Renewable and Sustainable Energy Reviews 73 (2017) 654–671

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Renewable and Sustainable Energy Reviews

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

Utilization of lignocellulosic biomass by oleaginous yeast and bacteria for MARK

production of biodiesel and renewable diesel
⁎
Dipesh Kumara, Bhaskar Singha, John Korstadb,
a
Centre for Environmental Sciences, Central University of Jharkhand, India
b
Dept. of Biology, Oral Roberts Univ., Tulsa, OK 74171, USA

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

Keywords: Development of biodiesel and renewable diesel is hampered by the high cost of feedstocks (60–70% of the total
Lignocellulose cost). In order for these biofuels to substitute or supplement petro-diesel, the utilization of cheap and
Pretreatment abundantly available feedstock is invaluable. Lignocellulose constitutes a significant proportion of agroindus-
Hydrolysis trial waste and it is the most abundant feedstock on earth for production of biofuels. Oleaginous microorgan-
Oleaginous microorganisms
isms, when grown on lignocellulosic hydrolysates under nitrogen-starved conditions, can utilize holocellulose-
Biodiesel
based sugars for significant lipid accumulation (sometimes up to 70% of Dry Cell Weight or DCW). Rapid
Renewable diesel
growth rate, high lipid accumulation capacity, well established mechanism for oil accumulation, and high
content of Triacylglycerols(TAGs) in oil and fatty acid profiles makes oil production from oleaginous organisms
very attractive. Some fungi and oleaginous bacteria can also utilize lignin-like compounds as substrate.
Identification of oleaginous microorganisms that are tolerant to toxic by-products; are capable of utilizing
pentose, hexose and lignin-like compounds simultaneously;have high neutral lipid accumulation capacity; and
have desirable fatty acid profiles are all critical. Biotechnology and genetic engineering for modifying the
structure and relative proportion of lignocellulosic components in the biomass, efficient pretreatment and
hydrolysis methods, and standard culture conditions are also important.

1. Introduction long chain alkanes. Renewable diesel can be produced by hydrotreat-

ment of lipids and biodiesel. Renewable diesel is a relatively clean fuel
Concerns about growing energy demand, energy independence, and has better cold-flow properties than biodiesel. Biofuels derived
limited reserves, and environmental degradation associated with con- from vegetable oils cannot significantly replace their fossil fuel-based
sumption of fossil fuels have spurred worldwide interest in the counterparts as the oil content is very low. Therefore, development and
development and utilization of sustainable, biomass-based liquid fuels. utilization of alternative feedstocks that can overcome these limitations
Biomass lipid-based liquid fuels like biodiesel and renewable diesel are is important. Single cell oil (SCO) from oleaginous microorganisms
widely hailed as clean and renewable fuels. These biofuels are (including some yeast, fungi, algae, and bacteria) have gained sig-
traditionally produced from vegetable oils. However, there are a nificant attention recently as these organisms can accumulate signifi-
number of challenges associated with the use of traditional vegetable cant quantities of lipids, mostly in the form of TAGs, and have fatty
oils as biofuel feedstock, including the high cost of feedstock; low acid (FA) profiles similar to those of vegetable oils traditionally used as
productivity; slow growth rate, low photosynthetic efficiency; competi- feedstock for biodiesel. Elimination of the food vs. fuel dilemma,
tion of feedstock plants for land, moisture and nutrients needed by insignificant changes in land use and land cover, short growth period,
food crops; rise in prices of food commodities; land use and land cover and high accumulation of lipids are among the major advantages
changes; and other factors. Biodiesel produced by transesterification of offered by oleaginous organisms over traditional vegetable oils.
triacylglycerols (TAGs) is miscible and can be blended with petroleum However, economic feasibility of biofuel production using SCO from
diesel in various proportions. Biodiesel is a renewable, non-toxic and oleaginous microorganisms is hampered by the high cost of sugar-
relatively clean burning fuel for combustion in compression ignition based growth substrate [1]. In order to achieve sustainable and
engines. Renewable diesel, also known as green diesel or hydrotreated economical production of lipids by oleaginous microorganisms, a cheap
vegetable oil, is similar to mineral diesel in composition, consisting of and abundantly available growth substrate is required. Lignocellulosic

⁎
Corresponding author.
E-mail addresses: rdsdipesh@gmail.com (D. Kumar), bhaskar.singh@cuj.ac.in (B. Singh), jkorstad@oru.edu (J. Korstad).

http://dx.doi.org/10.1016/j.rser.2017.01.022
Received 18 October 2015; Received in revised form 18 December 2016; Accepted 7 January 2017
Available online 03 February 2017
1364-0321/ © 2017 Elsevier Ltd. All rights reserved.
D. Kumar et al. Renewable and Sustainable Energy Reviews 73 (2017) 654–671

biomass is perhaps the most abundant and cheap natural polymer that constitute a minor fraction (1–5%) of lignocellulosic biomass [11].
can serve as an ideal source of sugar for culturing oleaginous micro- Cellulose has an unbranched and linear structure and is the major
organisms. It contains a significant proportion of polymeric carbohy- carbohydrate homopolymer of lignocellulosic biomass. Two D-glucose
drates (cellulose and hemicelluloses), and breakdown of these carbo- molecules, linked to each other (cellobiose) by β-(1,4) glycosidic bonds
hydrates into their respective monomers can produce hexose and via a condensation reaction, form the repeating units of cellulose with
pentose sugars which are readily utilized by oleaginous organisms to the degree of polymerization (number of repeating sub units) ranging
accumulate lipids. The recalcitrance of lignocellulosic biomass is from a few thousand to several thousand sub units. Several linear
attributed to the presence of lignin and its linkages with hemicelluloses cellulose chains (20–300) remain linked to each other by covalent
and cellulose. Therefore, a pretreatment and hydrolysis process is bonds, hydrogen bonds, and vander Waals force, forming cellulose
usually employed for reducing the recalcitrance of lignocelluloses and microfibrils. These microfibrils are bundled together to form cellulose
for obtaining the carbohydrate monomers in the hydrolysate. The fibers that give rise to the cellulose ultrastructure. Cellulose is mostly
pretreatment of lignocellulosic biomass can be achieved by physical, found in crystalline form and resists degradation. Hemicellulose and
chemical, or biological methods. Depending on the type of pretreat- lignin cover the cellulose microfibrils and reduce the accessible surface
ment method and operational conditions employed, degradation of area of cellulose to hydrolytic chemicals and enzymes. The proportion
lignocelluloses into toxic inhibitory compounds may take place. The of cellulose varies between plant types. Hardwood species such as
toxicants may have an inhibitory effect on the downstream processes of aspen, red maple, eucalyptus, white birch, oak, and Populus contain
enzymatic hydrolysis and lipid fermentation. Hydrolysis of lignocellu- approximately 39–54% of dry biomass as cellulose; for softwood
losic biomass can be achieved with or without pretreatment by means species such as pines and firs, cellulose content is between 41–50%;
of acid (dilute/concentrated acid hydrolysis) or enzymatic hydrolysis, while for agricultural residues like rice straw, cellulose constitutes 32–
but yield of pre-treated biomass upon hydrolysis is usually higher than 47% of the biomass.
that from untreated biomass. Lignocellulosic hydrolysates consist of After cellulose, hemicellulose is the most abundant (20–50%)
hexose sugar (from cellulose and hemicelluloses) and pentose (from carbohydrate in lignocellulosic biomass. Hemicelluloses have lower
hemicelluloses). Xylose, as the predominant pentose, is obtained from molecular weights than cellulose, and are heterogeneous and branched
hardwood biomass and agricultural residues while softwood degrada- carbohydrate polymers of pentoses, hexoses, and acetylated sugars.
tion products have mostly mannose [2]. When the growth media is The backbone of hemicelluloses can be a homopolymer or hetero-
starved of nitrogen, accumulation of citric acid takes place in the polymers with short lateral chains bonded mostly by β- (1,4) and
mitochondrial matrix of oleaginous microorganisms. The cleavage of occasionally by β- (1,3) glycosidic linkage [12] with some degree of
citric acid by isocitrate dehydrogenase enzymes produces Acetyl acetylation and are easily hydrolysable. Although hemicellulose co-
coenzyme A (acetyl-CoA), which serves as the fatty acid precursor crystalizes with cellulose, they never aggregate [13]. Hemicellulose is
and thus facilitates diversion of carbon towards lipid accumulation at the most thermo-chemically sensitive component of lignocellulose.
the expense of proteins and nucleic acids [3]. Glucose repression on Xylan is easily extracted and is the major component of hemicellulose
xylose accumulation has been reported (diauxic growth pattern) and in agricultural biomass and grasses, while softwood hemicellulose
this slows down the rate of lipid accumulation. However, a few species mostly consists of glucomannan and is difficult to extract. Under high
of oleaginous yeast have also been reported to accumulate both of the temperature conditions during pretreatment and/or hydrolysis, break-
sugars simultaneously [4,5]. down (and loss of fermentable sugar) of hemicellulose sugars into
Simultaneous utilization of both pentose and hexose sugars, along products (furfural, acetic acid, etc.) known to have inhibitory effect on
with relatively higher energy density of lipids (in comparison to downstream processes of hydrolysis and/or microbial fermentation
ethanol), makes the process of lipid fermentation by oleaginous yeast may take place [11,14]. Lignin, the third most abundant component of
cultured on lignocellulosic hydrolysates an attractive process [6]. lignocellulose, is an amorphous heteropolymer that imparts recalci-
Glycerol is a by-product (10% of initial lipid content) of the transester- trance, imperability, and structural support to lignocellulose [15].
ification of lipids to produce biodiesel; crude glycerol can also be used Lignin consists of three phenyl propionic alcohol monomer units:
as a substrate for lipid accumulation by oleaginous microorganisms coniferyl alcohol, coumaryl alcohol, and sinapyl alcohol. These mono-
[7,8]. Although limited studies on properties of biodiesel produced mers are linked together by alkyl-alkyl, aryl-aryl, and alkyl-aryl bonds.
from yeast oil are available, several statistical models have been used to Softwood biomass contains the highest proportion of lignin. The
predict the properties of biodiesel using fatty acid profiles and relative recalcitrance of lignocellulosic biomass is largely attributed to lignin
proportions of different fatty acids [9]. Some species of fungi and and its linkages with other components of the lignocelluloses [16].
bacteria have been reported to grow and accumulate lipids on model Generally, a decrease in lignin content improves the overall yield of
lignin compounds [10]. Since lignin constitutes a significant proportion fermentable sugars from carbohydrate polymers. After pretreatment
of lignocellulose, its utilization along with carbohydrates is important. and/or hydrolysis of lignocellulosic biomass, the recovered lignin is
The general scheme of lipid production by oleaginous microorganisms usually used as fuel for heating purposes. However, some species of
using lingocellulosic biomass for biodiesel and renewable diesel fungi and oleaginous bacteria can utilize lignin-like compounds for
production is depicted in Fig. 1. growth and thus it can potentially be used as a growth substrate [10].
Irreversible binding of cellulases to lignin can reduce the efficiency of
2. Composition of lignocellulosic biomass enzymatic hydrolysis of lignocellulosic biomass [16].

Lignocellulosic biomass is the most abundant raw material on earth 3. Pretreatment of lignocellulosic biomass
for producing biofuels. It is the principal building block of plant cell
walls, consisting of carbohydrates, lignin, extractives, and ashes. The features of an ideal pretreatment method for lipid synthesis by
Although the relative proportion of individual components varies with oleaginous organisms should include: maximum/complete saccharifi-
plant type, species, age, growth conditions, and plant parts, carbohy- cation of hemicellulose into its respective monomers, minimization of
drates and lignin constitute the major fraction of lignocellulosic sugar loss, minimum production of by-products and compounds
biomass. Carbohydrate components of lignocelluloses include cellulose, having inhibitory effects on the organisms employed for downstream
hemicelluloses, and small concentrations of pectin. Cellulose and processes, improved access of hydrolytic chemicals/enzymes to cellu-
hemicelluloses are collectively known as holocellulose. Extractives lose, and be economical and energetically less demanding. The
include lipophilic and hydrophilic components of lignocelluloses such lignocellulosic biomass, unless pretreated before hydrolysis, yields
as terpenes, steroids, fats, waxes, and phenolic constituents, and relatively low concentrations of monosaccharide sugars. Pretreatment

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D. Kumar et al. Renewable and Sustainable Energy Reviews 73 (2017) 654–671

Fig. 1. General scheme of de-novo lipid accumulation by oleaginous microorganisms using lingocellulosic biomass for biodiesel and renewable diesel production.

is done before it is hydrolyzed in order to obtain high concentrations of ization, and increase in surface area of the resulting biomass enhance
fermentable sugars in the hydrolysates [16]. The access of hydrolytic the yield upon hydrolysis by 5–25% and increase the rate of hydrolysis
enzyme and acid to cellulosic fibers is hindered by the presence of (corresponding to a reduction in time required for hydrolysis) by 23–
lignin and hemicellulose as they form a protective sheath around 59% when compared to untreated biomass [22,23]. Chang and
cellulose [17]. In order to achieve effective hydrolysis of cellulose the Holtzapple [24] reported a threshold particle size of 40 mesh, below
lignin and hemicellulose components of the biomass must be removed which the rate and yield of hydrolysis were not significant. The power
or rearranged by pretreatment of the biomass. Pretreatment of requirement for mechanical pretreatment of biomass is dependent on
lignocellulosic biomass reduces the crystalline nature of cellulose and the characteristics of the biomass and the particle size. Based on their
increases its porosity and facilitates its subsequent hydrolysis. study on agricultural waste and dicot angiospermic wood, Cadoche and
Depending on the pretreatment method, hemicellulose may disinte- Lopez [25]] reported that if the size of the resulting particle is kept
grate into its constituent monomers at this stage. However, pretreat- between 3–6 mm, then the energy required per ton of lignocellulosic
ment of lignocellulosic biomass can also lead to production of toxic biomass can be kept under 30 kWh. It results in a negative value for
inhibitory compounds [13]. Complete extraction of polysaccharides energy return on energy invested (EROI) as it exceeds the theoretical
amenable to pretreatment in monomeric form and minimization of energetic content for most types of biomass. Other methods like γ-ray
toxic compounds produced are desirable qualities of any pretreatment irradiation of cellulose, lead to breaking of glycosidic bonds between
process. the monomers and are too expensive. There are several biochemical
and biomass structural features that affect the specific energy require-
ment (SER) of mechanical size reduction of lignocellulosic biomass.
3.1. Mechanical pretreatment
Moisture content, content of cellulose and lignin and cellulose crystal-
linity negatively affect the SER, while the accessible surface area and
Mechanical pretreatment of lignocellulosic biomass consists of
ratio of arabinose/xylose positively affect the SER [26].
comminution of biomass in order to reduce the particle size of the
biomass and cellulosic crystallinity. Reduction in particle size leads to
reduction in degree of polymerization and an increase in available 3.2. Steam pretreatment and steam explosion pretreatment
surface area for hydrolytic chemicals and enzymes to act upon the
substrate [18]. Comminution of biomass is done by grinding, chipping, Steam pretreatment involves exposure of ligocellulosic biomass to
and milling. Chipping of lignocellulosic biomass leads to particles hot steam (up to 240 °C) in a high pressure vessel for a few minutes.
ranging in size from 10-30 mm, while particles after milling and This is followed by releasing the steam and allowing the biomass to cool
grinding measure about 0.2–2 mm [19]. Milling of biomass can be down. This process leads to solubilisation of hemicellulose and
achieved by simple ball milling, vibratory ball milling, colloid milling, improved exposure of cellulose to hydrolytic chemicals and enzymes
hammer milling, wet disk milling, and by other means. Milling of without formation of inhibitors [27]. Hemicellulose solubilisation leads
biomass assisted by chemical treatment has been reported for en- to formation of acids that are known to catalyze hydrolysis of soluble
hanced digestibility of glycan and xylan and also for reducing hydro- fractions of hemicellulose oligomers in a process known as auto-
lytic enzyme loading [20]. cleaving [28,29]. Generally, biomass having higher moisture content
When compared to ordinary milling, vibratory ball milling is more requires longer retention time for effective steam pretreatment [30].
effective and leads to improved digestibility of the biomass [21]. Steam explosion, on the other hand, involves explosive decompres-
Milling of biomass also leads to its shearing and consequent reduction sion of biomass due to its quick depressurization. Rapid cooling and
in crystallinity of cellulose. Shearing, reduction in degree of polymer- quick depressurization of the biomass causes the water trapped in the

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biomass to explode. Typically, a temperature of 160–260 °C and 3.4. Ammonia fiber explosion and ammonia recycle percolation
pressure of 0.69–4.38 MPa is maintained for a few minutes before
exposing the material to atmospheric pressure [19]. Steam explosion Ammonia fiber explosion (AFEX) pretreatment is similar to steam
leads to transformation of lignin and hemicellulose degradation [13]. explosion pretreatment and involves exposure of biomass to hot liquid
Acetic acid released during pretreatment leads to hydrolysis of hemi- ammonia under high pressure for a period of time followed by sudden
cellulose. Steam explosion pretreatment facilitated efficient (90%) release of the pressure. The AFEX process typically involves liquid
enzymatic hydrolysis of Poplar chips when compared to untreated ammonia at a dosage of 1–2 kg of ammonia/kg of lignocellulosic
biomass (15%) [31]. Although only limited removal of lignin occurs, biomass (dry wt.), and temperature of 90 °C for a period of 30 min
depolymerization/repolymerization of lignin components leading to its [13]. Yields close to the theoretical maximum have been attained at low
redistribution has been reported [32]. Steam explosion leads to enzyme loadings upon hydrolysis of lignocellulosic biomass pretreated
hemicellulose removal from the microfibrils, thus facilitating exposure with AFEX [47,48]. Herbaceous biomass and agricultural residues are
of cellulose and its subsequent hydrolysis by enzymes. Lignin redis- comparatively more amenable to AFEX pretreatment than hardwood,
tribution and hemicellulose removal increases the volume of the and it is not suitable for softwood biomass [16]. AFEX pretreatment
pretreated biomass which, in turn, leads to an increase in effective alters the structure of the material and increases the water-holding
surface area for hydrolytic process [33]. High pressure steam and capacity of the biomass and its digestibility [49]. AFEX pretreatment of
sudden decompression leads to modified cell wall structure and biomass led to a six-fold increase in yield upon hydrolysis when
produces slurry which is filtered to obtain hemicellulose-based sugars compared to untreated biomass [50]. Bariska et al.[51] and Kim
and filter cake rich in cellulose along with residual fractions of et al.[52] attributed the high yield of sugars by AFEX pretreatment
hemicelluloses and lignin [34]. to its delignification capacity and swelling of the cellulose.
Factors affecting steam explosion pretreatment include particle Bermudagrass and bagasse have lignin content of 5% and 15%,
size, moisture content, temperature, and residence time [35,36]. respectively, and when pretreated by the AFEX method led to cellulose
H2SO4, SO2, or CO2, when added during biomass pretreatment in and hemicellulose hydrolysis by over 90%. AFEX pretreatment led to
concentrations ranging from 0.3% to 3% (w/w), can effectively decrease changes in linkage between carbohydrates and lignin and altered the
residence time and temperature, leading to complete removal of crystallinity index in empty fruit bunch fiber of oil palm as it facilitated
hemicellulose, improved hydrolysis rate, and decrease in inhibitory partial removal and reallocation of lignin and resulted in increased
compounds production [37,38]. When compared to mechanical com- sugar yield [53].
minution, steam explosion requires lower energy input for pretreat- The AFEX pretreatment, however, was reported to be ineffective for
ment of biomass [13]. The limitations associated with steam explosion biomass with high lignin content such as aspen chips and newspaper
pretreatment include only partial disruption of lignin-carbohydrate (25% lignin content) and the yield reported was only between 40–50%
linkage, destruction of pentose polymers from hemicellulose, and [16].
generation of inhibitory compounds for downstream processes. Due da Costa Sousa et al. developed a new ammonia based pretreatment
to the presence of inhibitory compounds, the pretreated biomass must method called Extractive ammonia (EA), which uses low moisture
be washed with water in order to remove these compounds prior to biomass (around 10% of total weight) in three sequential steps of
using pretreated biomass for downstream processes (enzymatic hydro- reaction, extraction, and recovery of product/solvent. The process was
lysis, microbial growth, and lipid fermentation). The water wash may capable of selectively extracting lignin with simultaneous transforma-
lead to loss of soluble sugars generated during decomposition of tion of cellulose to an easily digestible allomorph. Yield comparable to
hemicellulose and hence decrease the overall yield [16]. that obtained from AFEX pretreatment was achieved for EA pretreated
biomass with 60% lower enzyme loading [54].
3.3. Hot water pretreatment AFEX pretreatment employs liquid ammonia while the Ammonia
Recycle Percolation (ARP) technique uses aqueous ammonia (5–15 wt
In hot water pretreatment instead of using steam, liquid hot water %) to effect pretreatment of lignocellulosic biomass. In the ARP process
is used to facilitate biomass pretreatment prior to hydrolysis. High aqueous ammonia is trickled down a reactor column packed with
pressure is employed to maintain the liquid state of water at high lignocellulosic biomass at a temperature of 160–180 °C, flow rate of
temperatures (200–230 °C). Hot water pretreatment is also termed 1 ml/cm2, and residence time of 15–90 min. After trickling down the
aquasolv [39], uncatalyzed solvolysis [29], aqueous fractionation [40], column the aqueous ammonia is collected, separated, and recycled.
and hydrothermolysis [41]. Praveen et al. reported that approximately ARP pretreatment promotes cleavage of lignin-cellulose and lignin-
40–60% of the initial biomass gets dissolved during hot water hemicellulose matrix and depolymerisation of lignin. Adjustable and
pretreatment (cellulose by 4–22%, lignin by 35–60% and entire significant degree of delignification for hardwood [55] and agriculture
hemicellulose) [13]. Biomass size reduction is not a prerequisite as residual waste [56] has been reported at 160–180 °C and 14 min of
cooking in water breaks apart the lignocellulosic particles [42]. residence time. After completion of the process the solid biomass is
Enzymatic hydrolysis of pruned biomass of olive tree released 45.5% washed extensively and boiling of the liquid fraction is carried out to
of the oligomers after hot water pretreatment (versus 76.5% for steam recover ammonia and precipitated lignin. Kim et al.reported ARP
explosion pretreatment) [43]. pretreatment to be less efficient for softwood pulp mill sludge [57].
Hot water pretreatment (4–8 min at 160–200 °C) followed by disk
milling of corn stover led to an increase in yields of xylose and glucose 3.5. Acid pretreatment
by 89% and 134%, respectively. The results were comparable to that of
dilute acid pretreatment followed by milling [44]. Acid pretreatment of lignocellulosic material is perhaps the most
Perez et al. studied the hot water pretreatment and enzymatic commonly used physiochemical pretreatment method. Although sul-
hydrolysis of wheat straw and concluded that the recovery of hemicellu- furic acid remains the most widely used acid for pretreatment of
lose-derived sugar is dependent on temperature, and yield increases as lignocellulosic biomass, other acids like nitric acid [58], hydrochloric
residence time and temperature increased [43]. Residual acetylated xylan acid [59], phosphoric acid [59], and acetic acid [60] have also been
components after pretreatment had a marked impact on digestibility of employed. Pretreatment acids can be used in diluted or concentrated
pretreated substrate. The effectiveness of this method is dependent on the form, but there are several problems associated with using concen-
lignin content of the biomass and is relatively ineffective for softwood. Hot trated acids such as high toxicity, hazardous nature, corrosiveness,
water pretreatment does not lead to formation of toxic inhibitory requirement of corrosion resistant infrastructure, and formation of
compounds if pH is maintained between 4 and 7 [45,46]. inhibitory products. Using concentrated acid also necessitates recovery

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and reuse of excess acid. When compared to the concentrated acid solubilisation and extraction of lignin [75]. Sodium hydroxide-based
method, dilute acid pretreatment is more suitable as it allows a more alkaline pretreatment has been extensively studied and is known to
efficient procedure. A range of feedstock types including hardwood, cause structural disruption of lignin.and thus increases the efficiency of
softwood, agro-industrial residue, herbaceous biomass, municipal solid enzymatic hydrolysis by enhancing the accessibility of enzymes to
waste, and waste paper have been reported as amenable to dilute acid cellulose and hemicellulose [76–79]. Sun et al. tested the effectiveness
pretreatment for high yields [61]. of different bases for pretreatment of wheat straw by analyzing
Sulfuric acid in the concentration range of 0.2–2.5 wt% can be dissolution of hemicellulose and its delignification [80]. They reported
added and mixed with biomass. The mixture is heated to a temperature that the optimal conditions for 60% removal of lignin and 80% release
of 130–210 °C with stirring to facilitate pretreatment. Dilute acid of hemicellulose sugar (mostly as xylose) was 1.5% NaOH solution,
pretreatment can be carried out at high temperature and short 144 h residence time, and a temperature of 20 °C. Effectiveness of
retention time (180 °C or above for 5 min) or at low temperature and NaOH has been reported for pretreatment of hardwood biomass,
high residence time (120 °C for 30–90 min) [62]. Acidic pretreatment switchgrass, and softwood biomass having lignin content less than
effectively hydrolyzes hemicellulose to its constituent monomers. The 26% [78]. Lime has also been studied extensively for pretreatment of
monomers may further degrade in acidic environment to form several switchgrass, corn stover, bagasse, rice, and wheat straw
products that have inhibitory effects on the downstream process of [8,19,27,81,82]. Alkaline pretreatment necessitates removal of dis-
enzymatic hydrolysis and lipid fermentation by oleaginous organisms. solved lignin, neutralization of excess alkali by acid, and removal of
Degradation of pentose sugar leads to formation of furfuryl, while 5- neutralization salt. Phenolic acids, aldehydes, and furfuryl that may
hydroxymethylfurfural (HMF) is produced by degradation of hexose form during alkaline pretreatment must be removed as they are
sugar [63,64]. Acidic pretreatment leads to partial lignin solublization; potential inhibitors for the downstream processes of enzymatic/che-
however, it condensates and forms a precipitate under acidic condi- mical hydrolysis and lipid fermentation by oleaginous organisms [69].
tions [65]. Removal of hemicellulose and lignin promotes efficient Although sodium hydroxide is the most commonly employed alkali,
hydrolysis of cellulose. Dilute sulfuric acid pretreatment is effective in pretreatment with calcium hydroxide is preferable as it is relatively
removal and recovery of hemicellulose as dissolved sugar, and in cheaper and safer than sodium hydroxide and it can be easily recovered
combination with efficient hydrolysis almost 100% recovery of sugar as calcium carbonate using waste CO2 stream [83]. Combining alkaline
from biomass is achievable [17]. Several surfactants such as Tween-80, pretreatment with other pretreatment methods like AFEX, ARP, wet
Tween-20, dodecylbenzene sulfonic acid, Triton X-100, and PEG 4000 oxidation, and radiofrequency-based dielectric heating have also been
have been used as an additive for dilute acid pretreatment and led to tested [78].
enhanced cellulose digestibility [66].
Dilute acid pretreatment combined with alkali pretreatment have 3.7. CO2 explosion
also been investigated for enhancing the yield and cellulose digest-
ibility. Using a combination of acid and alkaline pretreatment leads to At temperature and pressure above its critical point of 31 °C and
removal of non-cellulosic fractions and enhances the accessibility of 7.39 MPa, CO2 behaves as a supercritical fluid having a density similar
hydrolytic agents to cellulose [66]. Effective removal of hemicellulose to liquid CO2, while its penetration power and diffusibility are
and lignin has been reported using sulfuric acid and ammonium equivalent to that of a gas. At supercritical state, distinct gas and
hydroxide, respectively [66]. liquid phases do not exist and the fluid molecules can effuse through
A wide variety of feedstocks including hardwood, grasses, and solid biomass like a gas. The size of CO2 molecules is similar to H2O
agricultural residues have been tested and promising results have been and NH3; hence, it has access to pores in biomass accessible to water
reported [67,68]. Important limitations of dilute acid pretreatment and ammonia [13]. CO2 reacts with moisture to form carbonic acid,
include: 1) requirement ofexpensive corrosion-resistant reactors; 2) which further catalyzes biomass degradation.
degradation of monomeric sugar and formation of toxic inhibitory Super critical CO2 possesses the ability to penetrate through the
compounds during pretreatment like furfuryl, HMF, and other volatile available pores to the crystalline structure of the lignocellulosic
components; 3) requirement of a water wash to remove compounds biomass as it can overcome the mass-transfer limitations [84].
having inhibitory effects on downstream processes of hydrolysis and Explosive release of pressure leads to the disruption of hemicellulose
lipid fermentation which leads to loss of fermentable sugar; 4) and cellulose structure and increases the overall accessible area to
neutralization or recovery of excess acid; and 5) disposal problem of enzymes. Alina et al. studied the combination of supercritical CO2 and
waste gypsum generated during neutralization of excess acid by lime heated steam addition and reported the process to be efficient [85].
[17]. Dale and Moreira studied the effect of steam explosion, AFEX, and
supercritical CO2 on yield upon hydrolysis of alfalfa. Supercritical CO2
3.6. Alkaline pretreatment pretreatment resulted in 75% yield of glucose relative to its theoretical
yield [47]. Yields obtained were relatively low in comparison to that of
Alkaline pretreatment is the most common pretreatment method steam explosion and AFEX pretreatment, but higher than untreated
involving soaking lignocellulosic biomass in solution and mixing it at a biomass. In their comparative study on pretreatment of recycled paper,
target temperature. Bases such as hydroxides of calcium, sodium, Zheng et al. reported supercritical CO2 to be more cost-efficient than
potassium, and ammonia are commonly used [69]. Alkaline pretreat- AFEX pretreatment, and unlike steam explosion pretreatment, no toxic
ment promotes degradation of glycosidic side chains and ester linkages, products were reported for supercritical CO2 pretreatment [86]. Using
leading to structural alteration of lignin, partial decrystallization CO2 has several important advantages such as low cost, abundant
[70,71] and swelling of cellulose, and limited solvation of hemicellulose availability, non-flammability, environmental acceptability, and easy
[71,72]. The process leads to solvation as well as saponification of recovery, but requiring high pressure for supercritical CO2 pretreat-
intermolecular ester linkages, which leads to enhanced porosity of the ment proves to be a deterrent [87]. Delignification of biomass can be
biomass. enhanced by using a co-solvent such as ethanol during supercritical
Alkaline pretreatment of biomass can be conducted under ambient CO2 pretreatment [88].
conditions but pretreatment time can be greatly reduced by increasing
the pretreatment temperature [61]. Alkaline compounds specifically 3.8. Ozonolysis
target the lignin-cellulose/hemicellulose ester linkage and acetyl
groups of hemicellulose [73,74]. Alkaline pretreatment reduces the Ozone is a strong oxidant (E0 =2.07 V at 25 °C), which leads to
non-specific binding of hydrolytic enzymes to lignin as it leads to degradation of lignin and hemicellulose. The terminal oxygen of ozone

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is electron deficit and therefore selectively attacks substrates rich in 3.10. Biological pretreatment
electrons (e.g. lignin), while carbohydrates mostly remain unaffected
[80]. Unlike other pretreatment methods that require expensive instru-
Ozonolysis degrades lignin and hemicellulose present in several ments, have high-energy requirement, and involve addition of chemi-
lignocellulosic materials like bagasse, peanut, pine, green hay [89,90], cals, biological pretreatment is an environmentally friendly and
wheat straw [91], cotton straw, and poplar sawdust [92]. Degradation inexpensive method. Biological pretreatment takes advantage of the
is mostly limited to lignin, while hemicellulose degradation is slightly natural degradation mechanism of saprophytic fungi to effect pretreat-
affected and cellulose remains unaffected by ozonolysis [13]. ment of lignocellulosic biomass prior to its hydrolysis. Fungi such as
Ozonolysis is usually performed at ambient conditions of temperature brown, white, and soft rot fungi are capable of delignifying the
and pressure; however, since a large quantity of ozone is required, the lignocellulosic biomass [106]. White and soft rot fungi produce
process is somewhat costly. Vidal and Molinier reported a fivefold enzymes like lignin peroxidases, laccase, manganese-dependent prox-
increase in enzymatic hydrolysis rate of wheat straw after ozonolysis idases, and polyphenol oxidases that cause degradation of lignin and
that led to 60% removal of lignin [92]. An increase in hydrolysis rate cellulose, whereas the activity of brown rot fungi is primarily limited to
from 0% to 57% was reported with increase in lignin removal from 27% degradation of cellulose [15]. Rice husk subjected to simultaneous
to 8% in poplar sawdust. pretreatment and saccharification by the fungus Phanerochete chry-
Williams reported lignin removal of 49% after ozonolysis of corn sosporium had effective delignification through the release of cellulase,
stover [93]. Morrison and Akin [94], Euphrosine-Moy et al. [95], and lignin peroxidise, xylanase, aryl alcohol oxidase, and glyoxidase
Lasry et al. [96] reported the presence of several types of acids in the enzymes during pretreatment. Reducing sugar yield as high as
aqueous extract of ozone-treated hydrated sawdust and herbs like 895 mg/ml was reported on the 18thday of pretreatment [107].
oxalic acid, formic acid, glyoxylic acid, glyceric acid, succinic acid, p- Biological pretreatment of corn stalk using Irpex lacteus was studied
hydroxybenoic acid, propionic acid, caproic acid, vanillic acid, malonic by Du et al. who reported production of various oxidative and
acid, and aldehydes such as vanillin and p-hydroxybenzaldehyde, while hydrolytic (extracellular) enzymes and hydrolysis yield of about 82%
major inhibitors such as furfural and 5-hydroxymethylfurfural were after 28 days of pretreatment [108].
absent. Biological pretreatment has extensive delignification capability
[109]. A fungal cell typically has a C:N ratio of 30:1, while for bacteria
3.9. Organosolv pretreatment it is 10:1. As fungi are less dependent on nitrogen, they form an ideal
natural agent to promote decomposition of ligocellulosic biomass
The Organosolv process utilizes organic/aqueous organic solvents because the C:N ratio of lignocellulose is very high. Factors such as
such as ethanol, methanol, ethylene glycol, triethylene glycol, acetone, moisture content of the biomass, particle size, residence time, and
and tetrahydrofurfuryl alcohol [97,98] to bring about the pretreatment temperature affect the extent of biomass delignification.
of lignocellulosic biomass. The process may also involve acidic or Akin et al. reported white rot fungi mediated Bermudagrass stem
alkaline agents as catalysts. Inorganic acids (eg, H2SO4 and HCl) or delignification improvement of 29–32% byCeriporiopsis subvermis-
bases (eg, NaOH, NH4OH, and Ca(OH)2) are typically used [99]. pora and 63–77% by Cyathus stercoreus in 6 weeks [110].
Organic acids such as oxalic acid, acetylsalicylic acid, and salicylic Phanerochaete sordida [38], Pleurotus ostreatus [111], and
acids have also been used as catalysts [100]. Pycnoporus cinnabarinus [109] facilitated35% conversion of initial
Organosolv pretreatment breaks the internal lignin and hemicellu- biomass in 4–5 weeks. Biological pretreatment does not produce
lose bonds and is suitable for biomass having high lignin content [101]. compounds having inhibitory effects on downstream processes of
Lignin in relatively pure form can be obtained [102]. The process brings fermentation. However, biological pretreatment is a rather slow
about pre-hydrolysis of hemicellulose components and delignification process, and for large-scale operations, maintenance of sterile condi-
simultaneously. Hemicellulose can be easily broken down into pentose tions may be costly [112].
sugar if acid catalyst is employed [13]. Organosolv pretreatment Various combinations of biological pretreatment with other meth-
operates at a temperature range of 90–120 °C (for grasses) and 155– ods such as hot water treatment [113], steam explosion treatment
220 °C (for wood), with residence time between 25 and 100 min [103]. [114], and physical and chemical pretreatment [115], have been
Solvent and catalyst concentration varies with the type of feedstock. investigated and with promising results in terms of yield achieved
Pan et al. reported 90% hydrolysis of mixed softwood pulp cellulose and time required for pretreatment.
in 48 h using aqueous ethanol as solvent [104]. Organosolv pretreat-
ment causes fragmentation of cellulose that remains insoluble in 3.11. Ionic Liquid (IL) pretreatment
aqueous phase, while hemicellulose degrades into its oligomers,
monomers, and acetic acid that are soluble in aqueous phase. Ionic Liquid (IL) pretreatment of lignocellulosic biomass is a
Easily digestible cellulose and lignin of high purity can be obtained relatively new pretreatment approach and can simultaneously dissolve
from lignocellulosic biomass pretreated with ethanol during lignin and carbohydrates [101]. ILs are typically composed of large
Organosolv pretreatment. Ethanol as organic solvent can be used cations (organic) and small anions (inorganic/organic) [113], bonded
successfully for pretreatment of various lignocellulosic biomasses such together by a strong ionic bond that imparts certain valuable properties
as switchgrass, birch, lodgepole pin, wheat straw and eucalyptus [105]. to these liquids like high thermal stability, high polarity, low melting
Upto 95% conversion of switchgrass residues to glucose has been point ( < 100 °C), and negligible vapor pressure [14,115]. The most
reported [106]. Ethanol pretreatment under elevated temperature and commonly used ILs for pretreatment of lignocellulosic biomass are
pressure generates acetic acid from the acetyl groups present in the imidazonium salts. According to Zhang and Lynd [116] and Zhu et al.
biomass, which catalyzes the degradation of cellulose and lignin [117], AMIMCl (1-allyl-3-methylimidazonium chloride) and BMIMCl
dissolution. Auto-catalyzed Organosolv pretreatment minimizes input (1-butyl-3-methylimidazolium chloride) are potentially effective non-
of external acids for catalyzing the process. derivatizing solvents for dissolving cellulose.
Acetic acid formation reduces the pH of the medium and further In comparison to imidazolium ILs cholinium carboxylic acid ([Ch]
stimulates hydrolysis of hemicellulose [13]. Lignin is broken down into (CA) and cholinium amino acid ([Ch][AA]) are more environmentally
fragments having lower molecular weight. The residual solvent must be friendly because ILs can be derived from readily available renewable
extracted and recycled to minimize cost and eliminate any disturbance materials, have lower toxicity rates, and have relatively higher rates of
to the downstream processes [19]. Low boiling point and flammability biodegradation. They can also selectively extract lignin from the
of the solvents are problematic [105]. biomass [118]. Moulthrop et al. suggested a possible mechanism in

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which the ILs disrupt the 3D network of lignocelluloses components by product of H2O2), and highest delignification occurred at pH 11.5–
forming hydrogen bonds [119]. The formation of these bonds is 11.6.
facilitated by the anionic components of the ILs (eg, chlorides, acetate, Bamboo chips were pretreated with alkaline hydrogen peroxide (at
alkyl phosphonate, or formate) [103]. ILs as biomass fractionation pH 12.6) and it led to simultaneous solubilisation of hemicellulose and
solvents have attracted wider interest because they are powerful delignification of biomass and the pretreatment was able to enhance
solvents capable of dissolving the whole biomass (cellulose and lignin). the surface area and pore volume of the biomass; however, it resulted
After pretreatment with ILs, antisolvents are added, followed by in higher degree of crystallinity as lignin and hemicelluloses were
centrifugation and filtration. The process leads to separation of solubilized.
cellulose from the biomass matrix. The residual fractions (lignin and In addition to all of the above methods, different combinations of
hemicelluloses) can again be recovered by means of acid/antisolvent physical, chemical, and biological methods have also been tested to
addition [120]. overcome the limitations of individual methods. The advantages and
The recovered fraction of cellulose is easily amenable to hydrolysis. disadvantages of various pretreatment methods are summarized in
IL pretreatment employs temperatures ranging from 100 to 150 °C and Table 1.
a biomass to ionic liquid ratio of 1:10 (w/w) [103]. The residual
content of ionic liquids interferes with the enzymatic hydrolysis of the 4. Hydrolysis of lignocellulosic biomass
substrate and could decrease the amount of fermentable sugar released
and hence, the quantity of biofuel produced. Ionic liquids are expen- The process of hydrolysis causes the saccharification of carbohy-
sive, so their recycling and reuse is critical. Techniques such as drates to their constituent monomer sugar molecules. Hydrolysis is
pervaporation (pervaporative separation), ionic exchange, flash dis- usually preceded by pretreatment of lignocellulosic biomass in order to
tillation, and reverse osmosis can be used to recover the residual ionic efficiently convert carbohydrate polymers into fermentable sugars.
liquid. Ionic liquid pretreatment can significantly enhance cellulose Direct hydrolysis of lignocellulosic biomass is hampered by physical,
digestibility by more than 90%. chemical, and structural features of lignocellulosic biomass. Thus, the
IL (1-butyl-3-methylimidazolium chloride ([Bmim]Cl) was used for goal of any pretreatment method is to make the biomass amenable to
the pretreatment of mixed softwoods and it led to significant changes in hydrolysis for obtaining higher yield of fermentable sugars [19].
cellulose crystallinity, structure, composition, and surface character- Pretreatment of lignocellulosic biomass is usually preformed prior to
istics of the biomass with significant enhancement of biomass digest- its hydrolysis. Pretreatment is aimed at enhancing alteration of
ibility and > 90% conversion of cellulose to sugar [121]. cellulosic crystallinity, its degree of polymerization, available cellulose
Factors such as cations, anions, time, and temperature affect the surface area for hydrolytic enzymes or chemicals, and hemicelluloses
overall process of IL pretreatment of lignocellulosic biomass [120]. ILs and lignin content [16]. Therefore, efficiency of any hydrolysis method
are environmentally friendly, thermostable, non-volatile, and non- is greatly dependent on pretreatment method employed. Some of the
derivatizing solvents [15]. pretreatment methods and operating conditions leads to production of
by products that have an inhibitory effect on the downstream pro-
3.12. Oxidative pretreatment cesses. Hydrolysis of lignocellulosic biomass is usually performed by
cellulase enzymes or acids. The various advantages and disadvantages
The objective of oxidative pretreatment is the removal of hemi- associated with different hydrolysis methods are listed in Table 2.
celluloses and lignin in order to improve the accessibility of hydrolytic
agents to cellulose. Oxidative pretreatment involves the addition of 4.1. Enzymatic hydrolysis
oxidizing agents like O2 (wet oxidation), H2O2, or peracetic acid to the
biomass suspended in water. The reactions involved in oxidative Enzymatic hydrolysis of cellulose is performed by a collection of
pretreatment are side chain displacement of hemicelluloses, electro- enzymes commonly known as cellulase. These enzymes catalyze the
philic substitution, cleavage of aromatic nuclei of lignin, or the cleavage depolymerization of cellulose with high specificity [125]. Structural
of ether linkages between alkyl and aryl groups [121]. Non-selective features of the substrate including cellulose crystallinity, accessible
oxidants can cause loss of hemicelluloses and cellulose components. surface area of the substrate, its degree of polymerization, and lignin
High risk of inhibitor formation persists due to the oxidation of lignin content determine the susceptibility of the substrate to enzymatic
because it produces soluble aromatic fractions. During wet oxidation hydrolysis [16]. During enzymatic hydrolysis the enzymes are first
the phenolic fractions of lignin are degraded further to carboxylic acids. adsorbed on the cellulose surface, followed by its degradation into
In the presence of an alkali, hydrogen peroxide decomposes to constituent sugar monomer and desorption of the cellulase enzyme.
hydroxyl (*OH-), perhydroxyl anion (OOH-), and superoxide anion Cellulase represents a group of enzymes with members of protein from
(*O2-) radicals. These break the ether linkage between hemicelluloses at least 15 families and a few sub-families [126]. Synergistic action of a
and lignin [122]. minimum of three enzyme groups promotes the hydrolysis of cellulose.
Klinke et al.reported a relatively low concentration of inhibitors The enzymes belong to endo-glucanases, exo-glucanases, and β-gluco-
(furfural and hydroxymethyl furfural (HMF)) after wet oxidation, but sidases, and are collectively known as cellulase. The role of endo-
loss of hemicellulose fractions as it reacted with water and carbon glucanases is to attack amorphous cellulose and regions of low
dioxide [122]. When an alkali was used in combination with wet crysatallinity on cellulose fiber and create free chain-ends. These free
oxidation, the formation of inhibitory products was reduced and chain-ends are later attacked by exo-glucanases, which leads to
concentration of monosaccharide sugars increased [122]. removal of glucose dimers (cellubiose units). Cleavage of 1,4-D
Teixeira et al. studied the oxidative pretreatment of poplar and glycosidic bonds in cellobiose is performed by β-glucosidases, which
sugarcane bagasse using peracetic acid as oxidant and reported completes the saccharification of cellulose into glucose [127].
peracetic acid to be a very selective oxidant for lignin degradation as Cellobiose produced must be removed or converted to glucose (by β-
carbohydrate loss was insignificant [123]. Using peracetic acid (21% w/ glucosidases), because at higher concentrations it has inhibitory effects
w) as oxidant, enzymatic hydrolysis yield of poplar and sugarcane on cellulose. Degradation of hemicelluloses into its monomers (mostly
bagasse increased to a maximum of 98%, while it was only 6.8% for pentose and some hexose) can take place during pretreatment; the
untreated biomass. Similar results were obtained when a mixture of remainder can be degraded during hydrolysis while significant sac-
NaOH and preaceric acid was used [123]. Gould [124] studied the charification of cellulose is usually achieved during hydrolysis.
effect of H2O2 as oxidizing agent and reported that the delignification For saccharification of hemicellulose a different and complex set of
of biomass was probably carried out by the OH-ions (degradation enzymes are required, commonly referred to as hemicelluloses or

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Table 1
Major advantages and disadvantages associated with different pretreatment methods [13,19,27].

Pretreatment Method Advantages Disadvantages

Mechanical Increase in available specific surface area High energy consumption

Reduction in cellulosic crystallinity
Reduces the degree of polymerization (DP)
Steam Explosion Hemicellulose degradation Formation of inhibitory compounds
Lignin removal Partial degradation of xylan fraction
Cost effective Incomplete disruption of holocellulose-lignin matrix
AFEX Increases accessible cellulose surface area Inefficient for biomass with high lignin content e.g, softwood
Removal of hemicelluloses and partial lignin removal High cost of ammonia
Inhibitors not produced Only partial solubilisation of hemicellulose
ARP Significant removal of lignin High cost
Very effective for herbaceous biomass
CO2 Explosion Increases accessible cellulose surface area Lignin and hemicelluloses remain unaffected
Inhibitors not produced High pressure requirement
cost-effective
Low degradation of sugar
Hot Water Pure hemicelluloses is recovered High energy input
Catalyst not required Lignin and cellulose remain unaffected
Hydrolysis of hemicellulose High water requirement
Ozonolysis Removal of lignin Requires large quantity of ozone
Partial degradation of hemicellulose Expensive process
Inhibitory compounds not produced
Operates under ambient conditions
Acid High yield of fermentable sugars Requirement of expensive corrosion resistant reactors
Solubilisation of hemicelluloses Neutralization of excess acid is required
Alteration of lignin structure Formation of toxic inhibitors
Increases exposure of cellulose to hydrolytic chemical and enzymes Partial loss of hemicelluloses sugars
Partial hydrolysis of cellulose
Alkali Specifically target the lignin-cellulose/hemicellulose ester linkage and acetyl Long residence time
groups of hemicellulose Neutralization of excess alkali is required
Partial removal and alteration of lignin structure Neutralization salts gets incorporated into the pretreated
Removal of hemicellulose biomass
Increases accessible cellulose surface area High cost of alkaline catalyst
Low inhibitor formation
Organosolv Breaks the internal lignin and hemicellulose bonds High cost of solvents
simultaneously delignification and pre-hydrolysis of hemicellulose components Flammability of solvents
Mild processing conditions The residual solvents must be extracted to minimize cost and
Catalysts can greatly enhance the rate of pretreatment any disturbance to the process used downstream of
Relatively pure lignin can be fractionated for energy or other applications pretreatment
Suitable for biomass with high lignin content
Ionic liquid Simultaneous dissolution of lignin and carbohydrates Relatively new approach
Disrupts the 3D structure of lignocellulosic components by formation of Ionic liquids are very expensive
hydrogen bonds Residual content of ILs interfares with enzymatic hydrolysis of
Cellulosic fraction can be recovered by adding anti-solvents and easily hydrolyzed substrate
Uses environment friendly thermostable, non-volatile and nonderivatising
solvents
Biological Natural degradation mechanism Slow rate of degradation
Degrades lignin and hemicelluloses Careful and regular monitoring of culture conditions in
Relatively mild operational conditions required
Low energy requirement Maintenance of sterile conditions can be expensive
Oxidative Brings about reactions such as side chain displacement of hemicelluloses, High risk of lignin oxidation based formation of inhibitor
electrophilic substitution, cleavage of aromatic nuclei of lignin, or the cleavage formation
of ether linkages between alkyl and aryl groups to remove lignin and Partial loss of hemicellulose based sugar
hemicellulose
Peracetic acid is very selective oxidant and specifically attack lignin

xylanase. Saccharification of hemicelluloses is critical in the sense that cellulase. Bacterial species belonging to the generaClostridium,
it constitutes a significant proportion of lignocellulosic biomass and its Thermomonospora, Ruminococcus, Cellulomonas, Acetovibrio,
saccharification improves the overall yield of fermentable sugar. Xylose Bacillus, Bacteroides, Microbispora, Erwinia, and Streptomyces are
forms the repeating units of xylan which is a major group of known to produce cellulases [128]. Some of these species are aerobic
hemicelluloses. Complete saccharification of xylan requires several while others are anaerobic and are either mesophilic or thermophilic.
enzymes including endo-β-1,4-xylanase (produces xylo-oligosacchar- Among these, particularly Thermomonospora fusca and Cellulomonas
ides from xylan), β-xylosidase (catalyzes hydrolysis xylose chains to fimi have been studied extensively for their cellulose production
produce xylose), and several accessory enzymes such as α -glucuroni- efficiency. High specific activity of cellulose produced by anaerobic
dase acetylxylan esterase, ferulic acid esterase, α–galactosidase, and α species like Bacteroides cellulosolvens and Clostridium thermocellum
-L-arabinofuranosidase. have been reported but enzyme titres values are low [35]. The
Accessory enzymes constitute an important fraction of enzymatic requirements of maintaining anaerobic growth conditions and very
cocktail for achieving high yield of fermentable sugar. Additionally, slow growth rates have diverted most research on fungi for commer-
synergistic action of cellulases and hemicellulases has been reported for cially producing cellulose [35]. Several species of fungi, including P.
enhanced rate and high conversion of glucan to glucose as removal of chrysosporium, S. rolfsii, and members of the generaTrichoderma,
hemicelluloses enhances the accessibility of cellulases to cellulose Schizophyllum, Penicillium, and Aspergillus, have been reported to
fibrils. Certain species of bacteria and fungi can naturally produce produce hydrolytic enzyme cellulase [35,129,130]. Among these organ-

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Table 2
Advantages and disadvantages associated with different hydrolysis methods [19,111].

Hydrolysis Method Advantages Disadvantages

Enzymatic Hydrolysis Operates at low temperature (40–50 °C) High retention time
Optimal performance at only slightly acidic pH Product Inhibition
No corrosion problem Reversion of glucose
High yield Inhibition at high substrate to enzyme ratio
Partial recycling and reuse of enzymes Requires addition of surfactants to prevent Irreversible binding of enzymes to
Enzymes can be produced naturally lignin
Inhibitory effect of ionic surfactants
High cost of enzymes
Dilute Sulfuric Acid Hydrolysis Requires low concentration of acid Degradation of hemicelluloses sugar
Residence time is short Formation of inhibitory compounds
Mild hydrolysis conditions Requirement of high temperature
Low cost of acidic catalyst Corrosion of metallic reactors
Requirement of excess acid neutralization and detoxification of hydrolysates
Low sugar yield
Concentrated Sulfuric Acid Hydrolysis Operates at lower temperature Long residence time
High yield of sugar Requirement of expensive reactors resistant to corrosion
High acid consumption
Excess acid must be neutralized

isms, cellulases from T. reesi and T. viride are especially interesting fatty acid-based ester of sorbiton polyethoxylate arevery effective.
because they have entire complement cellulase production capacity Alkasrawi et al. [136], Kim et al. [137], and Borjesson et al. [138]
andthese enzymes are stable under conditions of enzymatic hydrolysis. tested the efficiency of polyethylene glycol and reported an increase in
These enzymes produced by the two species of Trichodermaremain conversion of carbohydrate from 42% to 78% in 16 h. Eriksson et al.
unaffected by the chemical inhibitors [127]. However, the sub-optimal [139] attributed this increase to binding of surfactant to lignin, which
level of production of β-glucosidases and its low activity are major prevented unproductive binding of cellulases to lignin. In order to
disadvantages of cellulases from Trichoderma. The overall activity of reduce the cost of enzymatic hydrolysis, the cellulases must be
cellulases derived from Trichoderma can be improved by supplement- recovered and recycled. Ramos et al. [140] reported five consecutive
ing the reaction mixture with β-glucosidases [131–134]. Aspergillus times recycling of commercial Celluclast and Novozym enzymes but
spp. are known to produce β-glucosidases. Blending of enzymes from with reduced efficiency each time. However, the efficiency of the
different microorganisms have also been reported [135]. recovered enzymes gradually decreased each time it was recycled.
Some studies have also investigated the prospects of cellulose and The intermediate product of hydrolysis cellubiose) and final product
hemicellulose isolation from pathogenic fungi capable of degrading (glucose) slightly inhibit the activity of cellulases. High enzymatic
plant cell walls. Hydrolytic enzymes obtained from Fusarium verticil- loading for efficient conversion of intermediates, supplementation of β-
lioides, Pycnoporus sanguineus, and Chrysoporthe cubensis have glucosidases in order to convert cellubiose to glucose, and removal of
shown promising results for saccharification [134]. end product glucose by simultaneous saccharification and fermentation
When attacked by cellulases, several physical changes ofcellulose or by ultrafiltration of the hydrolysates have been developed to
such as swelling, reduction in degree of polymerization, lowering of minimize inhibition of cellulases [19]. Reducing the cost of cellulases
tensile strength, fragmentation, and cracking precede the release of is an important challenge for enzymatic hydrolysis of lignocellulosic
fermentable sugar [135]. biomass. Cellulase coding sequences have been cloned into yeasts,
The optimum operational temperature and pH for enzymatic other fungi, bacteria, and plants in order to create new sources with
hydrolysis using different cellulases and hemicellulases are reported potential improvement opportunities [19].
to be between 40 and 50 °C and pH 4–5 [135]. Optimal temperature
and pH for functioning of cellulases are dependent on the source of the 4.2. Acid hydrolysis
enzymes, raw material composition, and hydrolysis duration. Initial
rate of hydrolysis and yield is dependent on the initial substrate Sulfuric acid has been the most widely investigated acid for
concentration. For low initial substrate concentration, the rate of catalyzing the hydrolysis of cellulose [141]; however, other acids such
hydrolysis increases with increase in substrate concentration. as hydrochloric acid have also been studied [142]. Depending on the
However, at high substrate concentration, the process is inhibited by concentration of acid used, the acid hydrolysis process can be
the substrate itself and results in low yield of fermentable sugars. The categorized into: 1) Dilute acid hydrolysis and 2) Concentrated acid
ratio of total cellulase enzymes to total substrate (cellulose and/or hydrolysis.
hemicelluloses) determines the extent of process inhibition [19]. At
high substrate concentration, inefficient mixing and problems asso- 4.2.1. Dilute acid hydrolysis
ciated with mass transfer can cause low yield of fermentable sugar. Dilute acid hydrolysis is a widely investigated and well-established
Cellulase loading in the range of 5–35 FPU per gram of substrate is hydrolysis technique. Dilute acid treatment of lignocellulosic biomass
usually employed with hydrolysis rate increasing in line with cellulase is also a pretreatment method that efficiently converts hemicelluloses
loading but at higher cost for extra enzyme [127]. Presence of lignin into its constituent sugar monomers with partial solubilisation of lignin
increases the non-specific and irreversible binding of cellulases and and cellulose. Sulfuric acid in the concentration range of 0.2–2.5 wt%
necessitates higher enzymatic loading for obtaining close to theoretical is usually employed in dilute acid hydrolysis of cellulose. Relatively
yield of sugar [16]. Cellulase activity and hydrolysis rate decreases over mild operational temperature conditions ( < 200 °C) are required for
time as cellulases partially bind irreversibly to cellulose [67]. hydrolysis of hemicelluloses, while recalcitrance of cellulose can only
Irreversible, non-specific, and unproductive binding of cellulases on be overcome at higher temperatures ( > 220 °C) for its efficient hydro-
lignin can be avoided by addition of surfactants. Ionic surfactants may lysis. Hemicellulose hydrolysis products (mostly pentose and some
have an inhibitory effect and therefore, non-ionic surfactantsare more hexose) are degraded at higher temperatures; some of the degradation
suited for the purpose [19]. Surfactants like polyethylene glycol and products have an inhibitory effect on the downstream processes.

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Degradation of sugar monomers leads to an overall reduction in yield should be selected carefully as metal and metal ions catalyze the
of fermentable sugar. Continued degradation of pentose and hexose decomposition of glucose [149].
sugars produces several potential inhibitors like furfural, 5-hydroxy-
methylfurfural (HMF), acetic acid, levulinic acid, uronic acid, vanillic 4.3. Other hydrolysis methods
acid, formic acid, 4-hydroxybenzoic acid, phenol, vanillin, cinnamal-
dehyde, and formaldehyde [11,14]. Taherzadeh et al. based on their In order to overcome the limitations of enzymatic and homogenous
kinetic study of single stage dilute sulfuric acid hydrolysis (H2SO4 sulfuric acid hydrolysis of cellulose, several other hydrolysis methods
concentration of 0.5%, temperature between 188 and 234 °C, and have been investigated with varying degree of success. Solid acid
7 min retention time), reported more than 80% hydrolysis of hemi- catalysts such as metal oxides, H zeolites, functionalized silica,
cellulose mannan into mannose at temperatures lower than 200 °C immobilized ionic liquids, heteropoly acids, acid resins, carbonaceous
[143]. The maximum yield of glucose from cellulose was obtained at and magnetic acids have been analyzed for their cellulose hydrolysis
temperatures greater than 220 °C, but without any significant change effectiveness. Major advantages associated with solid acid catalysts
in the concentration of total fermentable sugar (glucose from cellulose include lower residence time, easy recovery, and reusability prospects
and mannose from hemicelluloses glucomannan) because at higher [150]. Huang and Fu reviewed the various types of solid acid catalysts
temperature breakdown of mannose sugar commenced. In order to that have been used for hydrolysis of cellulose [151].
avoid loss of sugar and formation of potential inhibitors, dilute acid Glycerol-based solid carbon was utilized as a catalyst for hydrolysis
hydrolysis can be carried out in two stages. In the first stage, relatively of alkali pretreated and untreated raw rice straw. Total reducing sugar
mild temperature facilitates depolymerization of hemicelluloses into its released for alkali treated biomass in 4 h (at 140 °C) was 262 mg/g
constituent pentose and hexose sugar by 80–95%, and depolymeriza- with 31% efficiency, while for untreated biomass it was 147 mg/g with
tion of cellulose by 40–60% of its initial concentration. This stage 20% efficiency. Although the yield was relatively low, the catalysts were
prevents any further degradation of monosaccharides obtained in the reusable and the process did not produce fermentation inhibitors
hydrolysates. The aqueous hydrolysate is then separated from the solid [152]. Papanikolaou et al. [153] did an excellent review on the use of
phase (remainder cellulose). In the next stage of dilute acid hydrolysis, ionic liquids in degradation of lignocellulosic biomass. Application of
higher temperature facilitates the degradation of cellulose into glucose acidic ionic liquids for cellulose dissolution and its hydrolysis was first
[144]. Two-stage dilute acid hydrolysis can help in segregation of reported by Papanikolaou and Aggelis [154], who further studied
pentose and hexose sugars as simultaneous fermentation of both C5 different ionic liquids for their cellulose hydrolysis effectiveness.
and C6 sugar can be problematic. Most of the ionic liquids studied for cellulose hydrolysis are based on
pyridine, imidazole, triethylamine and triethanolammonium. Acidic
4.2.2. Concentrated acid hydrolysis ionic liquids (SO3-H) based on Imidazoline were used for hydrolyzing
Concentrated acid hydrolysis employs higher concentration of acids cellulose by Zhuo et al. [117] Total reducing sugar yield upto 85.1% in
(30–70%), but unlike dilute acid hydrolysis operates at low tempera- 1 h at 100 °C was reported.
ture (close to 40 °C) [127]. H2SO4 .remains the most widely investi-
gated acid for concentrated acid hydrolysis. HCl has rarely been used 5. Biosynthesis of lipids by oleaginous yeast and bacteria
due to its negative environmental impacts [127]. Under favorable
conditions, high yield of sugars (≥90% of theoretical yield) can be Oleaginous microorganisms accumulate greater than 20% of their
obtained. High temperature and dilution of acid can make the process dry cell weight (DCW) as lipids [150]. Accumulated lipids are mostly
corrosive and therefore requires costly reactors made of special alloys triacylglycerols (triglycerides, or TAGs) that are traditionally used as
or non-metallic constructions like carbon-brick lining or ceramics. In feedstock for lipid-based biofuels. Significant quantities of lipid can be
contrast to dilute acid pretreatment, the residence time for concen- accumulated by oleaginous microorganisms using a variety of sub-
trated acid hydrolysis is long (2–6 h). Since high concentration of acid strates under controlled and manipulated culture conditions.
is involved, its recovery after the hydrolysis is critical in order to Oleaginous microorganisms are of great industrial and commercial
improve the overall economics of the process. Acid recovery is an interest because, when cultured, they can produce significant quantities
energy intensive process. Acids must be neutralized and the salts of lipid-based biofuels along with other valuable biochemicals. Lipid-
should be separated before the hydrolysates can be used for down- based biofuels like biodiesel and renewable diesel are derived from
stream processes. Neutralization by lime is usually performed but the TAGs. Lipid accumulation properties (“oleaginicity”) have been dis-
process generates large quantity of gypsum. Thus, commercial interest covered in some yeast, bacteria, algae, and fungi. The focus of this
for concentrated acid hydrolysis is hampered by the high investment, review paper, however, is limited to oleaginous yeast and bacteria as
operation, and maintenance cost [134,145,146]. Van Groenestijn et al. they have higher lipid production rates than algae [150] and because
[147] reported the “Biosufurol process” in which impregnation of some of the oleaginous yeast and bacteria can simultaneously utilize
biomass with H2SO4 (70%) and addition of water initiates the process hexose and xylose sugars and are resistant to toxic chemicals generated
of hydrolysis and acid recovery is performed by anionic membranes during pretreatment and/or hydrolysis of lignocellulosic biomass.
and by anaerobic treatment of wastewater followed by recovery in the Furthermore, the scale-up of algal cultures for commercial operation
form of H2S to be cost effective for ethanol production. Substrate can be problematic.
properties such as particle size [143,148], its acid neutralizing capacity, When oleaginous organisms are cultured on hydrophobic sub-
degree of polymerization, linkage of cellulose with other constituent of strates (ex novo lipid accumulation) the accumulated lipid contains
the lignocellulosic biomass, proportion of easy to digest hemicelluloses lower quantity of TAGs relative to oleaginous organisms cultured on
and cellulose, and rate of hydrolysis of hydrolysis-resisting components sugar-based (hydrophilic) substrates (de novo lipid accumulation)
affect the acid hydrolysis process [127]. The overall acidity of the [151–153]. Therefore, lignocellulosic hydrolysates rich in C5 and C6
system is determined by the type of acid used, its concentration, sugar can be used to facilitate de novo lipid accumulation with higher
amount of acidic solution, and concentration of acids like acetic acid TAG content in oleaginous microorganisms. When hydrophobic sub-
released during hydrolysis, which affects the efficiency of the process strates are utilized by oleaginous microorganism, lipid accumulation is
and yield obtained. mostly a process associated with growth and remains unaffected by
The reversion of glucose to its dimers, anhydrosugars, and oligo- induced nitrogen limitation. However, substantial quantities of lipid
mers becomes important when the glucose concentration in the accumulation by oleaginous microorganisms have been reported when
hydrolysates exceeds 10%. The reverted forms of glucose remain using hydrophilic substrate under nitrogen-starved culture conditions
unavailable for fermentation [144]. The hydrolysis reactor material (high C/N ratio) [154]. Although a hyperactive system for fatty acid

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D. Kumar et al. Renewable and Sustainable Energy Reviews 73 (2017) 654–671

synthesis in oleaginous microorganisms is absent, they are capable of biomass-based sugar substrate an attractive process [6]. Several
producing significant quantities of acetyl-CoA (Fig. 1) which is the researchers have studied the effectiveness of oleaginous yeasts to
ultimate precursor for synthesis of fatty acids [155,156]. Exhaustion of utilize ligocellulosic hydrolysates for lipid accumulation. Huang et al.
some nutrients (particularly nitrogen) from the growth media causes [175] studied the effectiveness of Trichosporon fermentans for utiliz-
intracellular concentration of various metabolites to change [156– ing the detoxified hydrolysates of rice straw and reported a lipid
160]. Under nitrogen-starved conditions the activity of AMP-deami- content of 40% (DCW) and a cell density of 28.6 g L-1. Yu et al. [6]
nase cleaves the adenosine monophosphate (AMP) in inosine mono- examined the effectiveness of five oleaginous strains of yeast
phosphate (IMP) and ammonium ions (NH4+), leading to a rapid (Cryptococcus curvatus, Rhodosporidium toruloides, Rhodotorula
decline in the concentration of intracellular AMP. When nitrogen is glutinis, Yarrowia lipolytica, and Lipomyces starkeyi) at utilizing
deficient the ammonium ions produced by cleavage of AMP serves as a detoxified/non-detoxified dilute sulfuric acid pretreated wheat straw
complimentary source of nitrogen [161]. The activation of enzyme hydrolysates for lipid accumulation. All five species effectively utilized
NAD+-and NADP+- dependent isocitrate dehydrogenase responsible the detoxified hydrolysate for lipid accumulation, while all but
for transformation of isocitric acid to α-ketoglutaric acid is dependent Rhodosporidium toruloides also utilized the non-detoxified hydroly-
on intracellular concentration of AMP. Drastic reduction in AMP sate. Cryptococcus curvatus was most effective in accumulating lipid
concentration makes isocitrate dehydrogenase ineffective [162–165]. with lipid content of 33.5% and 27.1% in detoxified and non-detoxified
The concentration of isocitric acid within the mitochondria remains in hydrolysate, respectively. Pentose and hexose sugars were simulta-
equilibrium with citrate. After reaching a critical value, citrate is neously utilized and the fatty acid profile analysis revealed that oleic
transported to the cytoplasm in exchange of malate. The enzyme (C18:1), linoleic (C18:2), stearic (C18:0), and palmitic acid (C16:0)
ATP-citrate lyase (ATP-CL) cleaves citric acid into the fatty acid were predominantly present.
precursor acetyl-CoA and oxaloacetate [161,164,166]. The enzymatic Lipomyces starkeyi is an oleaginous yeast that can accumulate
complex ATP-CL is probably one of the most important factors that more than 70% of its biomass as intracellular lipids using a mixture of
accounts for the oleaginicity of various oleaginous microorganisms. Its hexose and penose sugar under controlled culture conditions
presence is only limited to oleaginous microorganisms, and when [32,176,177]. Lipid accumulation of 48.02% was reported when
cultured under similar conditions (nutrient starvation) non-oleaginous Yarrowia lipolytica was grown on hydrolysates of defatted rice bran
microorganisms transform the assimilated carbon in the form of [178]. Several species of oleaginous yeast are capable of utilizing
various polysaccharides such as glycogen, mannans, and glucans glucose and xylose (or other pentoses depending on the feedstock).
[160]. Fatty acids are produced from acetyl-CoA via a quasi-inverted Both have been identified for efficient utilization of lignocellulosic
β-oxidation process [157,159,167]. hydrolysates as simultaneous utilization of pentose and hexose sugar is
Fatty acid (FA) biosynthesis is performed by the action of enzymatic necessary for improving the overall economics of the process because
complex fatty acid synthetase (FAS), which facilitates the addition of degradation of hemicelluloses produces significant amounts of pentose
acetyl-CoA and malonyl-CoA to the growing FA chain and requires a sugars. Hu et al. [4] investigated the lipid accumulation by
pool of NADPH. Malase enzyme (ME) acts as the sole source of Trichosporon cutaneum when using both glucose and xylose as
NADPH for FAS and the extent of lipid accumulation in oleaginous substrate (2:1). They reported that as much as 59% of the total DCW
microorganisms correlates closely with the activity of ME unlike any of Trichosporon cutaneum consisted of lipids, and the obtained lipid
other enzyme [168]. FA synthesized by FAS usually contains 14–16 coefficient was up to 0.17 g g-1 of substrate sugar. Cell density of
carbon atoms in their framework and, based on the enzymatic arsenal 153 g l-1 and lipid content of up to 54% (DCW) in 140 h were reported
of individual organisms, further elongation and/or desaturation of the for Lipomyces starkeyi grown in fed-batch cultures [176].
chain can take place. Acyltransferase catalyzes the esterification of FAs Aqueous extract of Cassia fistula fruit pulp was used as the only
onto the glycerol backbone to produce storage lipid TAGs. growth substrate for culturing Rhodosporidium kratochvilovae
HIMPA1 and lipid content of 53.18% was achieved and fatty acids
6. Lipid accumulation by oleaginous yeast using largely consisted of palmitic acid (43.06%), stearic acid (28.74%), and
lignocellulosic hydrolysates oleic acid (17.34%). Although high content of saturated acids (72.58%)
was reported, its blending with unsaturated fatty acids derived from
Yeasts are unicellular, eukaryotic chemo-organotrophs with over other sources can be carried out for balancing a fatty acid profile
1600 described species classified in the Kingdom Fungi. Oleaginous required for producing biodiesel with better fuel properties [179].
(lipid content > 20% DCW) yeasts such as Trichosporon cutaneum, Cultures of Cryptococcus sp. SM5S05 grown on corncob hydro-
Rhodotorula glutinis, Rhodotorula rubra, Rhodosporidium toruloides, lysates containing glucose at 60 g l-1 led to lipid content, lipid yield, and
Cryptococcus albidum, and Lipomyces starkeyi have been extensively dry biomass of 60.2%, 7.6 g l-1, and 12.6 g l-1, respectively. The fatty
investigated for lipid accumulation [169]. Rhodosporidium sp., acid profile of oil was found to be similar to that obtained from
Lipomyces sp., and Rhodotorula sp. have high lipid accumulation conventional vegetable oil [180].
capacity (up to 70%) mostly in the form of TAGs, and some of these Fei et al. compared different fed batch cultivation modes for lipid
species can metabolize pentoses based on hemicellulose [7,170,171]. accumulation by R. toruloides grown on corn stover hydrolysate. They
Under conditions of nitrogen exhaustion, the flow of carbon in reported online sugar control to be the best mode as it led to the
oleaginous microorganisms is transferred towards accumulation of maximum lipid content (59 g l-1DCW) [181].
citric acid which is later transformed to acetyl-CoA, a precursor for Comprehensive screening of 1189 strains of oleaginous yeast was
fatty acid synthesis. When the availability of nitrogen is restricted, the carried out for their ability to simultaneously utilize a sugar mix
synthesis and accumulation of lipids continues unabated but the (glucose, xylose and L-arabinose). Out of 1189 strains, 12 were capable
synthesis of proteins and nucleic acids is curtailed and hence the of co-fermenting all of the sugars. The highest sugar utilization rate
concentration of lipids builds up [172,173]. (94.1%) was reported for Pseudozyma hubeiensis IPM1-10 [182].
Conversely, in a recent investigation C. curvatus ATCC 20509 was Based on 2008 figures, cost per ton for oil production from yeast
able to utilize acetate (generally considered as an inhibitor) and was reported to be around US$3000 (without the feedstock cost),
accumulated 73.4% of its total DCW as lipids under high availability which was about twice as high as petroleum [183]. However, the cost is
of nitrogen [174]. likely to come down to more reasonable levels due to technological
Ability of some of the yeasts to also utilize pentose sugar besides innovations. Huang et al. reported the cost of oil production in China to
hexose, and relatively higher energy density of lipids than ethanol, be around US$1230 per ton (including the feedstock cost) [184].
makes the lipid accumulation by oleaginous yeast using lignocellulosic The recorded lipid accumulation by several oleaginous yeasts

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utilizing lignocelluloses hydrolysates and representative sugars present

Reference in lignocellulosic biomass (glucose and xylose) under different condi-

[175]

[178]
[180]

[182]
[182]
[179]
[179]
[179]
tions are presented in Table 3.

[6]
[6]
[6]
[6]
[6]
Chen et al. [185] conducted a screening study to determine the

No external Nitrogen
impact of seven lignocellulosic degradation products (three organic
acids: acetic acid, formic acid, and levulinic acid; two furan com-
N- Limitation

pounds: furfural and 5-hydroxymethyl furfural; and two phenol

derivatives: vanillin and hydroxybenzaldehyde) on a few selected
species of oleaginous yeast and reported Trichosporon cutaneum
N
N
N
N
N
N
Y

Y
Y
Y
Y
Y
2.1374 to be the most tolerant yeast. Individually, acetic acid (from
acetyl group), formic acid and furfural (from xylose degradation), and
Lipid content (%)

vanillin (from lignin degradation) were reported to have strong

inhibitory effects while levulinic acid (from glucose oxidation), 5-
48.02%

HMF (from degradation of glucose) and 4-hydroxybenzaldehyde (from

44.55
38.94
32.00
32.30
33.5
27.1

29.1
31.2

lignin degradation) exhibited relatively weak inhibitory effect. The

40

59

45
–

combined effect of the full spectrum of inhibitory compounds produced

by steam pretreatment followed by enzymatic hydrolysis of corn stover
on Trichosporon cutaneum was found to be more pronounced than the
individual effects of the seven tested compounds. Some of the studies
Lipid Coefficient (g g-1

have shown that when both glucose and xylose are present in the
growth media, the utilization of sugar occurs sequentially and limited
utilization of xylose occurs until most of the glucose is exhausted. Hsiao
Recorded lipid accumulation by several oleaginous yeasts utilizing lignocelluloses hydrolysates and other representative sugars present in lignocellulosic biomass.

substrate)

et al. [186] used a mixture of xylose, glucose, xylulose, and xylitol as

0.0634

substrate for Rhodotorula toruloides and reported that the first sugar
0.194

0.115
0.095
0.145

0.119
0.19

0.17

to be utilized was glucose. Similarly, other oleaginous yeasts such as

–

–
–
–

Trichosporon fermentans [158], Lipomyces starkeyi [170], and

Acetic acid, furfural, HMF

Acetic acid, furfural, HMF

Acetic acid, furfural, HMF

Rhodotorula glutinis [187] were able to utilize both glucose and xylose,
Inhibitory compounds

but diauxic growth was observed and utilization of sugar occurred

sequentially (glucose first). Sequential utilization of pentose and hexose
can significantly increase the time required for effective lipid accumu-
Detoxified

Detoxified
detoxified
detoxified

lation. However, when Hu et al. [4] used a combination of glucose and

xylose (2:1) as lipid fermentation substrate for Trichosporon cuta-
–

–
–
–
–
–

neum, the intracellular lipid content and lipid coefficient were reported
to be 59% and 0.17 g g-1 sugar with absence of diauxic growth pattern.
This occurred because both sugars were utilized simultaneously and
individual substrate utilization was reported to be roughly proportional
to the concentration of sugars individually. When corn stover hydoly-
sate (after dilute acid pretreatment and enzymatic hydrolysis of corn
stover biomass) was used as substrate, the pentose and hexose sugars
Steam explosion pretreatment and

present in the hydolysates were utilized by T. cutaneum without any

preference and lipid content of 39.2% and lipid coefficient of 0.15 g g-1
Pretreatment and hydrolysis

Dilute H2SO4 pretreatment

Dilute H2SO4 pretreatment

Dilute H2SO4 pretreatment

Dilute H2SO4pretreatment

were achieved [4]. Joshua et al. [5] also reported the non-preference
behavior for Sulfolobus acidocaldarius while utilizing a pentose and
enzymatichydrolysis

hexose sugar mix. Slininger et al., in their comparative study, reported

Acid Hydrolysis

Acid hydrolysis

L. kononenkoae NRRL Y-7042, L. tetrasporus NRRL Y-11562, and R.

toruloides NRRL Y-1091 strains to be tolerant to toxic by-products
generated during AFEX and dilute acid pretreatment of corn stover and
switchgrass respectively. These strains were able to accumulate un-
–

–
–
–
–
–

precedented lipid titres at 39–45% of the theoretical values on

Defatted rice bran hydrolysate

lignocellulosic hydrolysates [188].

wheat straw hydrolysates
wheat straw hydrolysates

Wheat straw hydrolysate

Wheat straw hydrolysate

The activity of cellulases is known to be inhibited by glucose and

glucose and xylose (2:1)
rice straw hydrolysates

Corn cob hydrolysate

thus it must be removed from the hydrolysis reactors in order to

Glucose and xylose

achieve efficient hydrolysis of lignocellulosic biomass with minimum

use of costly enzymes. Simultaneous saccharification and lipid fermen-
tation (SSF) is an effective method that can overcome the impact of
Substrate

glucose

product inhibition on enzymatic hydrolysis. Liu et al. [189] reported

xylose
xylose
xylose

SSF to be more effective for lipid accumulation than separate hydro-

lysis and lipid fermentation steps (SHF) with partial cellulase recycling
Rhodosporidium toruloides
Trichosporon fermentans

capacity.
Trichosporon cutaneum
Cryptococcus curvatus
Cryptococcus curvatus

The recalcitrance of lignocelluloses is mostly attributed to lignin

Rhodotorula glutinis
Rhodotorula glutinis

Rhodotorula glutinis
Yarrowia lipolytica
Lipomyces starkeyi
Lipomyces starkeyi

Candida lipolytica

and its linkages with carbohydrates. Lignin constitutes a significant

Rhodotorula sp.

proportion of lignocellulose, and in order to utilize lignocellulosic

biomass efficiently for biofuel production, the identification and
utilization of lignin to biofuels routes must be developed. Natural
Species
Table 3

degradation of lignin is limited to fungi, but some species of bacteria

can also degrade compounds related to lignin using the β-ketoadipate

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D. Kumar et al. Renewable and Sustainable Energy Reviews 73 (2017) 654–671

pathway. Studies on utilization of lignin by oleaginous organisms hold Maximum lipid content reported was 93% and 96% for R. opacus
much promise for achieving overall efficiency and sustainable utiliza- and Gordonia sp. respectively when they were grown on sugarcane
tion of lignocellulosic biomass for biofuel production. Biotechnology molasses. Maximum TAGs content of 57.8 mg l-1 and 88.9 mg l-1 were
and genetic engineering-based alteration of lignin content, its consti- obtained for Gordonia sp. and R. opacus, respectively when grown on
tuent structures, identification, and development of lignin to biofuel orange and carob waste [195].
routes under well-defined conditions are important future study areas Bacterial species have been reported to be capable of digesting
for efficient and sustainable utilization of lignocellulosic biomass. aromatic compounds (similar to lignin) aerobically through a β-
Screening of high lipid producing and robust organisms capable of ketoadipate pathway. Various species of Rhodococcus are oleaginous
growing under a wide range of environmental conditions and on with lipid accumulation of more than 20%. Rhodococcus opacus DSM
several types of carbon substrates in combination with strategies for 1069 and PD 630 strains have been shown to convert lignin model
the strategic manipulation of TAG production pathways in oleaginous compounds (4-hydroxybenzoic and vanillic acid) to triacylglycerols.
organisms along with implementation of developed practices of meta- The strain DSM 1069 of Rhodococcus opacus showed no dependence
bolic engineering has tremendous potential to revolutionize biofuel on source of nitrogen. The growth of both strains showed a positive
production from microorganisms. Overexpression of certain genes in influence with increase in pH. The strain PD 630 accumulated a high
model yeast (Saccharomyces cerevisiae) has been performed for concentration of unsaturated fatty acid (~50%) that is considered to be
studying their effect on biosynthesis of fatty acids and TAGs. advantageous for biodiesel if good, low temperature properties are
According to Beopoulos et al., overexpression of three FA biosynthesis desired [196]. A bacterium, Bacillus thuringiensis, has been reported
genes (FAS1, FAS2, and ACC1) led to an increase of about 17% in to be suitable for bioconversion of crude glycerol to polyhydroxyalk-
cellular lipid content in S. cerevisiae. They further reported increase an anoate under non-limiting nitrogen conditions. The bacterium, EGU45,
in lipid production when native promoters of FA synthesis genes were was reported to produce1.5–3.5 g polyhydroxyalkanoate L-1 without
replaced with strong constitutive promoter genes (e.g. TEF1). Deletion any acclimatization. PHA was produced by the bacterium at the rate of
of the GUT2 gene (coding for glycerol-3-phosphate dehydrogenase, 1.54–1.83 g L-1 [197].
serving as the major link between lipid and carbohydrate metabolism) The consortium of Streptomyces coelicolor with Ralstonia eutro-
in Y. lipolytica translated to a threefold enhanced lipid induction [190]. pha has been studied and found to produce 114 mg L-1 fatty acids
Deletion of GUT2 gene prevented the transformation of G3P (glycerol- [198]. The bacteria contained medium, long chain, and very long chain
3-phosphate) to DHAP (dihydrogen acetone phosphate), which led to fatty acids. The fuel properties of the biodiesel obtained was reported to
enhanced accumulation of neutral lipids. Glycerol in the form of possess good characteristics in terms of cetane number (65) and
glycerol-3-phosphate is required for esterification of FAs onto the oxidation stability (76 h).
glycerol backbone for the production of TAGs. Over expression of the De novo and ex novo lipid accumulation by oleaginous organisms
GPD1 gene (coding glycerol-3-phosphate dehydrogenese1, catalyzing takes place when grown on hydrophilic and hydrophobic substrates,
the transformation of DHAP to G3P) led to a fourfold enhancement in respectively. Ex novo lipid accumulation by oleaginous organisms occurs
lipid accumulation and when combined with deletion β-oxidation when grown on hydrophobic substrates such as fatty acids, TAGs, and
encoding genes, yeasts were capable of accumulating > 80% of their other fatty esters, but microbial oils produced usually contained lower
mass as lipids were obtained [191]. levels TAGs than those obtained from de novo lipid accumulation. When
The last step in TAG biosynthesis (conversion of fatty acyl CoA and compared to ex novo lipid accumulation, de novo lipid accumulation can
diacylglycerol to TAG) is catalyzed by diacylglycerol acyltransferase utilize a variety of simple sugars (such as glucose, fructose, xylose, lactose,
(DGAT) and introduction of Arabidopsis DGAT in yeast led to a 3–9 and sucrose) as well as complex sugars (polysaccharides); these sugars can
fold increase in accumulation of TAG. Other FA and TAG biosynthesis be derived from various types of lignocellulosic biomass. First generation
genes manipulated for enhanced lipid accumulation include genes feedstocks based on edible crops can only gain acceptance in countries
encoding diacylglycerol acyltransferase, ATP-citrate lyase, d9-desatur- that have excess produce and are considered unsustainable in the long
ase, and acetyl-CoA carboxylase [192]. run. Second-generation feedstocks, on the other hand, include either non-
edible lignocellulosic biomass or non-edible plant parts of edibles crops
6.1. Lipid accumulation by bacteria using lignocellulosic such as corn stover. With an annual cellulose and hemicellulose produc-
hydrolysates tion of over 85×109 tons, lignocellulosic biomass appears to be the most
attractive and abundant feedstock for bioenergy production [199].
Bacteria and other heterotrophic microorganisms (yeast, fungi, and Monomeric sugars for culturing oleaginous organisms can be obtained
heterotrophically grown algae) can play an important role in produc- via pretreatment and hydrolysis of lignocellulosic biomass. C/N ratio of
tion of biodiesel. Fatty acid biosynthesis in bacteria is accomplished by the feed biomass should be carefully monitored as it plays an important
acetyl-CoA, which then acts as a substrate for KSa (β-ketoacyl-ACP role during de novo lipid accumulation. Several types of agro-industrial
synthase) [193]. Gram positive and gram negative species of bacteria wastes such as wheat bran, wheat straw, rice husk, rice straw, corn cob,
use Acetyl-ACP in different ways. The gram positive bacteria use PlsX corn stalk, tomato waste, pear pomace, pitch pine [200], saw dust,
from acyl-CoA to form acyl-phosphate. This is then transferred to grinding dust, bark, and cutter chips have been utilized for microbial
glycerol-3-phosphate (G3P) through PlsY. The gram negative species of lipid production. Prior to their use as growth substrate, these feedstocks
bacteria instead use PlsB acyltransferase from acyl-CoA to load an acyl have been subjected to different pretreatment processes. Biomass with
group on G3P. low lignin content such as herbs and softwood are inherently more
Compared to fungi, yeast, plants, and animals, lipids of bacteria desirable as their carbohydrates are relatively easily amenable to hydro-
have been studied less extensively. Members of certain genera of lysis. Biomass composition is a critical factor as it affects several
bacteria such as Nocardia, Streptomyces, Mycobacterium, and operations along the biomass supply chain. In order to ensure year-round
Rhodococcus have been studied for biosynthesis of TAGs. Many of operation, availing biomass from different sources is just as important as
the bacterial species belonging to these genera have shown oleaginicity, ensuring sufficient supply of feedstock for biofuel production. Dedicated
but they produce neutral lipids in variable amounts when grown on bioenergy plantations such as short rotation coppice (poplar, willow, etc.)
different carbon sources [194]. Several agro-industrial waste materials and perennial grasses (Miscanthus, switchgrass, etc.) are increasingly
such as wheat bran, vegetable and fruit wastes, potato infusion, barley gaining popularity for bioethanol production. These plants are fast
seeds, and molasses were used as carbon source for culturing Gordonia growing and produce substantial amount of their biomass as carbohy-
sp. and Rhodococcus opacus PD630. Both were able to accumulate > drates and thus can potentially be utilized as feedstock for microbial oil
50% of their biomass as lipids for most biomass types studied. production. An ideal biofuel feedstock plant should have the following

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D. Kumar et al. Renewable and Sustainable Energy Reviews 73 (2017) 654–671

characteristics: high rate of growth, low concentration of lignin, low performance of renewable diesel in required. In terms of emissions of
degree of cellulose crystallinity, low degree of cellulose polymerization, particulate matter and carbon monoxide, biodiesel performs better
balanced amount of cellulose and hemicellulose, amenability to easy while when it comes to emission of NOx and hydrocarbons, renewable
hydrolysis of polymeric carbohydrates to fermentable sugars, and non- diesel is comparatively cleaner than biodiesel [207].
competitive for human consumption.

7. Biodiesel and renewable diesel 8. Conclusion

Biodiesel is derived from TAG-based feedstocks such as vegetable oil, Cost of the feedstock is considered the single largest limiting factors
animal fat and microbial oil/single cell oil. The feedstock is subjected to in commercialization of biofuels. Lignocellulosic biomass can serve as
transesterification reaction involving a monohydric alcohol (often an abundant and cheap feedstock for the production of biofuels. In
methanol) and is usually a catalyst-driven process. Three moles of fatty addition to the fast growing dedicated bioenergy plants, several types of
acid alkyl esters (biodiesel) and one mole of glycerol are produced per agroindustrial wastes can also be utilized for biofuel production and it
mole of TAG. Viscosity of biodiesel is substantially lower than the can also ensure year round availability of feedstock for continuous
feedstock material (TAG) and is comparable to that of mineral diesel. operation of the biomass conversion/production facility.
Comparable viscosity and similar fuel properties facilitates use of Lignocellulosic biomass rich in holocellulose sugars can facilitate
biodiesel as mineral diesel substitute/supplement. Biodiesel is consid- heterotrophic growth of oleaginous organisms. However, recalcitrance
ered to be a safe and renewable fuel that can be used in existing inherent in lignocellulosic biomass renders its simple hydrolysis
compression ignition engines. It is cleaner than conventional mineral ineffective, and biomass needs to be pretreated prior to its hydrolysis
diesel and hence safeguards the environment and maintains its serenity. for yielding simple fermentable sugars by physical, chemical, and
However, the limited availability of feedstock is a major constraint in its biological means or by using a combination of pretreatment strategies.
commercialization and usage in transportation vehicles. This has led to Several approaches of pretreatment and hydrolysis have been reported
exploration of various non-edible plants and microalgae as feedstock for in the literature and the effectiveness of individual methods or
extraction of oil and their subsequent conversion to biodiesel. combinations depends on a multitude of factors. Complete hydrolysis
Microalgae have been extensively studied for single cell oil production of holocellulose and minimization of inhibitory compounds formed are
as well as for the remediation of nutrients such as nitrogen and critical for efficient utilization of lignocellulosic biomass for single cell
phosphorus from contaminated streams [201] and a variety of products oil production. Biosynthesis of lipids by oleaginous organisms appears
can be obtained from algal biomass after subjecting it to appropriate to be an attractive pathway for large scale production of biofuels.
treatment techniques [202]. The acceptability and marketability of Lignocellulosic hydrolysates can facilitate de novo lipid accumulation
biodiesel is hampered by the high production cost. As much as 75– in oleaginous yeasts and bacteria. Disproportionately large levels of
85% of the overall production cost of biodiesel is attributed to the cost of lipid accumulation in certain yeasts have been reported when grown on
feedstock. Apart from edible/non-edible vegetable oils, animal fats and lignocellulosic hydrolysates under limited availability of nitrogen in the
recycled cooking oil, single cell oils derived from oleaginous micro- culture media as lipid accumulation occurs at the expense of proteins
organisms such as microalgae, yeast and bacteria are increasingly and nucleic acids. Unprecedented high levels (39–45% of theoretical
attracting attention as potential biodiesel feedstock. Lignocellulosic yield) of lipid titres have been reported recently for a few strains of
biomass hydrolysate consists of pentose and hexose sugars which can oleaginous yeasts grown on non-detoxified lignocellulosic hydrolysates.
be utilized by oleaginous organisms for accumulation of TAGs. A Additionally, some recent studies have reported the utility of acetate
detailed review on biodiesel production using hydrolysates of various (generally considered as an inhibitor) as growth substrate for oleagi-
non-edible lignocellulosic biomass has been reported by Patel et al. [203] nous yeast. Traits such as tolerance to inhibitory compounds produced
Glycerol as a by-product of TAG transesterification to biodiesel has also during biomass pretreatment and/or hydrolysis, ability to uptake
been shown to have excellent growth substrate characteristics for lignin-based compounds, and ability to simultaneously uptake pentose
production of SCO. As the lignocellulosic biomass is abundant; its and hexose sugars are invaluable for biofuel production using oleagi-
conversion to biofuels (eg, biodiesel and renewable diesel) has the nous yeast and bacteria. Genetic regulation for lipid accumulation in
potential to revolutionize the transportation fuel industry. An excellent oleaginous organisms has been examined and several genetic engineer-
review on the prospects and challenges of biodiesel derived from ing based modifications have been realized with promising results.
lignocellulosic biomass has been described by Yousuf [204]. These studies have focused on selective insertion/deletion and/or
Renewable diesel is the terminology used for ‘diesel-like alkanes’ overexpression of genes of interest. Genetic engineering appears to
that is produced from vegetable oil. It is often interchangeably referred be a fairly attractive opportunity for maximizing the production of
to as green diesel, second generation biodiesel, or hydrotreated lipids and consequently can also improve the overall sustainability and
vegetable oil (HVO). According to Knothe, the term renewable diesel economic feasibility of biofuel production. Single cell oils have been
appears to be the most appropriate term for diesel-like alkanes derived examined extensively for the production of biofuels (particularly
from hydrodeoxygenation of renewable sources such as vegetable oil, biodiesel). Biodiesel (fatty acid methyl ester) produced via transester-
animal fat, SCO, or even biodiesel [205]. Hydrodeoxygenation is one ification of single cell oil has a fatty acid profile similar to that of
among the five technical routes for lowering the viscosity of TAGs. The traditional biodiesel feedstock (vegetable oil) and can be used in
other four include: pyrolysis, microemulsion, blending, and transester- existing diesel engines with little or no modification. Renewable diesel
ification. Hydrodeoxygenation is carried out under high temperature produced by hydrotreatment of single cell oil can also be used as a
and elevated pressure in the presence of H2 and involves a catalyst. drop-in fuel. Biodiesel and renewable diesel thus have a promising
Double bonds and ester moieties (OR) are replaced in the process with future as alternatives to fossil diesel.
hydrogen which leads to alkane like end products. NiMo/γ-Al2O3,
CoMo/γ-Al2O3, CoMo/C, Rh-Al2O3, CoMo/Si, Pt/C, Pd/SiO2, Pd/C,
and Ni/SiO2have been used as hydrodeoxygenation catalysts. Acknowledgements
Renewable diesel has better oxidative stability and cold flow properties
than biodiesel and can be produced and distributed using existing Dipesh Kumar is thankful to University Grants Commission
petroleum refining and distribution infrastructure [206]. In spite of (UGC), India for his Senior Research Fellowship. John Korstad
these advantages, more research based on life cycle assessment based wishes to recognize Joe Moore and Bob Warwick for their faithful
approaches to ascertain the economic, energetic, and environmental support of biofuel rememediation research in Tulsa, Oklahoma (USA).

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D. Kumar et al. Renewable and Sustainable Energy Reviews 73 (2017) 654–671

References hot compressed liquid water. Ind Eng Chem Res 1992;31:1157–61.
[30] Brownell HH, Yu EK, Saddler JN. Steam-explosion pretreatment of wood: effect of
chip size, acid, moisture content and pressure drop. Biotechnol Bioeng
[1] Lin H, Cheng W, Ding HT, Chen XJ, Zhou QF, Zhao YH. Direct microbial 1986;28:792–801.
conversion of wheat straw into lipid by a cellulolytic fungus of Aspergillus oryzae [31] Grous WR, Converse AO, Grethlein HE. Effect of steam explosion pretreatment on
A-4 in solid-state fermentation. Bioresour Technol 2010;101:7556–62. pore size and enzymatic hydrolysis of poplar. Enzym Microbial Technol
[2] Galbe M, Zacchi G. A review of the production of ethanol from softwood. Appl 1986;8:274–80.
Microbiol Biotechnol 2002;59:618–28. [32] Li J, Henriksson G, Gellerstedt G. Lignin depolymerization/repolymerization and
[3] Ratledge C. Fatty acid biosynthesis in microorganisms being used for single cell oil its critical role for delignification of aspen wood by steam explosion. Bioresour
production. Biochimie 2004;86:807–15. Technol 2007;98:3061–8.
[4] Hu C, Wu S, Wang Q, Jin G, Shen H, Zhao ZK. Simultaneous utilization of glucose [33] Kabel MA, Bos G, Zeevalking J, Voragen AGJ, Schols HA. Effect of pretreatment
and xylose for lipid production by Trichosporon cutaneum. Biotechnol Biofuels severity on xylan solubility and enzymatic breakdown of the remaining cellulose
2011;4(1):25. from wheat straw. Bioresour Technol 2007;98:2034–42.
[5] Joshua CJ, Dahl R, Benke PI, Keasling JD. Absence of diauxie during simultaneous [34] Jönsson LJ, Martín C. Pretreatment of lignocellulose: formation of inhibitory by-
utilization of glucose and xylose by Sulfolobus acidocaldarius. J Bacteriol products and strategies for minimizing their effects. Bioresour Technol
2011;193(6):1293–301. 2016;199:103–12.
[6] Yu X, Zheng Y, Dorgan KM, Chen S. Oil production by oleaginous yeasts using the [35] Duff SJB, Murray WD. Bioconversion of forest products industry waste cellulosics
hydrolysate from pretreatment of wheat straw with dilute sulfuric acid. Bioresour to fuel ethanol: a review. Bioresour Technol 1996;55:1–33.
Technol 2011;102:6134–40. [36] Wright JD. Ethanol from biomass by enzymatic hydrolysis. Chem Eng Prog
[7] PAEP Meesters, Huijberts , Eggink GNM, High-cell-density G. cultivation of the 1998;84:62–74.
lipid accumulating yeast Cryptococcus curvatus using glycerol as a carbon source. [37] Tengborg C, Stenberg K, Galbe M, Zacchi G, Larsson S, Palmqvist E, et al.
Appl Microbiol Biotechnol 1996;45:575–9. Comparison of SO2 and H2SO4 impregnation of softwood prior to steam
[8] Liang Y, Siddaramu T, Yesuf J, Sarkany N. Fermentable sugar release from pretreatment on ethanol production. In: Finkelstein M, Davison BH, editors.
Jatropha seed cakes following lime pretreatment and enzymatic hydrolysis. Proceedings of the ninet. symp. biotechnol. fuels chem. Held May 4–8. 1997,
Bioresour Technol 2010;101(16):6417–24. Color. Springs, Color., Totowa, NJ: Humana Press; 1998, p. 3–15.
[9] Pinzi S, Leiva D, Arzamendi G, Gandia LM, Dorado MP. Multiple response [38] Ballesteros I, Negro MJ, Oliva JM, Cabañas A, Manzanares P, Ballesteros M.
optimization of vegetable oils fatty acid composition to improve biodiesel physical Ethanol production from steam-explosion pretreated wheat straw. In: McMillan
properties. Bioresour Technol 2011;102:7280–8. JD, Adney WS, Mielenz JR, Klasson KT, editors. Proceedings of the twenty-
[10] Kosa M, Ragauskas AJ. Bioconversion of lignin model compounds with oleaginous seventh symp. biotechnol. fuels chem. Totowa, NJ: Humana Press; 2006, p. 496–
Rhodococci. Appl Microbiol Biotechnol 2012;93:891–900. 508.
[11] Taherzadeh MJ. Ethanol from lignocellulose: physiological effects of inhibitors [39] Allen SG, Kam LC, Zemann AJ, Antal MJ. Fractionation of sugar cane with hot,
and fermentation strategies. Goteborg, Sweden: Chemical Reaction Engineering, compressed, liquid water. Ind Eng Chem Res 1996;35:2709–15.
Chalmers Univesity of Technology; 1999. [40] Bouchard J, Nguyen TS, Chornet E, Overend RP. Analytical methodology for
[12] Kuhad RC, Singh A, Eriksson K-EL. Microorganisms and enzymes involved in the biomass pretreatment. Part 2: characterization of the filtrates and cumulative
degradation of plant fiber cell walls. In: Eriksson K-EL, Babel W, Blanch HW, product distribution as a function of treatment severity. Bioresour Technol
Cooney CL, Enfors S-O, Eriksson K-EL, editors. Biotechnol. Pulp Pap. Ind.. Berlin, 1991;36:121–31.
Heidelberg: Springer Berlin Heidelberg; 1997. p. 45–125. [41] Bobleter O, Binder H, Concin R, Burtscher E. The conversion of biomass to fuel
[13] Kumar P, Barrett DM, Delwiche MJ, Stroeve P. Methods for pretreatment of raw material by hydrothermal pretreatment. In: Palz W, Chartier P, Hall DO,
lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind Eng editors. Energy from biomass. London: Applied Science Publishers; 1981. p.
Chem Res 2009;48(8):3713–29. 554–62.
[14] Larsson S, Alexis Quintana S, Anders Reimann R, Nils-Olof N, Leif J. Influence of [42] Weil J, Sarikaya A, Rau S-L, Goetz J, Ladisch CM, Brewer M, et al. Pretreatment of
lignocellulose-derived aromatic compounds on oxygen-limited growth and etha- yellow poplar sawdust by pressure cooking in water. Appl Biochem Biotechnol
nolic fermentation by Saccharomyces cerevisiae. In: Proceedings of the twenty- 1997;68:21–40.
first symposium on biotechnology for fuels and chemicals, Humana Press; 2000. p. [43] Pérez JA, González A, Oliva JM, Ballesteros I, Manzanares P. Effect of process
617–32 variables on liquid hot water pretreatment of wheat straw for bioconversion to
[15] Agbor VB, Cicek N, Sparling R, Berlin A, Levin DB. Biomass pretreatment: fuel-ethanol in a batch reactor. J Chem Technol Biotechnol 2007;82:929–38.
fundamentals toward application. Biotechnol Adv 2011;29(6):675–85. [44] Kim SM, Dien BS, Tumbleson ME, Rausch KD, Singh V. Improvement of sugar
[16] McMillan JD. Pretreatment of lignocellulosic biomass. enzym. convers. biomass yields from corn stover using sequential hot water pretreatment and disk milling.
fuels prod., vol. 566, American Chemical Society; 1994. p. 15–292. Bioresour Technol 2016;216:706–13.
[17] Mosier NS, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch MR. [45] Weil J, Westgate P, Kohlmann K, Ladisch MR. Cellulose pretreaments of
Features of promising technologies for pretreatment of lignocellulosic biomass. lignocellulosic substrates. Enzym Micro Technol 1994;16:1002–4.
Bioresour Technol 2005;96:673–86. [46] Schell DJ, Farmer J, Newman M, McMillan JD. Dilute-sulfuric acid pretreatment
[18] Palmowski L, Muller J. Influence of the size reduction of organic waste on their of corn stover in pilot-scale reactor. In: Davison BH, Lee JW, Finkelstein M,
anaerobic digestion. In: Proceedings of II international symposium on anaerobic McMillan JD, editors. Proceedings of biotechnol. fuels chem. twenty-fourth symp.,
digestion of solid waste, Barcelona; 15–17 June 1999. p.137–44. Totowa, NJ: Humana Press; 2003, p. 69–85.
[19] Sun Y, Cheng J. Hydrolysis of lignocellulosic materials for ethanol production: a [47] Dale BE, Moreira MJ. A freeze-explosion technique for increasing cellulose
review. Bioresour Technol 2002;83:1–11. hydrolysis. Biotechnol Bioeng Symp 1982;12:31–43.
[20] Barakat A, Monlau F, Solhy A, Carrere H. Mechanical dissociation and fragmen- [48] Holtzapple MT, Jun J-H, Ashok G, Patibandla SL, Dale BE. The ammonia freeze
tation of lignocellulosic biomass: effect of initial moisture, biochemical and explosion (AFEX) process. Appl Biochem Biotechnol 1991;28:59–74.
structural proprieties on energy requirement. Appl Energy 2015;142:240–6. [49] Galbe M, Zacchi G. Pretreatment of lignocellulosic materials for efficient
[21] Millet MA, Baker JA, Scatter LD. Physical and chemical pretreatment for bioethanol production. In: Olsson L, editor. Biofuels. Berlin, Heidelberg: Springer
enhancing cellulose saccharification. Biotechnol Bioeng Symp 1976;6:125–53. Berlin Heidelberg; 2007. p. 41–65.
[22] Delgenés JP, Penaud V, Moletta R. Pretreatments for the enhancement of [50] Alizadeh H, Teymouri F, Gilbert TI, Dale BE. Pretreatment of switchgrass by
anaerobic digestion of solid wastes. In: Mata-Alvarez J, editor. Biomethanization ammonia fiber explosion (AFEX). Appl Biochem Biotechnol 2005;124:1133–41.
of the organic fraction of municipal solid wastes. IWA Publishing; 2002. p. [51] Bariska M. Collapse phenomena in beechwood during and after NH3-impregna-
201–28. tion. Wood Sci Technol 1975;9:293–306.
[23] Hartmann H, Angelidaki I, Ahring BK. Increase of anaerobic degradation of [52] Kim TH, Lee YY. Pretreatment of corn stover by soaking in aqueous ammonia. In:
particulate organic matter in full-scale biogas plants by mechanical maceration. Davison BH, Evans BR, Finkelstein M, McMillan JD, editors. Proceedings of the
In: Mata-Alvarez J, Tilche A, Cecchi F, editors. Proceedings of the second twenty-sixth symp. biotechnol. fuels chem., Totowa, NJ: Humana Press; 2005, p.
international symposium on anaerobic digestion of solid wastes, Barcelona: 1119–31.
Grafiques; 1999, p. 129–36. [53] Abdul PM, Jahim JM, Harun S, Markom M, Lutpi NA, Hassan O, et al. Effects of
[24] Chang VS, Holtzapple MT. Fundamental factors affecting biomass enzymatic changes in chemical and structural characteristic of ammonia fibre expansion
reactivity. In: Finkelstein M, Davison BH, editors. Proceedings of the twenty-first (AFEX) pretreated oil palm empty fruit bunch fibre on enzymatic saccharification
symp. biotechnol. fuels chem. Held May 2–6, 1999, Fort Collins, Color., Totowa, and fermentability for biohydrogen. Bioresour Technol 2016;211:200–8.
NJ: Humana Press; 2000. p. 5–37. [54] da Costa Sousa L, Jin M, Chundawat SPS, Bokade V, Tang X, Azarpira A, et al.
[25] Cadoche L, Lopez GD. Assessment of size reduction as a preliminary step in the Next-generation ammonia pretreatment enhances cellulosic biofuel production.
production of ethanol from lignocellulosic wastes. Biol Waste 1989;30:153–7. Energy Environ Sci 2016;9:1215–23.
[26] Barakat A, Monlau F, Solhy A, Carrere H. Mechanical dissociation and fragmen- [55] Yoon HH, Wu ZW, Lee YY. Ammonia-recycled percolation process for pretreat-
tation of lignocellulosic biomass: effect of initial moisture, biochemical and ment of biomass feedstock. Appl Biochem Biotechnol 1995;51:5–19.
structural proprieties on energy requirement. Appl Energy 2015;142:240–6. [56] Iyer P V, Wu Z-W, Kim SB, Lee YY. Ammonia recycled percolation process for
[27] Hendriks ATWM, Zeeman G. Pretreatments to enhance the digestibility of pretreatment of herbaceous biomass. In: Wyman CE, Davison BH, editors.
lignocellulosic biomass. Bioresour Technol 2009;100:10–8. Proceedindgs of the seventeenth symp. biotechnol. fuels chem. Present. as Vol. 57
[28] Bobleter O, Bonn G, Prutsch W. Steam explosion-hydrothermolysis-organosolv: a 58 Appl. Biochem. Biotechnol., Totowa, NJ: Humana Press; 1996, p. 121–32.
comparison. In: Focher B, Marzetti A, Crescenzi V, editors. Steam explosion [57] Kim JS, Lee YY, Park SC. Pretreatment of wastepaper and pulp mill sludge by
techniques. Philadelphia: Gordon and Breach; 1991. p. 59–82. aqueous ammonia and hydrogen peroxide. In: Finkelstein M, Davison BH, editors.
[29] Mok WSL, Antal MJ. Uncatalyzed solvolysis of whole biomass hemicellulose by Proceedings of the twenty-first symp. biotechnol. fuels chem. Held May 2–6, 1999,

668
D. Kumar et al. Renewable and Sustainable Energy Reviews 73 (2017) 654–671

Fort Collins, Color., Totowa, NJ: Humana Press; 2000. p. 129–39. Biotechnol Bioeng 1984;26:59–65.
[58] Xiao W, Clarkson WW. Acid solubilization of lignin and bioconversion of treated [91] Ben-Ghedalia D, Miron J. The effect of combined chemical and enzyme treatments
newsprint to methane. Biodegradation 1997;8:61–6. on the saccharification and in vitro digestion rate of wheat straw. Biotechnol
[59] Israilides CJ, Grant GA, Han YW. Sugar level, fermentability, and acceptability of Bioeng 1981;23:823–31.
straw treated with different acids. Appl Environ Microbiol 1978;36:43–6. [92] Vidal PF, Molinier J. Ozonolysis of lignin—improvement of in vitro digestibility of
[60] Xiao W, Clarkson WW. Acid solubilization of lignin and bioconversion of treated poplar sawdust. Biomass 1988;16:1–17.
newsprint to methane. Biodegradation 1997;8:61–6. [93] Williams KC. Subcritical water and chemical pretreatments of cotton stalk for the
[61] Chung Y-C, Bakalinsky A, Penner MH. Enzymatic saccharification and fermenta- production of ethanol [Master's thesis]. Raleigh, NC, USA: North Carolina State
tion of xylose-optimized dilute acid-treated lignocellulosics. Appl Biochem University; 2006.
Biotechnol 2005;124:947–61. [94] Morrison WH, Akin DE. Water soluble reaction products from ozonolysis of
[62] Taherzadeh MJ, Karimi K. Pretreatment of lignocellulosic wastes to improve grasses. J Agric Food Chem 1990;38:678–81.
ethanol and biogas production: a review. Int J Mol Sci 2008;9:1621–51. [95] Euphrosine-Moy V, Lasry T, Bes RS, Molinier J, Mathieu J. Degradation of poplar
[63] Fengel D, Wegener G. Wood: chemistry, ultrastructure, reactions. Walter de lignin with ozone. Ozone Sci Eng 1991;13:239–48.
Gruyter; 1983. [96] Lasry T, Laurent JL, Euphrosine-Moy V, Bes RS, Molinier J, Mathieu I.
[64] Ramos LP. The chemistry involved in the steam treatment of lignocellulosic Identification and evaluation of polar sawdust ozonation products. Analysis
materials. Quim Nova 2003;26:863–71. 1990;18:192–9.
[65] Liu C, Wyman CE. The effect of flow rate of compressed hot water on Xylan, [97] Chum HL, Johnson DK, Black S, Baker J, Grohmann K, Sarkanen KV, et al.
Lignin, and total mass removal from corn stover. Ind Eng Chem Res Organosolv pretreatment for enzymatic hydrolysis of poplars: i. Enzyme hydro-
2003;42:5409–16. lysis of cellulosic residues. Biotechnol Bioeng 1988;31:643–9.
[66] Singh J, Suhag M, Dhaka A. Augmented digestion of lignocellulose by steam [98] Thring RW, Chornet E, Overend RP. Recovery of a solvolytic lignin: effects of
explosion, acid and alkaline pretreatment methods: a review. Carbohydr Polym spent liquor/acid volume ratio, acid concentration and temperature. Biomass
2015;117:624–31. 1990;23:289–305.
[67] Knappert D, Grethlein H, Converse A. Partial acid hydrolysis of poplar wood as a [99] Zhao X, Cheng K, Liu D. Organosolv pretreatment of lignocellulosic biomass for
pretreatment for enzymatic hydrolysis. In: Proceedings of biotechnol. bioeng. enzymatic hydrolysis. Appl Microbiol Biotechnol 2009;82:815.
symp., United States, vol. 11, 1981. [100] Sarkanen KV. Acid catalyzed delignification of lignocellulosics in organic solvents.
[68] Converse AO, Matsuno R, Tanaka M, Taniguchi M. A model of enzyme adsorption In: Sarkanen KV, Tillman DA, editors. Progress biomass conv 1980; 1980. p.
and hydrolysis of microcrystalline cellulose with slow deactivation of the adsorbed 127–44.
enzyme. Biotechnol Bioeng 1988;32:38–45. [101] Haghighi Mood S, Hossein Golfeshan A, Tabatabaei M, Salehi Jouzani G, Najafi
[69] Brodeur G, Yau E, Badal K, Collier J, Ramachandran KB, Ramakrishnan S. GH, Gholami M, et al. Lignocellulosic biomass to bioethanol, a comprehensive
Chemical and physicochemical pretreatment of lignocellulosic biomass: a review. review with a focus on pretreatment. Renew Sustain Energy Rev 2013;27:77–93.
Enzym Res 2011;2011:1–17. [102] Mesa L, González E, Cara C, González M, Castro E, Mussatto SI. The effect of
[70] Cheng Y-S, Zheng Y, Yu CW, Dooley TM, Jenkins BM, VanderGheynst JS. organosolv pretreatment variables on enzymatic hydrolysis of sugarcane bagasse.
Evaluation of high solids alkaline pretreatment of rice straw. Appl Biochem Chem Eng J 2011;168:1157–62.
Biotechnol 2010;162:1768–84. [103] da Costa Sousa L, Chundawat SPS, Balan V, Dale BE. “Cradle-to-grave” assess-
[71] McIntosh S, Vancov T. Enhanced enzyme saccharification of Sorghum bicolor ment of existing lignocellulose pretreatment technologies. Curr Opin Biotechnol
straw using dilute alkali pretreatment. Bioresour Technol 2010;101:6718–27. 2009;20:339–47.
[72] Sills DL, Gossett JM. Assessment of commercial hemicellulases for saccharifica- [104] Pan X, Arato C, Gilkes N, Gregg D, Mabee W, Pye K, et al. Biorefining of softwoods
tion of alkaline pretreated perennial biomass. Bioresour Technol using ethanol organosolv pulping: preliminary evaluation of process streams for
2011;102:1389–98. manufacture of fuel-grade ethanol and co-products. Biotechnol Bioeng
[73] Chang VS, Holtzapple MT. Fundamental factors affecting biomass enzymatic 2005;90:473–81.
reactivity. In: Finkelstein M, Davison BH, editors. Proceedings of the twenty-first [105] Sun F, Chen H. Organosolv pretreatment by crude glycerol from oleochemicals
symp. biotechnol. fuels chem. Held May 2–6, 1999, Fort Collins, Color., Totowa, industry for enzymatic hydrolysis of wheat straw. Bioresour Technol
NJ: Humana Press; 2000, p. 5–37 2008;99:5474–9.
[74] Laureano-Perez L, Teymouri F, Alizadeh H, Dale BE. Understanding factors that [106] Shi J, Sharma-Shivappa RR, Chinn M, Howell N. Effect of microbial pretreatment
limit enzymatic hydrolysis of biomass. Appl Biochem Biotechnol on enzymatic hydrolysis and fermentation of cotton stalks for ethanol production.
2005;124:1081–99. Biomass- Bioenergy 2009;33:88–96.
[75] Kim J, Yu Y, Lee C. Thermo-alkaline pretreatment of waste activated sludge at [107] Potumarthi R, Baadhe RR, Nayak P, Jetty A. Simultaneous pretreatment and
low-temperatures: effects on sludge disintegration, methane production, and saccharification of rice husk by Phanerochete chrysosporium for improved
methanogen community structure. Bioresour Technol 2013;144:194–201. production of reducing sugars. Bioresour Technol 2013;128:113–7.
[76] MacDonald DG, Bakhshi NN, Mathews JF, Roychowdhury A, Bajpai P, Moo- [108] Du W, Yu H, Song L, Zhang J, Weng C, Ma F, et al. The promoting effect of
Young M. Alkali treatment of corn stover to improve sugar production by byproducts from Irpex lacteus on subsequent enzymatic hydrolysis of bio-
enzymatic hydrolysis. Biotechnol Bioeng 1983;25:2067–76. pretreated cornstalks. Biotechnol Biofuels 2011;4:37.
[77] Soto ML, Domínguez H, Núñez MJ, Lema JM. Enzymatic saccharification of [109] Okano K, Kitagawa M, Sasaki Y, Watanabe T. Conversion of Japanese red cedar
alkali-treated sunflower hulls. Bioresour Technol 1994;49:53–9. (Cryptomeria japonica) into a feed for ruminants by white-rot basidiomycetes.
[78] Zhao Y, Wang Y, Zhu JY, Ragauskas A, Deng Y. Enhanced enzymatic hydrolysis of Anim Feed Sci Technol 2005;120:235–43.
spruce by alkaline pretreatment at low temperature. Biotechnol Bioeng [110] Akin D, Rigsby L, Sethuraman A, Morrison W, 3rd, Gamble G, Eriksson K.
2008;99:1320–8. Alterations in structure, chemistry, and biodegradability of grass lignocellulose
[79] Zhu J, Wan C, Li Y. Enhanced solid-state anaerobic digestion of corn stover by treated with the white rot fungi Ceriporiopsis subvermispora and Cyathus
alkaline pretreatment. Bioresour Technol 2010;101:7523–8. stercoreus. Appl Environ Microbiol 1995;61:1591–8.
[80] Sun R, Lawther JM, Banks WB. Influence of alkaline pre-treatments on the cell [111] Hatakka AI. Pretreatment of wheat straw by white-rot fungi for enzymic
wall components of wheat straw. Ind Crops Prod 1995;4:127–45. saccharification of cellulose. Eur J Appl Microbiol Biotechnol 1983;18:350–7.
[81] Hu Z, Wang Y, Wen Z. Alkali (NaOH) pretreatment of switchgrass by radio [112] Sindhu R, Binod P, Pandey A. Biological pretreatment of lignocellulosic biomass –
frequency-based dielectric heating. Appl Biochem Biotechnol 2008;148:71–81. an overview. Bioresour Technol 2016;199:76–82.
[82] Park J, Shiroma R, Al-Haq MI, Zhang Y, Ike M, Arai-Sanoh Y, et al. A novel lime [113] Alvira P, Tomás-Pejó E, Ballesteros M, Negro MJ. Pretreatment technologies for
pretreatment for subsequent bioethanol production from rice straw – calcium an efficient bioethanol production process based on enzymatic hydrolysis: a
capturing by carbonation (CaCCO) process. Bioresour Technol review. Bioresour Technol 2010;101:4851–61.
2010;101:6805–11. [114] Wasserscheid P, Keim W. Ionic liquids—new “Solutions” for transition metal
[83] Chen B-Y, Chen S-W, Wang H-T. Use of different alkaline pretreatments and catalysis. Angew Chemie Int Ed 2000;39:3772–89.
enzyme models to improve low-cost cellulosic biomass conversion. Biomass- [115] Zavrel M, Bross D, Funke M, Büchs J, Spiess AC. High-throughput screening for
Bioenergy 2012;39:182–91. ionic liquids dissolving (ligno-)cellulose. Bioresour Technol 2009;100:2580–7.
[84] Sawan SP, Sawan SP. Supercritical fluid cleaning: fundamentals, technology and [116] Zhang Y-HP, Lynd LR. A functionally based model for hydrolysis of cellulose by
applications. In: Mchardy J, Sawan. SPElsevier Science; 1998 fungal cellulase. Biotechnol Bioeng 2006;94:888–98.
[85] Alinia R, Zabihi S, Esmaeilzadeh F, Kalajahi JF. Pretreatment of wheat straw by [117] Zhu S, Wu Y, Chen Q, Yu Z, Wang C, Jin S, et al. Dissolution of cellulose with ionic
supercritical CO2 and its enzymatic hydrolysis for sugar production. Biosyst Eng liquids and its application: a mini-review. Green Chem 2006;8:325–7.
2010;107:61–6. [118] Hou X-D, Xu J, Li N, Zong M-H. Effect of anion structures on cholinium ionic
[86] Zheng Y, Lin H, Tsao G. Pretreatment for cellulose hydrolysis by carbon dioxide liquids pretreatment of rice straw and the subsequent enzymatic hydrolysis.
explosion. Biotechnol Prog 1998;14:890–6. Biotechnol Bioeng 2015;112:65–73.
[87] Schacht C, Zetzl C, Brunner G. From plant materials to ethanol by means of [119] Moulthrop JS, Swatloski RP, Moyna G, Rogers RD. High-resolution 13C NMR
supercritical fluid technology. J Supercrit Fluids 2008;46:299–321. studies of cellulose and cellulose oligomers in ionic liquid solutions. Chem
[88] Maurya DP, Singla A, Negi S. An overview of key pretreatment processes for Commun 2005:1557–9.
biological conversion of lignocellulosic biomass to bioethanol. 3 Biotech [120] Singh S, Simmons BA, Vogel KP. Visualization of biomass solubilization and
2015;5:597–609. cellulose regeneration during ionic liquid pretreatment of switchgrass. Biotechnol
[89] Li C, Wang L, Chen Z, Li Y, Wang R, Luo X, et al. Ozonolysis pretreatment of Bioeng 2009;104:68–75.
maize stover: the interactive effect of sample particle size and moisture on [121] Hon DNS, Shiraishi N. Wood and cellulosic chemistry. Taylor & Francis; 2000,
ozonolysis process. Bioresour Technol 2015;183:240–7. Second edition [Revised and Expanded].
[90] Neely WC. Factors affecting the pretreatment of biomass with gaseous ozone. [122] Klinke HB, Ahring BK, Schmidt AS, Thomsen AB. Characterization of degradation

669
D. Kumar et al. Renewable and Sustainable Energy Reviews 73 (2017) 654–671

products from alkaline wet oxidation of wheat straw. Bioresour Technol [156] Ratledge C. Yeasts, moulds, algae and bacteria as sources of lipids. In: Kamel BS,
2002;82:15–26. Kakuda Y, editors. Technol. Adv. Improv. Altern. Sources Lipids. Boston, MA:
[123] Teixeira LC, Linden JC, Schroeder HA. Alkaline and peracetic acid pretreatments Springer US; 1994. p. 235–91.
of biomass for ethanol production. Appl Biochem Biotechnol 1999;77:19–34. [157] Ratledge C, Wynn J. The biochemistry and molecular biology of lipid accumula-
[124] Gould JM. Alkaline peroxide delignification of agricultural residues to enhance tion in oleaginous microorganisms. In: Laskin AI, Bennett JW, Gadd GM, editors.
enzymatic saccharification. Biotechnol Bioeng 1984;26:46–52. Advances in applied microbiology. Elsevier Science; 2002. p. 1–51.
[125] Béguin P, Aubert J-P. The biological degradation of cellulose. FEMS Microbiol [158] Ratledge C. Microbial lipids. Biotechnology. Wiley-VCH Verlag GmbH; 2008. p.
Rev 1994;13, [25 LP-58]. 133–97.
[126] Rabinovich ML, Melnik MS, Bolobova AV. Dedicated to the memory of I.V. [159] Papanikolaou S, Aggelis G. Biotechnological valorization of biodiesel derived
Berezin and R.V. Feniksova Microbial Cellulases (Review). Appl Biochem glycerol waste through production of single cell oil and citric acid by Yarrowia
Microbiol 2002;38:305–22. lipolytica. Lipid Technol 2009;21:83–7.
[127] Taherzadeh MJ, Karimi K. Enzyme-based hydrolysis processes for ethanol from [160] Ratledge C. Biochemistry, stoichiometry, substrates and economics. In: Moreton
lignocellulosic materials: a review. BioResources 2007;2(3):707–38. RS, editor. Single Cell Oil. Harlow, UK: Longman Scientific & Technical; 1988. p.
[128] Bisaria VS, Martin AM, et al. Bioprocessing of agro-residues to glucose and 33–70.
chemicals. Bioconversion Waste Mater Ind Prod 1991:187–223. [161] Evans CT, Ratledge C. The role of the mitochondrial NAD+: isocitrate dehydro-
[129] Sternberg D. Production of cellulase by Trichoderma. Biotechnol Bioeng Symp genase in lipid accumulation by the oleaginous yeast Rhodosporidium toruloides
1976:35–53. CBS 14. Can J Microbiol 1985;31:845–50.
[130] Fan L, Gharpuray MM, Lee YH. Cellulose hydrolysis. Springer Berlin Heidelberg; [162] Papanikolaou S, Galiotou-Panayotou M, Fakas S, Komaitis M, Aggelis G. Lipid
2012. production by oleaginous Mucorales cultivated on renewable carbon sources. Eur
[131] Hari Krishna S, Janardhan Reddy T, Chowdary GV. Simultaneous saccharification J Lipid Sci Technol 2007;109:1060–70.
and fermentation of lignocellulosic wastes to ethanol using a thermotolerant yeast. [163] Boulton CA, Ratledge C. Regulatory studies on citrate synthase in Candida 107, an
Bioresour Technol 2001;77:193–6. oleaginous yeast. Microbiology 1980;121:441–7.
[132] Itoh H, Wada M, Honda Y, Kuwahara M, Watanabe T. Bioorganosolve pretreat- [164] Boulton CA, Ratledge C. Correlation of lipid accumulation in yeasts with
ments for simultaneous saccharification and fermentation of beech wood by possession of ATP: citrate lyase. Microbiology 1981;127:169–76.
ethanolysis and white rot fungi. J Biotechnol 2003;103:273–80. [165] Wynn JP, Hamid AA, Li Y, Ratledge C. Biochemical events leading to the diversion
[133] Ortega N, D. Busto M, Perez-Mateos M. Kinetics of cellulose saccharification by of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and
Trichoderma reesei cellulases. Int Biodeterior Biodegrad 2001;47:7–14. Mortierella alpina. Microbiology 2001;147:2857–64.
[134] Wyman C. Handbook on bioethanol: production and utilization. Taylor & [166] Botham PA, Ratledge C. A biochemical explanation for lipid accumulation in
Francis; 1996. Candida 107 and other oleaginous micro-organisms. Microbiology
[135] Olsson L, Hahn-Hägerdal B. Fermentation of lignocellulosic hydrolysates for 1979;114:361–75.
ethanol production. Enzym Micro Technol 1996;18:312–31. [167] Fakas S, Papanikolaou S, Panayotou MG, Komaitis M, Aggelis G. Biochemistry
[136] Alkasrawi M, Eriksson T, Börjesson J, Wingren A, Galbe M, Tjerneld F, et al. The and biotechnology of single cell oil. In: Pandey A, Larroche C, editors. New
effect of Tween-20 on simultaneous saccharification and fermentation of softwood horizons in biotechnology. New Delhi, India: AsiaTech Publishers Inc.; 2009. p.
to ethanol. Enzym Micro Technol 2003;33:71–8. 38–60.
[137] Kim SB, Kim HJ, Kim CJ. Enhancement of the enzymatic digestibility of waste [168] Ratledge C. Regulation of lipid accumulation in oleaginous micro-organisms.
newspaper using tween. In: McMillan JD, Adney WS, Mielenz JR, Klasson KT, Biochem Soc Trans 2002;30:1047–50.
editors. Proceedings of the twenty-seventh symp. biotechnol. fuels chem., Totowa, [169] Ageitos JM, Vallejo JA, Veiga-Crespo P, Villa TG. Oily yeasts as oleaginous cell
NJ: Humana Press; 2006. p. 486–95. factories. Appl Microbiol Biotechnol 2011;90:1219–27.
[138] Börjesson J, Peterson R, Tjerneld F. Enhanced enzymatic conversion of softwood [170] Zongbao Z. Toward cheaper microbial oil for biodiesel oil. J Chin Biotechnol
lignocellulose by poly(ethylene glycol) addition. Enzym Microbiol Technol 2005;25:8–11.
2007;40:754–62. [171] Papanikolaou S, Chevalot I, Komaitis M, Marc I, Aggelis G. Single cell oil
[139] Eriksson T, Börjesson J, Tjerneld F. Mechanism of surfactant effect in enzymatic production by Yarrowia lipolytica growing on an industrial derivative of animal fat
hydrolysis of lignocellulose. Enzym Microbiol Technol 2002;31:353–64. in batch cultures. Appl Microbiol Biotechnol 2002;58:308–12.
[140] Ramos LP, Breuil C, Saddler JN. The use of enzyme recycling and the influence of [172] Kessell RHJ. Fatty acids of Rhodotorula gracilis: fat production in submerged
sugar accumulation on cellulose hydrolysis by Trichoderma cellulases. Enzym culture and the particular effect of pH value. J Appl Bacteriol 1968;31:220–31.
Microbiol Technol 1993;15:19–25. [173] Gill CO, Hall MJ, Ratledge C. Lipid accumulation in an oleaginous yeast (Candida
[141] Harris EE, Beglinger E. Madison wood sugar process. Ind Eng Chem 107) growing on glucose in single-stage continuous culture. Appl Environ
1946;38:890–5. Microbiol 1977;33:231–9.
[142] Hashem AM, Rahshad MM. Production of ethanol by yeasts grown on hydrolysate [174] Gong Z, Shen H, Zhou W, Wang Y, Yang X, Zhao ZK. Efficient conversion of
of Egyptian sweet potato. Egypt J Food Sci 1993;21(2):171–80. acetate into lipids by the oleaginous yeast Cryptococcus curvatus. Biotechnol
[143] Taherzadeh MJ, Eklund R, Gustafsson L, Niklasson C, Lidén G. Characterization Biofuels 2015;8:189.
and Fermentation of Dilute-Acid Hydrolyzates from Wood. Ind Eng Chem Res [175] Huang C, Zong M, Wu H, Liu Q. Microbial oil production from rice straw
1997;36:4659–65. hydrolysate by Trichosporon fermentans. Bioresour Technol 2009;100:4535–8.
[144] Harris J, Baker A, Zerbe J. Two stage, dilute sulfuric acid hydrolysis of hardwood [176] Yamauchi H, Mori H, Kobayashi T, Shimizu S. Mass production of lipids by
for ethanol production. Energy Biomass- Wastes 1984;8:1151–70. Lipomyces starkeyi in microcomputer-aided fed-batch culture. J Ferment
[145] Jones JL, Semrau KT. Wood hydrolysis for ethanol production—previous ex- Technol 1983;61:275–80.
perience and the economics of selected processes. Biomass 1984;5:109–35. [177] Angerbauer C, Siebenhofer M, Mittelbach M, Guebitz GM. Conversion of sewage
[146] Katzen R, Madson PW, Monceaux DA. Use of cellulosic feedstocks for alcohol sludge into lipids by Lipomyces starkeyi for biodiesel production. Bioresour
production. The Alcohols Textbook, Nothingham University Press; 1995. Technol 2008;99:3051–6.
[147] Groenestijn JV, Hazewinkel O, Bakker R. Pretreatment of lignocelluloses with [178] Tsigie YA, Wang CY, Kasim NS, Diem QD, Huynh LH, Ho QP, Ju YH. Oil
biological acid recovery. (Biosulfurol Process) Zuckerind 2006;131(9):639–41. production from Yarrowia lipolytica Po1g using rice bran hydrolysate. J Biomed
[148] Kosaric N, Wieczorirek A, Cosentono GP, Magee RJ. Ethanol fermentation. In: Biotechnol 2012, 10 pages, doi:http://dx.doi.org/10.1155/2012/378384.
Rehm HJ, Reed G, Dellweg H, editors. Biotechnology 1983; 1983. p. 257–386. [179] Patel A, Sindhu DK, Arora N, Singh RP, Pruthi V, Pruthi PA. Biodiesel production
[149] Xiang Q, Lee YY, Torget RW. Kinetics of glucose decomposition during dilute-acid from non-edible lignocellulosic biomass of Cassia fistula L. fruit pulp using
hydrolysis of lignocellulosic biomass. In: Finkelstein M, McMillan JD, Davison oleaginous yeast Rhodosporidium kratochvilovae HIMPA1. Bioresour Technol
BH, Evans B, editors. Proceedings of the twenty-fifth symp. biotechnol. fuels 2015;197:91–8.
chem. Held May 4–7, 2003, Breckenridge, CO, Totowa, NJ: Humana Press; 2004, [180] Kahr H, Pointner M, Krennhuber K, Wallner B, Jäger A. Lipid production from
p. 1127–38. diverse oleaginous yeasts from steam exploded corn cobs. Agron Res
[150] Xu J, Du W, Zhao X, Zhang G, Liu D. Microbial oil production from various 2015;13(2):318–27.
carbon sources and its use for biodiesel preparation. Biofuels Bioprod Bioref [181] Fei Q, O’Brien M, Nelson R, Chen X, Lowell A, Dowe N. Enhanced lipid
2013;7:65–77. production by Rhodosporidium toruloides using different fed-batch feeding
[151] Fakas S, Papanikolaou S, Galiotou-Panayotou M, Komaitis M, Aggelis G. Lipids of strategies with lignocellulosic hydrolysate as the sole carbon source. Biotechnol
Cunninghamella echinulata with emphasis to γ-linolenic acid distribution among Biofuels 2016;9:130.
lipid classes. Appl Microbiol Biotechnol 2006;73:676–83. [182] Tanimura A, Takashima M, Sugita T, Endoh R, Ohkuma M, Kishino S, et al. Lipid
[152] Fakas S, Papanikolaou S, Galiotou-Panayotou M, Komaitis M, Aggelis G. Organic production through simultaneous utilization of glucose, xylose, and l-arabinose by
nitrogen of tomato waste hydrolysate enhances glucose uptake and lipid accu- Pseudozyma hubeiensis: a comparative screening study. AMB Express 2016;6:58.
mulation in Cunninghamella echinulata. J Appl Microbiol 2008;105:1062–70. [183] Ratledge C, Cohen Z. Microbial and algal oils: do they have a future for biodiesel
[153] Papanikolaou S, Chevalot I, Komaitis M, Aggelis G, Marc I. Kinetic profile of the or as commodity oils?. Lipid Technol 2008;20:155–60.
cellular lipid composition in an oleaginous Yarrowia lipolytica capable of [184] Huang C, Chen X, Xiong L, Chen X, Ma L, Chen Y. Single cell oil production from
producing a cocoa-butter substitute from industrial fats. Antonie Van low-cost substrates: the possibility and potential of its industrialization.
Leeuwenhoek 2001;80:215–24. Biotechnol Adv 2013;31:129–39.
[154] Papanikolaou S, Aggelis G. Lipids of oleaginous yeasts. Part I: biochemistry of [185] Chen X, Li Z, Zhang X, Hu F, Ryu DDY, Bao J. Screening of oleaginous yeast
single cell oil production. Eur J Lipid Sci Technol 2011;113:1031–51. http:// strains tolerant to lignocellulose degradation compounds. Appl Biochem
dx.doi.org/10.1002/ejlt.201100014. Biotechnol 2009;159:591.
[155] Davies RJ, Holdsworth JE. Synthesis of lipids in yeasts: biochemistry, physiology [186] Hsiao HY, Chiang LC, Ueng PP, Tsao GT. Sequential utilization of mixed
and production. Adv Appl Lipid Res 1992;1:119–59. monosaccharides by yeasts. Appl Environ Microbiol 1982;43:840–5.

670
D. Kumar et al. Renewable and Sustainable Energy Reviews 73 (2017) 654–671

[187] Dai CC, Tao J, Xie F, Dai YJ, Zhao M. Biodiesel generation from oleaginous yeast conditions. Int J Biol Macromol 2015;78:9–16.
Rhodotorula glutinis with xylose assimilating capacity. Afr J Biotechnol [198] Bhatia SK, Yi D-H, Kim Y-H, Kim H-J, Seo H-M, Lee J-H, et al. Development of
2007;6(18):2130–4. semi-synthetic microbial consortia of Streptomyces coelicolor for increased
[188] Slininger PJ, Dien BS, Kurtzman CP, Moser BR, Bakota EL, Thompson SR, et al. production of biodiesel (fatty acid methyl esters). Fuel 2015;159:189–96.
Comparative lipid production by oleaginous yeasts in hydrolyzates of lignocellu- [199] Roth P. Myslin H, Bachofen R, editors. New trends in research and utilization of
losic biomass and process strategy for high titers. Biotechnol Bioeng solar energy through biological systems. Basel–Boston–Stuttgart: Birkhäuser
2016;9(2):430–46. Verlag; 1982. p. 278.
[189] Liu W, Wang Y, Yu Z, Bao J. Simultaneous saccharification and microbial lipid [200] Anwar Z, Gulfraz M, Irshad M. Agro-industrial lignocellulosic biomass a key to
fermentation of corn stover by oleaginous yeast Trichosporon cutaneum. unlock the future bio-energy: a brief review. J Radiat Res Appl Sci
Bioresour Technol 2012;118:13–8. 2014;7:163–73.
[190] Beopoulos A, Chardot T, Nicaud J-M. Yarrowia lipolytica: a model and a tool to [201] Ji F, Zhou Y, Pang A, Ning L, Rodgers K, Liu Y, et al. Fed-batch cultivation of
understand the mechanisms implicated in lipid accumulation. Biochimie Desmodesmus sp. in anaerobic digestion wastewater for improved nutrient
2009;91:692–6. removal and biodiesel production. Bioresour Technol 2015;184:116–22.
[191] Beopoulos A, Mrozova Z, Thevenieau F, Le Dall M-T, Hapala I, Papanikolaou S, [202] Li G, Zhou Y, Ji F, Liu Y, Adhikari B, Tian L, et al. Yield and characteristics of
et al. Control of Lipid Accumulation in the Yeast Yarrowia lipolytica. Appl Environ pyrolysis products obtained from Schizochytrium limacinum under different
Microbiol 2008;74:7779–89. temperature regimes. Energies 2013;6:3339–52.
[192] Courchesne NMD, Parisien A, Wang B, Lan CQ. Enhancement of lipid production [203] Patel A, Arora N, Sartaj K, Pruthi V, Pruthi PA. Sustainable biodiesel production
using biochemical, genetic and transcription factor engineering approaches. J from oleaginous yeasts utilizing hydrolysates of various non-edible lignocellulosic
Biotechnol 2009;141:31–41. biomasses. Renew Sustain Energy Rev 2016;62:836–55.
[193] Kosa M, Ragauskas AJ. Lipids from heterotrophic microbes: advances in [204] Yousuf A. Biodiesel from lignocellulosic biomass – prospects and challenges.
metabolism research. Trends Biotechnol 2011;29:53–61. Waste Manag 2012;32:2061–7.
[194] Alvarez H, Steinbüchel A. Triacylglycerols in prokaryotic microorganisms. Appl [205] Knothe G. Biodiesel and renewable diesel: a comparison. Prog Energy Combust
Microbiol Biotechnol 2002;60:367–76. Sci 2010;36:364–73.
[195] Gouda MK, Omar SH, Aouad LM. Single cell oil production by Gordonia sp. DG [206] Srifa A, Faungnawakij K, Itthibenchapong V, Assabumrungrat S. Roles of
using agro-industrial wastes. World J Microbiol Biotechnol 2008;24:1703. monometallic catalysts in hydrodeoxygenation of palm oil to green diesel. Chem
[196] Kosa M, Ragauskas AJ. Lignin to lipid bioconversion by heterotrophic oleaginous Eng J 2015;278:249–58.
Rhodococci. Renew Chem 2013;15, [2070–61]. [207] Arun N, Sharma RV, Dalai AK. Green diesel synthesis by hydrodeoxygenation of
[197] Kumar P, Ray S, Patel SKS, Lee J-K, Kalia VC. Bioconversion of crude glycerol to bio-based feedstocks: strategies for catalyst design and development. Renew
polyhydroxyalkanoate by Bacillus thuringiensis under non-limiting nitrogen Sustain Energy Rev 2015;48:240–55.

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