and thus prove to be a major obstacle for large scale Enzyme performance, reduction of enzyme costs, cofermentation of
production [7,13]. Ligninolytic fungi and their enzymes including C5 and C6 sugars, reduction of investment costs, reduction of process
manganese peroxidase, laccase, and lignin peroxidase, have been time and released toxins during pretreatment are the main challenges

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M. Rastogi, S. Shrivastava Renewable and Sustainable Energy Reviews 80 (2017) 330–340

associated with biochemical process [28]. In order to produce bioetha- 3. Hexose and pentose utilization in different
nol from lignocellulosic biomass, that is economically feasible, sustain- microorganisms
able, and competitive with petroleum-based fuels, conventional process
steps need to be integrated into consolidated process to avoid A variety of microbes are capable of fermenting hexose and pentose
maximum production of inhibitory sugar derivatives and achieve high sugars both separately as well as simultaneously. Bacterial genera like
ethanol titres. In the present review, an overview of various strategies Aerobacter, Bacillus, Klebsiella, Thermoanerobacter, Aeromonas;
for enhancement of second generation ethanol production has been yeasts like Candidashehatae, Pichiastipitis, Pachysolen tannophilus
discussed giving elaborate description of pretreatment, saccharification and fungi like Fusarium, Mucor, Neurospora, Monilia, Rhizopus are
and fermentation processes. Research methodologies designed for successfully capable of fermenting xylose to ethanol [45–47]. In all
development of efficient physico-chemical techniques leading to max- microorganisms, the metabolic pathway of D-xylose involves its con-
imal utilization of hexose and pentose sugars and various consolidated version to D-xylulose followed by xylulose kinase (XK) reaction to D-
bioprocess system leading to improved bio-ethanol production has also xylulose-5-phosphate. Once converted to xylulose-5-phosphate, these
been discussed in detail [29]. sugars are directed to the native pentose phosphate pathway (PPP)
(Fig. 1). Bacteria generally convert D-xylose directly to D-xylulose via
xylose isomerase (XI) enzyme, whereas yeasts and fungi employ a two-
2. Lignocellulosic biomass step oxidation-reduction pathway [48,49]. In fungi and yeasts, the D-
xylose is first reduced to xylitol by D-xylose reductase (XR) followed
Lignocellulosic biomass can be either byproducts of agriculture or by subsequent oxidation to D-xylulose by xylitol dehydrogenase
its related industry, such as cotton stalks, bagasse, wheat and rice (XDH), which is sequentially converted to D-xylulose-5-phosphate.
straw, maize cobs, coconut shells, jute sticks, and rice husks or organic Phosphopentose epimerase (PPE), transaldolase (TAL) and transketo-
fraction of municipal solid waste (MSW), sewage treatment sludge, or lase (TK) further metabolize D-xylose-5-phosphate into glyceraldehyde-
forestry waste including wood chips, sawdust, and bark. 3-phosphate and fructose-6-phoshate by non-oxidative rearrangement
Lignocelluloses are mainly composed of cellulose (40– 60% of the leading to fermentation of sugars to ethanol by Embden-Meyerhof-
total dry weight), hemicellulose (20–40%), and lignin (10–25%) [30]. Parnas (EMP) Pathway [49,50]. Alternatively, phosphoketolase (PKL)
Cellulose consists of long chains of β-glucose monomers packed into (important in lipid producing yeast) can split D-xylose–5-phosphate
microfibril bundles, which are attached to each other by hemicelluloses into glycerealdehyde-3-phosphate and acetyl phosphate. In most fungi,
(mostly xyloglucans or xylans) by hydrogen bonds. Hemicellulose has a the initial conversion of L-arabinose to D-xylulose-5-phosphate pro-
lower molecular weight than cellulose and is composed of mainly ceeds through a series of reduction and oxidation steps via arabinose
pentoses (like xylose and arabinose) and hexoses (like mannose, reductase (AR) and L-arabitol dehydrogenase (LAD) involving the
glucose, and galactose). Hemicelluloses are linked to lignin by covalent cofactors NAD(P)+/NAD(P)H [51]. Alternatively, in bacteria arabinose
bonds. Lignins are phenolic compounds which are formed by poly- isomerise (AI) converts arabinose to ribulose followed by its conversion
merization of three types of monomers (p-coumaryl, coniferyl, and to ribulose-5-phosphate via ribulokinase (RK). Subsequently, ribulose-
synapyl alcohols); that is responsible for providing rigidity to the plant 5-phosphate-4-epimerase (R5PE) catalyzes the final epimerization of
cell wall and resistance against microbial attack [31]. ribulose-5-phosphate to D-xylulose-5-phosphate, an intermediary in
The crystalline nature of cellulose fibrils makes it highly resistant to the pentose phosphate pathway [52,53].
enzymatic digestion and thus cellulose is more susceptible to enzymatic Yeasts can produce ethanol both aerobically and anaerobically
hydrolysis in its non-crystalline form. Moreover, lignin can irreversibly along with adaptive regulatory mechanisms. Irrespective of the carbon
adsorb enzymes inhibiting their action on the cellulose chains and this source, D-xylose-fermenting yeasts require oxygen for growth and
process is rapid at higher temperatures [32]. Addition of surfactants sufficient ATP are not produced. In aerobic conditions even in the
[33] and certain pre-treatments have been found to reduce the enzyme presence of significant xylose concentrations, yeasts utilize the formed
adsorption onto lignin. For efficient enzymatic hydrolysis of biomass, ethanol as a carbon source to further yield cell mass, CO2 and acetate.
the crystalline structure of cellulose needs to be disintegrated so that Thus ethanol production first tends to increase sharply followed by its
the accessible area is increased, leading to efficient separation of lignin declination and boost in the biomass yields [20,46,50]. However, in
and hemicellulose. Composition of various lignocellulosic biomasses anaerobic xylose fermentation the tricarboxylic acid (TCA) cycle does
available in abundant that can be effectively converted to various value not function and ethanol uptake is not seen.
added products is elaborated in Table 1. In fungi and yeasts (Fig. 1), specifically S. cerevisiae, following

Table 1
Composition of various lignocellulosic biomasses.

Biomass Cellulose (%) Hemicellulose (%) Lignin (%) Ash (%) References

Sugarcane bagasse 32–48 19–24 23–32 1.5–5 [34–37]

Corn stalk 39–47 26–31 3–5 12–16 [34–37]
Rice husk 31.3 24.3 14.3 23.5 [38,39]
Rice straw 28–36 23–28 12–14 14–20 [34–37]
Wheat Straw 33–38 26–32 17–19 6–8 [34–37]
Groundnut shell 35.7 18.7 30.2 5.9 [39,40]
Coconut shell 29.7 NA 44.0 0.5 [41]
Corn stover 38–40 28 7–21 3.6–7.0 [34–37]
Cotton waste 80–95 5–20 – – [34–37]
Softwoods 45–50 25–35 25–35 NA [42–44]
Hardwoods 40–55 24–40 18–25 NA [42–44]
Newspaper 40–55 25–40 18–30 8.8–1.8 [43,44]
Algae (green) 20–40 20–50 NA NA [34–37]

NA: Not available. Composition is represented in percent weight on dry weight of the samples

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Fig. 1. Comparison of hexose (glucose) and pentose (xylose and arabinose) sugar utilization pathways by different microbes. L-XuR: L-xylulose reductase.

uptake by the hexose transporters, glucose is metabolized to pyruvate intermediates and finally into pyruvate. First, 3-phosphate dehydro-
via the glycolytic pathway. In the initial step through the action of genase (GDH) converts glyceraldehydes-3-phosphate into 1,3-dipho-
hexokinases (HK), glucose is phosphorylated to glucose-6-phosphate, sphoglycerate, which is in turn converted to 3-phosphoglycerate by the
which by the action of phosphoglucose isomerase (PI) further iso- action of phosphoglycerate kinase (PGK). Phosphoglycerate mutase
merizes to fructose-6-phosphate. Phosphofructokinase (PFK) converts (PGM) promotes the relocation of the phosphate group from C3 to C2,
fructose-6-phosphate to fructose-1,6-biphosphate, which in turn is allowing the dehydration by the enolase (Eno), resulting in the
reversibly converted by yeast fructose bisphosphate aldolase (FA) into phosphoenolpyruvate. Then the pyruvate kinase (PK) converts this
glyceraldehydes-3-phosphate and dihydroxyacetone phosphate, that highly energetic molecule to pyruvate, having several fates depending
are interconvertible by triosephosphate isomerase (TPI). on the environmental conditions suitable for the microorganism.
Glyceraldehyde 3-phosphate is sequentially converted to form various Under anaerobic conditions, pyruvate undergoes a decarboxylation

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 Table 2
Comparative analysis of various pre-treatment techniques.

Pretreatment Mode of action Advantages Disadvantages References

M. Rastogi, S. Shrivastava

Mechanical Milling, grinding, shredding or chipping reduces particle size • Increase in specific surface area and High energy consumption renders this method [73,74]
digestibility of biomass economically inefficient.
• Reduced crystallinity and degree of
polymerization of cellulose
Extrusion/ Pyrolysis Treatment at high temperature ( > 300 °C) following by mixing and shearing Parameters in bioreactor need to be highly efficient. [74]
causes defibrillation, fibrillation and shortening of the fiber
Liquid Hot water Liquid hot water (160–240 °C) under high pressure ( > 5 MPa) for time ranging • Better pH control minimizes non-specific • High energy requirement and water demand. [75,76]
from few minutes up to an hour removes hemicellulose from lignocellulosic degradation of polysaccharides. • Not feasible for commercial scale.
biomass making the cellulose more accessible. • High pentose recovery and lower formation of
inhibitors.
• No chemicals and corrosion resistant
materials are required.
Steam explosion Exposure of chopped biomass to hot steam (160–260 °C) under high pressure • Improved enzymatic hydrolysis • Less effective for softwoods [77,78]
(Autohydrolysis) for specific period of time followed by sudden release in pressure causes environmental impact of inhibitory products (eg furfural and
a
autohydrolysis of acetyl groups of hemicellulose. Individual fibers are separated
• Lowerhazardous chemicals required
• Formation
HMF )
disrupting the cell wall structure.
• Less sugar yield degradation of hemicelluloses and lignin
• High • Partial equipment requirement for acid addition
• Feasible for industries • Additional
cost
Ammonia Fiber Expansion Treatment with liquid ammonia at moderate temperature (60–100 °C) for 30– the surface area accessible for
• High
Not very effective for the biomass with high lignin [73,74]
(AFEX) 60 min at high pressure (250–300 psi) followed by sudden pressure release
• Increases
enzymes and thus enhanced digestibility.
• content.
causes disruption of biomass fibers and partial decrystallization of cellulose. • Less inhibitory or toxic compound formation. • High cost of large amount of ammonia

334
• Hemicelluloses are not significantly reduced
affecting the sugar yield.
Acid Dilute rather than concentrated acid is used either at high temperature (e.g., • Enhanced hydrolysis of hemicelluloses and • High energy input so cost is high. [74,79,80]
180 °C) for short period of time or lower temperature (e.g., 120 °C) for longer amount of amorphous cellulose • Acids are corrosive so the process requires specific
retention time (30–90 min) to solubilize hemicelluloses and lignin • High sugar yield reaction vessels.
of inhibitory compounds (furfural, 5-
a
• Formation
HMF , phenolic acids and aldehydes).
Alkali Treatment with alkali such as sodium, potassium, calcium and ammonium • Efficient removal of all lignin. • Downstream processing costs are high. [56,81]
hydroxides disrupts the ester and glycosidic side chains causing alteration in • Increased accessibility of hemicelluloses- • Not efficient for industrial scale.
lignin structure, cellulose swelling and its partial decrystallization and partial degrading enzyme.
solubilization of hemicelluloses • Decrease in the degree of polymerization and
crystallinity of cellulose.
Organosolv Organic or aqueous organic solvent mixtures (such as ethanol, ethylene glycol, • Improvement in enzymatic digestibility of • Cost of solvent and the catalysts are high. [74]
acetone, methanol, etc.) with inorganic acid catalysts are used to extract lignin. lignin and hemicelluloses. • Risk of fires and explosions as organic solvents are
inflammable.
Ozonolysis Ozone treatment degrades lignin by attacking aromatic rings structure, hardly • No toxic residues produced. • Highly expensive due to requirement of large [82]
affecting cellulose and hemicelluloses. • Reaction is carried out at room temperature amount of ozone.
and pressure
Biological Microorganisms specifically white rot fungi such as P. chrysosporium, C. • Low capital cost • Slow rate of hydrolysis so inefficient for industrial [74,83,84]
lacerata, C. stercolerus, etcproduce lignin peroxidases and manganese- • No chemicals required purposes
dependent peroxidases and laccase that causes lignin degradation • Low energy requirement • Operational costs increase in large scale operation
• Mild environmental conditions required • More microbes need to be identified and isolated to
• Improved productivity delignify the plant material quickly and efficiently.
• Control of pH during sugar utilization
a
HMF: hydroxymethylfurfural
Renewable and Sustainable Energy Reviews 80 (2017) 330–340
M. Rastogi, S. Shrivastava Renewable and Sustainable Energy Reviews 80 (2017) 330–340

reaction to yield acetaldehyde and carbon dioxide through the action of hydroxide) is the most conventional method on industrial scale for
the pyruvate decarboxylase (PDC) Acetaldehyde is further reduced by treating lignocellulosic biomass. Unlike alkali treatment which is
alcohol dehydrogenase (ADH) to form ethanol [54]. E. coli however performed at ambient conditions, acid pretreatment is usually em-
contains a gene encoding for pyruvate formate lyase instead of pyruvate ployed either at high ( > 180 °C) or low temperature ( < 120 °C) for
decarboxylase for conversion of pyruvate to ethanol [55]. short or longer durations, respectively leading to hemicelluloses and
Most bacteria have the glycolytic and pentose phosphate pathways, lignin removal [58,59]. Alkalis work by solubilizing hemicellulose,
but some substitute the Entner-Doudoroff pathway (EDP) for glyco- modify the lignin structure through degradation of the side chains of
lysis. The pathway is generally found in Pseudomonas, Rhizobium, esters and glycosides, cellulose swelling and cellulose decrystallization
Agrobacterium various gram-negative bacteria but the facultative [60,61]. Both these treatments follow extensive washing to remove the
anaerobe, Zymomonas mobilis (formerly Pseudomonas lindneri) dis- acid or salts before fermentation, which is a cumbersome process. Ionic
similating glucose to ethanol representing a major alcoholic fermenta- liquids (ILs) have recently emerged to be of remarkable importance
tion reaction in a bacterium. EDP (Fig. 1) begins with the same due to their unique properties such as low melting points (below
reactions as the pentose phosphate pathway with the formation of 100 °C), negligible vapor pressure, wide liquid range, high thermal and
glucose-6-phosphate. Glucose-6-phosphate is oxidized by glucose-6- chemical stability, high polarity and good solvation property [62,63].
phosphate dehydrogenase (GPDH) to 6-phosphoglucono-δ-lactone, ILs typically comprise of large organic cation and a small anion and can
which is further hydrolyzed by lactonase (Lac) to 6-phosphogluconate. effectively pretreat biomass by dissolving cellulose (especially), redu-
6-phosphogluconate is then dehydrated by 6-phosphogluconate dehy- cing its crystallinity, thereby enhancing the enzymatic hydrolysis of
dratase (PGD) to form 2-keto-3-deoxy-6-phosphogluconate (KDPG), cellulose [63]. Imidazolium salts are the most frequently used ILs but
which is the key intermediate in this pathway. KDPG is then cleaved by the difficulty in recycling and reuse, generation of inhibitors and the
2-keto-3-deoxy-6-phosphogluconate aldolase (KDPGA) to pyruvate high cost of ionic liquids remain the areas that need further exploration
and glyceraldehyde 3-phosphate. The glyceraldehyde 3-phosphate is [58]. Deep eutectic solvents (DESs) show similarity to ILs in relation to
converted to pyruvate similar to the reactions of the glycolytic pathway. physical behaviour and properties because of their capability of self-
Pyruvate is converted to ethanol in a similar way through the formation association forming a eutectic mixture of low melting point than its
of acetaldehyde [54]. each individual constituent. DESs can also be made from non-ionic
species [58,64,65]. Recently, natural products such as choline, sugars,
4. Pretreatment of lignocellulosic biomass urea, amino acids and many organic acids termed as natural deep
eutectic solvents (NADES) [66] have been utilized for solubilizing and
Several pretreatment strategies have been developed to boost the extracting lignin (of high purity) from agricultural waste as they show
reactivity of cellulose and enhance the resulting fermentable sugars. no toxicity, biocompatibility and high biodegradability, are cost effec-
Major objectives of pre-treatment include: (1) reduction in the crystal- tive and can be synthesized easily [67].
linity and degree of polymerization of cellulose as well as increase in Physico-chemical processes combine the physical forces and che-
the porosity of the lignocelluloses to enhance sugar yields during mical effects to release cellulose from lignocellulosic feedstocks. During
enzymatic hydrolysis, (2) avert the degradation of sugars (particularly steam explosion, the saturated steam enters the biomass expanding the
pentoses) as well as those derived from hemicellulose, (3) minimize the walls of fibers leading to autohydrolysis of acetyl groups of hemicellu-
formation of inhibitory products for subsequent fermentation pro- lose and increasing the cellulose accessibility by the enzymes. Steam
cesses, (4) recover lignin for conversion into valuable coproducts, and pretreatment is highly beneficial for pretreating hardwoods and
(5) minimize energy inputs to be cost effective and ease of operation agricultural residues and is affected by temperature, biomass size,
[56,57]. The choice of the pretreatment process varies with the type of residence time and moisture content [68]. Wet oxidation on the other
feedstock due to difference in their physical characteristics and hand employs the use of air/oxygen along with water or hydrogen
chemical composition. The removal of toxic compounds (such as peroxide for treating biomass at elevated temperatures (above 120 °C
phenolics, furans, aliphatic acids and inorganic compounds) generated for 30 min) during which water behaves like an acid (above 170 °C),
during the process also add to the cost of pretreatment [22]. breaking the hemicelluloses into smaller pentose monomers and
Physico-chemical and biological processes are being used for oxidizing lignin while least affecting the cellulose content [69]. The
pretreatment of lignocellulosic biomass. Different pretreatment tech- efficiency of wet oxidation depends on oxygen pressure, temperature
niques have been briefly discussed in Table 2. Physical pretreatment and reaction time. However, this method is not feasible at industrial
include milling or grinding to breakdown biomass size and crystal scale due to combustible nature of pure oxygen and high cost of
structure to increase the surface area. Grinding or milling reduces the hydrogen peroxide [70]. Sulfite pretreatment to overcome recalcitrance
biomass size and crystallinity of cellulose. In mechanical extrusion, the of lignocellulose (SPORL), another popular method of pretreatment for
combined action of high heat ( > 300 °C) and shear mixing disrupts the lignocellulosic feedstocks, requires the subjection of biomass to cal-
crystalline cellulose matrix but due to intensive energy input is difficult cium or magnesium sulfite (to remove hemicellulose and lignin
to scale up. In contrast, pyrolysis process employs the exposure of fractions) followed by significant reduction in size of pretreated
biomass at high temperatures of 500–800 °C in the absence of biomass via mechanical disk miller. SPORL pretreatment is highly
oxidizing agent. Under these circumstances, cellulose decomposes versatile, efficient and simple, requires less energy and has excellent
rapidly to form certain end products (pyrolysis oil, gaseous substances scalability at industrial level but sugar degradation, extensive washing
and charcoal). Pyrolysis treatment is used for production of bio-oil after treatment and high cost of recovering pretreatment chemicals are
from lignocellulosic feedstocks. Pulsed-electric field, another physical added issues [70].
method, employs the subjection of feedstock to a sudden burst of high Liquid ammonia has been widely used in the past in different
voltage between 5.0–20.0 kV/cm for short durations (nano to milli- processes like ammonia fiber explosion (AFEX), ammonia recycle
seconds) creating pores in the cell membrane thereby allowing the percolation (ARP) and soaking aqueous ammonia (SAA). AFEX is
entry of agents that will break the cellulose into constituent sugars. Not performed at ambient temperature while ARP proceeds at high
only it utilizes low energy but also the treatment can be carried out at temperatures [71]. Ammonia treatment at high pressure and given
ambient conditions [58]. temperature modifies the lignin structure, increasing the water holding
Chemical pretreatment methods remove and/or disrupt hemicellu- capacity, causes the swelling and phase change in the crystallinity of
loses and lignin, thereby unravelling the lignin hemicellulose network. cellulose in the biomass leading to increased digestibility and reactivity
Pretreatment using dilute acid (sulphuric acid, oxalic and maleic acid) of leftover carbohydrates after pretreatment [72]. Biological pretreat-
or mild alkali (sodium hydroxide, potassium hydroxide and ammonium ment involves the use of microorganisms such as white, brown and soft

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rot-fungi for the delignification of lignocellulosic materials. The slow enzymatic hydrolysis have attracted increasing research in this tech-
rate of hydrolysis associated with this method is the major drawback nology [90].
and currently alternatives like combining other pretreatment technol-
ogies with biological treatment and developing novel strains for rapid 5.4. Consolidated bioprocessing (CBP)
hydrolysis are under thorough investigation [58].
Direct microbial conversion (DMC) or consolidated bioprocessing
5. Current methodologies adopted for bioethanol production integrates all the reactions required for the transformation of biomass
from lignocellulosic biomass into ethanol. This strategy differs from others as only a single microbial
community carries out both the production of enzymes and fermenta-
Pretreatment of lignocellulosic biomass is necessary to boost the tion, i.e., enzymes production, enzymatic hydrolysis and fermentation
cellulose availability, that is confined within the layers of hemicellu- are carried out in a single step. This process has several advantages
loses and lignin and make it accessible for the enzymatic saccharifica- since no capital or operational costs are required for enzyme produc-
tion. Different cellulases and hemicellulases act on the cellulose and tion, whole substrate is utilized for release of sugars and no part of it
hemicellulose component, released from the biomass and further goes for cellulase production. Moreover, the enzymatic and fermenta-
converts them into glucose and xylose, respectively. Either these tion processes are entirely compatible. Thermophilic microbes have a
enzymes are produced in a separate reactor or externally bought. distinct advantage over conventional yeasts as they can directly use
These monomeric sugars (hexoses and pentoses) so formed as a result number of inexpensive biomass feedstocks and also withstand high
of saccharification are subsequently converted into bioethanol by a temperatures. However, they have low bioethanol tolerance ( < 2% v/
variety of microorganisms via the process of fermentation [30]. In an v), which is a major obstacle for their industrial application. Despite
attempt to increase the efficacy of bioethanol production different that, a thermotolerant yeast strain K. marxianus has been engineered
methods to integrate hydrolysis and fermentation have been proposed. for the co-display of endoglucanase and β-glucosidase on its cell
surface. It can grow well at temperatures as high as 48 °C, and
5.1. Separate hydrolysis and fermentation (SHF) produced ethanol from β-glucan (cellulosic material) with a yield of
0.47 g ethanol per gram of consumed carbohydrate. Other proposed
This is a two-stage process wherein hydrolysis and fermentation approach is the utilization of mixed cultures so that the hydrolysis and
processes operate distinctly, that is, the pretreated lignocellulosic fermentation of lignocellulosic biomass is carried out simultaneously
biomass is first degraded into monomer units (glucose and xylose) [85].
through enzymes, followed by fermentation of these sugars into
ethanol. The main advantage of this process is that both enzymatic 6. Recent advances in bioethanol production using
hydrolysis and fermentation function at their respective optimum molecular tools and genetically modified organisms
conditions, nevertheless, accumulation of sugars inhibiting the enzyme
activity remains a major drawback that ultimately affects the ethanol In recent years, several hosts have been engineered to produce
yield [85,86]. varieties of biofuels from lignocellulosic feedstocks. CBP organisms can
be generated by improving biofuel yield of naturally existing cellulolytic
5.2. Simultaneous saccharification and fermentation (SSF) microorganisms or by engineering cellulolytic microbes to be ethano-
logenic or ethanologenic microbes to be cellulolytic [27,91]. Different
This approach combines the saccharification of biomass with species of bacteria like Clostridium thermocellum [92–94], C. phyto-
simultaneous fermentation of released sugars in a single reactor. The fermentans [95], C. cellulolyticum [96], Thermoanaerobacterium
key feature of this process is that as soon as the sugars are formed from saccharolyticum [97], Caldicellulosiruptor bescii [98], Escherichia
biomass, they are rapidly converted into ethanol, thus diminishing the coli [99,100], Zymomonas mobilis [101]; fungi such as Fusarium
accumulation of inhibitory sugars in the medium. Ease of operation, oxysporum [102], Trichoderma reesei [103], Paecilomyces variotii
low equipment requirement than the SHF process and the presence of [104], Aspergillus oryzae [105]; and yeasts including Kluyveromyces
ethanol in the broth making the medium less vulnerable to contamina- marxianus [106,107], Clavispora [108], Pichia stipitis [109,110] and
tion, are some of the benefits of this process [85,87,88]. The main Saccharomyces cerevisiae [111–113] have been studied for their
drawback of this process is difficulty in optimization of process widespread use in bioethanol production [Table 3]. These strains have
parameters considering both enzymes and microorganisms at the same been modified by means of adaptive evolution strategies and recombi-
time. For instance, cellulolytic enzymes for enzyme hydrolysis work nant tools such as directed mutagenesis, genetic and metabolic
best at around 50 °C but the optimal conditions for microbes for engineering to enhance ethanol yield as well as tolerance, but no single
ethanol fermentation is between 28 °C and 37 °C. Lowering the commercially viable CBP organism has yet been reported.
optimum temperature of enzymes through protein engineering would Rather than a single microbe, a microbial consortium could be used
be practically difficult and therefore, thermotolerant strains are re- to perform CBP that could not only utilize substrate more efficiently
quired that could grow well and efficiently produce ethanol at high and increase product yield but also avoid the loss of activity and
temperature [89]. robustness of biocatalyst that occurs due to introduction of genes for
exogenous functions. A consortium may consist of a strain producing
5.3. Simultaneous Saccharification and Co-Fermentation (SSCF) enzymes for hydrolysis of biomass and two different strains each for
fermenting C5 and C6 sugars into ethanol. One such model was
This integration is oriented to the microbial assimilation of all the proposed by Brethauer and Studer (2014) using Trichoderma reesei,
sugars formerly released during the pretreatment and hydrolytic for enzyme secretion; Saccharomyces cerevisiae, to ferment hexoses;
processes of lignocellulosic biomass. Using mixed cultures of yeasts and Scheffersomyces stipitis, to utilize pentose sugars, and convert
that can assimilate both hexoses and pentoses is another option but the lignocellulose to ethanol in a biofilm membrane reactor. The aerobic
only problem is higher conversion of hexoses to ethanol as hexose- fungi grow directly on the dense oxygen permeable membrane,
utilizing microorganisms grow faster than pentose-utilizing microor- producing enzymes which hydrolyze the cellulose and hemicellulose
ganisms. Another variant is to utilize a single microorganism that can in the biomass to release sugars. Secreted enzyme is only transported
optimally assimilate both hexoses and pentoses to allow high conver- from the gas phase through the membrane creating an oxygen gradient
sion and ethanol yield [7]. Advantages like low cost, shorter operation and leaving the upper part of the biofilm oxygen depleted. The released
time, lower contamination risk and fewer inhibitory effects during sugars are then fermented to ethanol by the anaerobically growing

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yeast cells. They were able to produce ethanol from whole slurry dilute
References acid pretreated wheat straw with a 69% yield (9.8 g/L) [117]. This
appears to be a reasonable approach even so the major challenge here
[114]

[113]

[115]

[110]

[103]

[116]
[102]
[94]

[98]

[96]
is the control of consortium and difficulty to find identical fermentation
conditions for all concerned strains. Still, the system could be enhanced

22.4 ± 1.4 g/l (75% of the theoretical maximum)

by the application of other strains that will utilize a broader range of
substrate, produce more enzymes and efficiently utilize C5 and C6
sugars.

62% and 73% (theoretical maximum)

Conventional genomic, transcriptomic, proteomic techniques fail to
provide accurate insights of functional cellular processes and regula-
Ethanol concentration/ yield

33.8% (theoretical maximum)

tory networks in organisms. Knowledge of these is not only required to
identify new organisms and enzymes with desired biofuel-relevant
capabilities but also for genetically modifying organisms for improved
yields [118]. Activity-based protein profiling, popularly ABPP is a
62.61 mg/g cell/h
56.37 mg/g cell/h

chemical biology probe-based technology to study enzymatic activity in

complex proteomes. The key feature of ABPP is selective identification
2.44 g/l/h
3.13 g/l/h

of only the active forms of target enzyme using chemical probes, which
4.63 g/l
2.3 mM
1.6 mM
2.7 g/l

9.7 g/l
3.1 g/l

2.7 g/l react with the active site of a specific target protein (or protein family)
in a mechanism-based manner. Besides this, ABPP strategies can also
be employed to identify and characterize unknown protein functions, to
Mutation: adaptive evolution strategy. Apparent changes in Clo1313_1831–2, AdhE and GapDH genes

Co-expression of endoglucanase (eg3) and β-glucosidase (bgl1) were obtained from Trichoderma viride
Expression of bi-functional acetaldehyde/alcohol dehydrogenase (AdhE) from T. pseudethanolicus 39E

Co-expression of endoxylanase and endoglucanase from Aspergillus niger and Aspergillus aculeatus
Cell-displayed minicellulosome consisting of endo- and exo-glucanase with intracellular cellodextrin

Integration of codon optimized variants of T. lanuginosusglucoamylase (TLG1) and S. fibuligeraα-

Genome shuffling and mutagenesis to improve production of ethanol under aerobic condition and
marxianus respectively, with repression of proteinase gene PEP4 and switch between haploid and

study the alterations in enzymatic activity under different conditions, to

Co-expression of exo-inulinase (InuMK1) and endo-inulinase (InuB) genes from A. niger and K.

spot the protein targets of covalently binding natural products, and to

discover and assess putative new enzymes thereby facilitating the
Inactivation of L-lactate dehydrogenase (ldh) and L-malate dehydrogenase (mdh) genes

discovery of new biofuel pathways [119]. Glycoside hydrolases (GHs)

are necessary for hydrolysis of lignocellulose. Thus, a GH activity-based
probe (GH-ABP) that can directly identify and quantify functionally
Post-translational gene silencing of the sugar transporter (Hxt) in the fungus

active cellulolytic enzymes in native secreted proteomes as well as allow

study of synergistic interactions between GH enzymes in vitro, in situ
and in vivo will be highly beneficial.
Chauvigné-Hines and co-workers (2012) used a suite of GH-ABPs
(difluoromethylphenyl aglycone, 2-deoxy-2-fluoroglycoside, and N-
utilization pathway mimicking the one in C. thermocellum

halogenated glycosylacetamide) to directly label the cell-adherent

in the C. bescii strain lacking lactate dehydrogenase gene

cellulosome of the model anaerobic thermophilic organism

Clostridium thermocellum. In this study, not only 177 glycoside
hydrolases were quantitatively identified but also the off-target labeling
incurred as a result of the reactivity of two of the probe types employed,
provided a broader knowledge of sugar catabolism by the microorgan-
increased ethanol tolerance (4% v/v)

ism [120]. Another study by Anderson et al. (2013) [121] included the
use of GH-probe in Trichoderma reesei wild-type strain QM6a and
mutant strains NG14 and RUT-C30. Alterations to enzyme function
in adapted strain (LL1210).

due to growth variables (pH, temperature, salts, etc.) could be rapidly

amylase (SFA1) genes

identified, including both slight and remarkable changes. This is largely

applicable to distinguish existing and recently identified microorgan-
diploid strains.

isms with highly valuable biomass degrading properties. This could be

Description

respectively

employed to enhance and optimize enzyme cocktails for lignocellulosic

biomass processing [122].
One of the major limitations of bioethanol production is the
recalcitrance of biomass to hydrolysis, which is primarily due to lignin.
Genetically modified microbes for consolidated bioprocess system.

C. thermocellum strain AG553

S. cerevisiae strains M2n and

In this case, manipulation of the lignin biosynthesis pathway to reduce

S. stipitis strain BCC15191

recalcitrance can be a better approach for improving the production of

bioethanol from lignocellulosic biomass. Wuddineh et al. (2016)
T. reesei CICC 40360

recently published a study where they observed that ectopic over-

Microorganism

C. cellulolyticum

expression of Knotted1 transcription factor, PvKN1 (a putative ortho-

F. oxysporum
S. cerevisiae

S. cerevisiae

S. cerevisiae

log of maize KN1) in switchgrass altered growth, especially in early

C. bescii

developmental stages. Their study suggests that PvKN1 broadly

MEL2

regulates the lignin biosynthesis pathway via modulation of GA

(gibberellic acid) signaling. In the transgenic lines the overexpression
Jerusalem artichoke tuber

20 g/l galactose + 10 g/l CMC

of PvKN1 not only reduced the lignin content but also altered the
50 g/l Sugarcane bagasse
20 g/l galactose + PASC

10 g/l crystalline cellulose

expression of cellulose and hemicellulose biosynthetic genes thereby

2% (w/v) Switchgrass
60 g/l cellulose (Avicel

increasing the efficiency of sugar release [123]. Similar work was

Xylan and β-glucan

published before by Voorend et al. (2016) where they observed that

Natural sorghum

2% (w/v) Avicel

overexpression of GA20-OXIDASE1 in maize increased the sacchar-

50 g/l Glucose

Wheat straw
20 g/L CMC
Triticale
Substrate

ification of modified biomass after NaOH pretreatment [124].

PH105)
(10 g/l)
powder

Therefore, apart from developing pretreatment technologies and

Table 3

Inulin

composing enzyme consortiums, lignocellulose feedstock improvement

also seems to be a feasible strategy to enhance bioethanol yields.

337
M. Rastogi, S. Shrivastava Renewable and Sustainable Energy Reviews 80 (2017) 330–340

Xuan Li et al. (2010), developed a two-phase simultaneous sacchar- pretreatment process, that improves digestibility of celluloses and hemi-
ification and fermentation (TPSSF) process for cost effective conversion celluloses, thus facilitating its further conversion. The review discusses
of aqueous ammonia pretreated corn stover to ethanol. In a single various strategies have been employed to extract maximal amount of
reactor, they achieved the highest ethanol concentration of 22.3 g/L monosaccharide sugars from all possible lignocellulosic wastes available in
(84% theoretical yield) via integrating both pentoses (xylan) and abundance. It highlights the importance of highly functional enzymes in
hexoses (glucose) utilization by E. coli KO11 and S. cerevisiae D5A fermentation process and discusses in details recent advances for
in the first and second phases, respectively. Thus, they not only saccharification and fermentation of lignocellulosic waste for production
increased the yield of bioethanol but also overcame the limitation of of ethanol with details on strategies like simultaneous saccharification and
glucose inhibition on fermentation of xylose in a single process [125]. fermentation, separate hydrolysis and fermentation, separate hydrolysis
A weed Lantana camara, available abundantly in India was and co-fermentation and consolidated saccharification and fermentation.
successfully converted to bioethanol with twofold benefits of energy Problems and developmental potential associated with all techniques
production as well as weed management. The delignification of acid involved in complete hydrolysis and fermentation of lignocelluloses have
pretreated biomass by using sodium sulfite (5.0% w/v) and sodium been discussed. In conclusion, lignocelluloses hold considerable potential
chlorite (3.0% w/v) resulted in about 87.2% lignin removal. The to meet the current energy demand of the modern world that are also
fermentation of enzymatic and acid hydrolysates yielded 17.7 and essential to overcome our excessive dependence on petroleum for liquid
5.16 g/L of ethanol with Saccharomyces cerevisiae and Pichia stipitis fuels that cause environmental pollution or global climate change.
after 16 and 24 h, respectively [126].
In an another study, Karagöz et al. (2012) were able to obtain Acknowledgement
14.07 g/100 g raw straw and 5.73 g ethanol from liquid (additionally)
obtained after 5% alkaline peroxide (at 50 °C for 1 h) pretreatment of Authors are thankful to Science and Engineering Research Board,
rapeseed straw through SVSCF (Same Vessel Saccharification and Co- New Delhi, India, for providing financial assistance to carry project File
fermentation) processes from Saccharomyces cerevisiae and Pichia number: YSS/2015/002072.
stipitis [127].
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