Renewable and Sustainable Energy Reviews 62 (2016) 836–855

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Renewable and Sustainable Energy Reviews

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

Sustainable biodiesel production from oleaginous yeasts utilizing

hydrolysates of various non-edible lignocellulosic biomasses
Alok Patel n, Neha Arora, Km Sartaj, Vikas Pruthi, Parul A. Pruthi
Molecular Microbiology Laboratory, Biotechnology Department, Indian Institute of Technology Roorkee (IIT-R), Roorkee 247667, Uttarakhand, India

art ic l e i nf o a b s t r a c t

Article history: Biodiesel, as one of the best alternative fuels, has a number of advantages over petro diesel, such as
Received 22 November 2015 originating from a renewable and domestic feedstock which reduces the net production cost of biodiesel.
Received in revised form In the recent years, biodiesel has received increasing interest due to energy crisis worldwide along with
24 February 2016
exhausting reserves and the shortage of oil supplies. The major problem behind the use of vegetable oil
Accepted 3 May 2016
for biodiesel production is sustainability because it directly competes with human food. To combat this
problem, the other renewable sources have been developed as microbial oils have similarity to vegetable
Keywords: oils and extensively used for biodiesel production. Oleaginous yeasts have recently been suggested as
Non-edible lignocellulosic biomass microscopic biofactories and alternative lipid producer to vegetable oil for a more sustainable biodiesel
Oleaginous yeast
industry. It is a potential novel technology where non-edible lignocellulosic biomasses are exploited as
Fermentable sugars
raw materials for biodiesel production from oleaginous yeasts which drop net greenhouse gas emissions
Triacylglycerols (TAG)
Biodiesel by substituting the practice of fossil fuels and would convey benefits to rural economies and national
energy security. The usage of oleaginous yeasts have many advantages over other renewable sources like
faster growth rate, shorter life cycle, easier scale-up, with no effects from the season and climate var-
iation, and can serve as the excellent oil accumulating renewable feedstocks which are non-competitive
to food resources and do not require arable land. Non-edible lignocellulosic biomass, consists of three
different types of natural polymers, namely cellulose, hemicellulose, and lignin, is the most abundant
renewable bioresource in the biosphere. The production of fermentable sugars from hydrolysates of
various non-edible lignocellulosic biomass, either by physical, chemical or enzymatic hydrolysis has been
utilized as feedstock in bioethanol or biodiesel production, extensively. During hydrolysis generation of
non-carbohydrate compounds, such as 5- hydroxymethylfurfural (HMF), furfural acetic acid and phenolic
compounds have various effects on the growth of microorganisms, their metabolism, as well as on final
products, presenting a key challenge in the biological conversion of biomasses.
& 2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Types of lignocellulosic biomass (LB) for saccharification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1. Agricultural residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2. Non-edible crops or energy crops and forest residues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.3. Industrial residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3. Pretreatment of lignocellulosic biomasses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.1. Mechanical pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.2. Thermal pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.3. Acid and alkali pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.4. Oxidative pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.5. Steam explosion/steam pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.6. Liquid hot water (LHW) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

n
Corresponding author. Mobile: þ 9196 75453418.
E-mail address: biotechalok@gmail.com (A. Patel).

http://dx.doi.org/10.1016/j.rser.2016.05.014
1364-0321/& 2016 Elsevier Ltd. All rights reserved.
 A. Patel et al. / Renewable and Sustainable Energy Reviews 62 (2016) 836–855 837

3.7. Carbon dioxide (CO2) pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3.8. Ozonolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.9. Green solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.10. Biological pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.11. Microwave pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4. Oleaginous yeasts: a promising feedstock for biodiesel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5. Comprehensive procedure for biodiesel production using oleaginous yeasts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.1. Screening of high triacylglycerol (TAG) accumulating oleaginous yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.2. Optimization of different parameter for higher lipid yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.2.1. Selection of carbon sources for fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.2.2. Optimization of dissolved oxygen, aeration rate and pH of the fermentation medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.3. Estimation of TAG accumulation and efficient lipid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
5.4. Transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
6. Lipid accumulation by oleaginous yeasts utilizing hydrolysates of various non-edible lignocellulosic biomasses . . . . . . . . . . . . . . . . . . . . . . . . . 12
7. Effect of inhibitors generated during hydrolysis of LB on growth and lipid accumulation by oleaginous yeast. . . . . . . . . . . . . . . . . . . . . . . . . . . 12
8. Fatty acids profile of oleaginous yeasts grown on various non-edible lignocellulosic biomasses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
9. Estimation of biodiesel properties on the basis of fatty acids profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
10. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

1. Introduction fermentation process of lipid production and decline the yield of

The availability of petroleum-based resources (fossil fuels)
continues to decline with increasing demand for energy world-
wide [1]. In order to diminish dependence on fossil fuels, many
countries have received extensive interest in biomass-based bio-
fuels, which are usually derived from renewable and domestic
feedstocks. In India and other developing countries, direct com-
bustion of lignocellulosic biomasses (LB) is routinely used for heat
generation and cooking purposes. Direct combustion of LB has led
to many problems such as environmental pollution and low
energy efficiency. In order to substitute the direct combustion of
LB, it can be converted into high-quality bio-products and pro-
duced energy by other means. A lot of lignocellulosic biomasses is
obtained from forest woody feedstocks, agricultural residues,
herbaceous and municipal solid wastes and non-edible energy
crops [2]. Sugarcane bagasse, sugar cane husk, wheat and rice
straws and corn stover are most promising plant residues derived
LB which are used for energy generation in U.S., Asia and Europe
[3–5]. Lignocellulosic biomasses are mainly composed of three
different polymers such as cellulose, hemicellulose, and lignin.
These polymers are toughly intertwined and associated with non-
covalent bonds and covalent cross-linkages [6]. Cell walls of plants
have lignocellulose as a primary building block in which cellulose
plays a major structural role. Celluloses are organized as micro-
fibrils that are surrounded by hemicellulose and lignin (Fig. 3). The
clubhouse is the repeat unit of cellulose containing D-glucose
subunits linked to each other by β-(1,4)-glycosidic bonds [7]. A
suitable pretreatment is required to break the lignocellulosic bio-
masses to obtain cellulose units and further treated with enzyme
cocktails or sulfuric acid to generate sugars [8]. The treated cel-
lulose releases mono and oligosaccharides composed of pentose
and hexose sugars that are also known as hydrolysates. These
hydrolysates are utilized by oleaginous microorganisms as
potential carbon sources for lipid synthesis. From last decade,
various researches have been performed to improve the efficiency
of hydrolysis. The efficiency is determined by presence of high
concentration of monosaccharides in hydrolysates. However, the
hydrolysis of LB is restricted by the production of toxic compounds
such as furfural (pentose sugars by-product), hydroxyl methyl
furfural (HMF; hexose sugar by-product), phenolic acid (lignin by-
product) and acetate (deacetylation product of hemicellulose).
These by-products show the various inhibitory effect on the
final products [9,10].
Biodiesel is renewable, non-hazardous, biodegradable, sus-
tainable, nonflammable, eco-friendly and free from sulfur and
aromatic contents. It has higher cetane number and higher flash
point than conventional diesel. Its inherent lubricity increases the
engine efficiency, so no need to add extra lubricant for engines
[11]. It emits almost 78% less net carbon dioxide on a lifecycle basis
compared to conventional diesel fuel. These properties of biodiesel
make it an ideal fuel for polluted metro cities and play a major role
in the aspect of climatic change as it is climatic neutral [12–16].
Biodiesel is mainly produced by a transesterification reaction with
vegetable oils (edible or non-edible), waste cooking oils and ani-
mal fats [17]. Due to global food securities, oil derived from food
sources cannot fulfill the requirement for biodiesel production in
large scale. Therefore, it is necessary to search sustainable feed-
stocks for biodiesel production such as non-edible lignocellulosic
energy crops [18]. However, direct utilization of these substrates to
produce biodiesel involve high production cost. Out of which 75%
of the total cost is spent on raw materials which are the major
obstacle for its large scale production and widespread application
[19]. The use of animal fats, waste cooking oils, and oils from non-
food crops as feedstocks are a good choice to reduce the produc-
tion cost [20,21]. Moreover, this strategy also cannot fulfill the
requirement of renewable fuels for current energy demand.
Therefore, researchers are looking for novel oil resources to pro-
duce biodiesel. Among various resources, lipid produced by
microorganisms, involving yeasts, bacteria, molds and algae
known as single cell oil (SCOs) are considered as promising feed-
stock for biodiesel production due to its similarity with vegetable
oils in fatty acid compositions [22–25]. These microorganisms can
utilize organic carbon to synthesize oils in their cellular com-
partment. Moreover, the productivity of many microorganisms is
reported more than the oil producing crops. It has been reported
that only a minor population of yeasts accumulate more than 25%
of lipids [26,27]. The genera of yeasts which considered as olea-
ginous are Rhodosporidium, Rhodotorula, Yarrowia, Cryptococcus,
Candida, Lipomyces and Trichosporon. Among these oleaginous
yeast genera, Rhodosporidium is found to produce the highest
amount of lipid in its cellular compartment [28].
The advantage of oleaginous yeast culture is the independence
of weather conditions that is more prone in the case of plants.
It takes hardly 5–9 days to achieve lipid accumulation (stationary
838 A. Patel et al. / Renewable and Sustainable Energy Reviews 62 (2016) 836–855

Nomenclature HHV high heating value

HMF hydroxyl methyl furfural
ACL ATP–citrate lyase IV iodine value
ASTM American Society of the International Association for KV kinematic viscosity
Testing and Materials LB lignocellulosic biomass
CCR carbon catabolite repression LCSF long chain saturation factor
CDW cell dry weight LD lipid droplets
CFPP cold filter plugging point LHW liquid hot water
CN cetane number MUFA mono-unsaturated fatty acids
COD chemical oxygen demand OS oxidative stability
D density PA phosphatidic acid
DAG diacylglycerol PAP phosphatidate phosphatase
DHAP dihydroxyacetone phosphate PHB polyhydroxybutyrate
DU degree of unsaturation PUFA poly-unsaturated fatty acids
ETC electron transport chain SFA saturated fatty acids
ETC electron transport chain SHV sweetpotato vines hydrolysate
EU European Union SV saponification value
FAME fatty acid methyl esters TAG triacylglycerols
G-3-P glycerol-3-phosphate UFA unsaturated fatty acids
h hours UN united nations

phase) in its cellular compartment depending on the species of
yeasts. In addition, oleaginous yeasts have specific properties to
synthesize lipids while consuming a large number of renewable
substrates, even inexpensive material, such as nutritional residues
from agriculture and industries [29–31]. Most of these micro-
organisms utilize preferably glucose over other sugars and do not
shift to other sugars until glucose is consumed, but simultaneous
utilization of glucose and other sugar sources by microorganisms
are rarely observed [32,33]. However, it is an essential considera-
tion for biofuels production from a variety of substrates such as a
mixture of sugars derived from non- edible lignocellulosic bio-
masses. Most of the microorganisms have their own carbon cata-
bolite repression (CCR) mechanism, which normally metabolize
sugars in a sequential manner (first glucose and then xylose)
meanwhile glucose suppresses other sugar utilization. Further-
more, it delays the utilization of mixed sugars obtained from
hydrolysis of lignocellulosic biomasses that results in a delay of the
cultivation period and also reduce the efficacy of the overall pro-
cess [34]. In the case of lipid production by oleaginous yeast,
glucose is also most commonly supplied sugar source [35]. Studies
on the red oleaginous yeast Rhodosporidium toruloides and R. glu-
tinis have been revealed the utilization of mix sugar hydrolysates
as they utilize both pentoses and hexoses in a simultaneous
manner that are present in LB hydrolysates [36]. Rhodosporidium
species have shown to produce the highest amount of lipids uti-
lizing various renewable substrates and multiple carbon sources
simultaneously, such as molasses, glycerol, and complex carbo-
hydrates like rice and wheat hydrolysates, starch, inulin, Jatropha,
wheat straw, levoglucosan, waste oils, animal fats, Jerusalem
artichoke hydrolysates, hydrolysate of cassava starch, wastewater
and mixtures of glucose, xylose and glycerol [37–41]. The idea to
explore the non-edible lignocellulosic biomasses as feedstocks for
oleaginous yeasts may greatly reduce the raw materials cost along
with overall production cost (Fig. 1).
2. Types of lignocellulosic biomass (LB) for saccharification
For biodiesel production, lignocellulosic biomasses constitute
world's largest renewable resources [35,42–46]. Ethanol and
butanol production through bioconversion of lignocellulosic bio-
masses have been already successfully achieved by many
researchers worldwide either laboratory scale or industrial scale
[13,47–53]. However, the technology to convert these residues into
biodiesel has yet to progress from laboratory scale to the pilot
scale. The bulk amount of these residues is obtained from agri-
cultural, industrial, and forest sources (Fig. 2) [54]. It is to be noted
that all available residues are usually not utilized for biodiesel
production, some of these are used for fodder, manure production,
and paper industries or directly utilized for burning purposes as
fuels [55]. The main obstacle behind this issue is the statistical
analysis of various lignocellulosic biomasses available for biodiesel
production.
2.1. Agricultural residues
Various types of agricultural residues are used extensively as
lignocellulosic biomass feedstocks to obtain a lot of sugars for lipid
production experiments. Generally, these residues are allocated
into two groups such as the crop residues and the agro-industrial
residues [56]. The crop residues are the remaining parts of the
crops after collection of useful materials such as grains while agro-
industrial residues are the by-products of post-harvesting pro-
cesses. The most common agricultural wastes such as sugarcane
bagasse, corn stover, corn stalks, rice and wheat straws are
extensively used for microbial lipid production [57,58].
2.2. Non-edible crops or energy crops and forest residues
World's most fertile lands are used for cultivation of food crops.
However, with the rapidly increasing population, we will have to
face the problem of food shortage and energy crisis in the near
future. Earlier researchers were more focused toward agricultural
residues and some valuable energy crops such as corn stover and
switchgrass, however, woody biomass obtained from forest
remain important feedstocks for biofuel production [59–62]. These
residues constitute second largest non-edible lignocellulosic bio-
mass next to agricultural residues, that are available after removal
of extraneous materials to get proper shape and size of trees [63].
These residues cover a large area of the world in huge quantity
without affecting the sustainability of environment [64]. The
important forest residues are those that are not utilized for saw
logs, such as dead wood and top thin branches. The other impor-
tant sources of lignocellulosic biomasses are derived from wood
 A. Patel et al. / Renewable and Sustainable Energy Reviews 62 (2016) 836–855 839

Fig. 1. (a) Flow chart of biodiesel production from oleaginous yeast utilizing non-edible lignocellulosic biomasses as feedstock. (b) Determination of lipid accumulation in
oleaginous yeast cells. Representative images for lipid droplets formation in oleaginous yeast using fluorescence microscopy of live cells stained with BODIPY495/505 and the
estimation of FAME profile through various techniques.

processing industries (sawmill rejects and sawdust) and recycled
woods that are resulting from destruction of buildings, pallets,
cardboard boxes and wooden packing crates [18,65].
2.3. Industrial residues
The waste or by-products generated by industrial activities
have very harmful impact on the environment in various aspects if
they are not properly disposed off. In many cases, the treatment of
these wastes are often more costly than the production cost and
are dealt with environment unfriendly ways [66]. Furthermore,
the treatment of organic wastes through anaerobic digestion
requires skills to ensure safety due to the accumulation of harmful
gases. A substitute to anaerobic digestion, the waste products can
be treated by oleaginous microorganisms to metabolize in its the
cellular compartment as lipid droplets that are further converted
into biodiesel [60]. Industries related to palm oil, pulp and paper,
natural rubber, and biodiesel produce a large amount of processing
wastes that contain abundant organic materials [67]. Biological
processes are commonly used for the treatment of the organic
contaminants present in the waste waters or effluents released
from the industries. These are basically of two types, aerobic and
anaerobic. However, the biological treatment including micro-
organisms is more suitable to reduce the toxic organic compound
load in treated effluents. Currently, bacteria, fungi, yeasts, and
algae are extensively used for same purposes. The treatment
process can be integrated with high-quality biodiesel production
840 A. Patel et al. / Renewable and Sustainable Energy Reviews 62 (2016) 836–855
Fig. 2. Different types of non-edible lignocellulosic biomasses and their exploita-
tion as cultivating and lipid production medium for oleaginous yeasts.
by oleaginous yeast (OY) utilizing organic materials present in
industrial effluents [68].
3. Pretreatment of lignocellulosic biomasses
To boost the digestibility of lignocellulosic materials, different
pretreatment methods such as mechanical, thermal, acid, alkaline
and oxidative pretreatment have been taken into consideration.
The other pretreatment methods currently used consists of ozo-
nolysis, liquid hot water, uncatalysed steam explosion, acid acti-
vated steam explosion, ammonia fiber explosion, and CO2 explo-
sion and the selected method is based on the nature of the initial
content of the taken materials [69]. All existing methods have a
different mode of action on cellulosic materials and require dif-
ferent types of neutralization reactions to remove the generated
inhibitors present in the fermenting medium used by the oleagi-
nous microorganisms. Overview of pretreatment of lignocellulosic
biomass is presented in Fig. 3. Various pretreatment methods for
digestibility of lignocellulosic biomasses utilized by oleaginous
yeasts are reported in Table 1.
3.1. Mechanical pretreatment
Milling is the best mechanical pretreatment method of lig-
nocellulosic biomasses to minimize the particles size of biomass
and destruct the crystallinity [70]. Due to shearing of biomass
specific surface area increases with reduction in the degree of
polymerization that increases overall hydrolysis process [71]. The
absence of inhibitors (like furfural and HMF, hydro-
xymethylfurfural) after milling is advantageous for growing olea-
ginous microorganisms in fermenting media. However, milling is
still not economical due to the high energy requirement in the
overall process.
3.2. Thermal pretreatment
In thermal pretreatment, lignocellulosic biomasses are heated
above 150 °C so that hemicellulosic part of biomass become
solubilize followed by lignin [72]. Xylan and glucomannan are two
main components of hemicellulose in which xylan is least ther-
mally stable [73]. The heating of lignocellulosic biomasses at ele-
vated temperature (4160 °C) causes the production of inhibitors
that inhibit the growth of bacteria and yeasts [74].
3.3. Acid and alkali pretreatment
To enhance the solubility of hemicellulose, lignocellulosic bio-
masses can be treated with dilute or strong acids at ambient
temperature. Strong acid pretreatment is more pronounced than
dilute acid, however, strong acid pretreatment generates more
volatile products and inhibitors [19,75]. For enzymatic hydrolysis
acid pretreatment is required to enhance the solubility of hemi-
cellulose. In the process of alkaline pretreatment dissolved poly-
saccharides are converted into xylan but the temperature is kept
low in order to minimize peeling off end groups which result into
polysaccharides lost. During alkaline pretreatment, biomass itself
consumes some of the alkalies and remaining alkali used for fur-
ther reactions [74]. Both acid and alkali pretreatment methods are
used by industries routinely. The major difference between these
two methods are solubilising of materials as in acid pretreatment,
hemicellulose is solubilised rapidly, whereas solubilization of lig-
nin fraction is observed in alkali pretreatment. This is the advan-
tage of using an alkali pretreatment method where lignin fraction
removed without degrading the other part of biomass [69]. Salt
formation is a major drawback of using this pretreatment method,
which affects the hydrolysates composition.
3.4. Oxidative pretreatment
To increase the accessibility of cellulose from hemicellulose and
lignin, various oxidizing agents such as H2O2 and peracetic acid are
added to biomass. The treatment with the oxidizing compound is
very selective due to certain chemical reactions such as cleavage of
alkyl aryl ether linkages, electrophilic substitution or displacement
of side chains [60]. If the selection of oxidant is not proper, a large
amount of hemicellulose and lignin are lost and also due to oxi-
dation of lignin, many soluble aromatic compounds and inhibitors
are formed.
3.5. Steam explosion/steam pretreatment
To combat the problem associated with inhibitors formation
and to increase the accessibility of cellulose for enzymatic
hydrolysis, steam pretreatment is frequently used. The treatment
is performed in the largely closed vessel at elevated temperature
(up to 240 °C) and pressure. After few minutes of reaction, the
steam is released and treated biomass is rapidly cooled. The pre-
sence of moisture content in the biomasses hinder the overall
process, therefore, requires long steam pretreatment duration [76].
3.6. Liquid hot water (LHW)
The other way of biomass treatment is liquid hot water, where
water is held at supercritical temperature and pressure (374 °C at
22 MPa). The operating temperature of the water should be
around 180–230 °C and pressure at 27.6 MPa before rapid cooling.
The major advantages of using the LHW treatment over steam
pretreatment is the presence of solubilized hemicellulose and
lignin products in lower concentrations due to higher water con-
tent and it reduce the formation of degradation products like
furfural [77].
 A. Patel et al. / Renewable and Sustainable Energy Reviews 62 (2016) 836–855
Fig. 3. Overview of pretreatment of lignocellulosic biomasses [6].
3.7. Carbon dioxide (CO2) pretreatment
LB treatment with CO2 is similar to the liquid hot water treat-
ment in which supercritical fluid is used at a temperature range
between 110 and 170 °C with a pressure range of 3100–4000 psi. It
can be used for substrate digestibility to remove lignin proportion
from biomass. CO2 reacts with H2O forms carbonic acid (H2CO3)
which assists hydrolysis of polymers. Under high pressure, CO2
becomes acidic and disrupt the structure of cellulose and hemi-
cellulose. Moreover, treatment under low temperature is more
favorable due to the prevention of monosaccharides degradation.
Under high pressure, it penetrates into small pores of lig-
nocellulose which can improve delignification [78]. For an exam-
ple, supercritical carbon dioxide pretreatment of southern yellow
pine (soft wood) and Aspen (hardwood) were showed highest
sugar yields compared to the thermal pretreatment [4].
3.8. Ozonolysis
Ozonolysis is an efficient oxidant that is used for delignification
of biomass [66]. The pretreatment is performed at room
841
temperature and normal pressure to avoid the formation of toxic
compounds [79]. It can be used to degrade lignin without affecting
the cellulose proportion of different lignocellulosic materials such
as wheat straw, bagasse, cotton straw and poplar sawdust [80].
The major drawback of using this pretreatment is the large
requirement of ozone which makes it expensive. For last decade,
utilization of ionic liquids (IL) and other green solvents for pro-
cessing of lignocellulosic biomass have been increased [81]. Due to
its large solubility range, a wide variety of biomasses can be
treated such as cotton [82], corn stover [83], bagasse [84]
switchgrass [85], wheat straw [86].
3.9. Green solvents
Green solvents are made-up of salts which have a small anion
and a large organic cation. Due to the existence in liquid form at
room temperature and having very low vapor pressure it can be
regenerated after a large number of treatment operations and thus
reducing the cost of processing. The formation of inhibitor is
reduced during the pretreatment process and its reusable property
makes it a greener solvent.
 842
Table 1
Utilization of various pretreated non-edible lignocellulosic biomass by oleaginous yeasts for lipid production.

A. Patel et al. / Renewable and Sustainable Energy Reviews 62 (2016) 836–855

Oleaginous yeasts Utilized Carbon sources Pretreatment methods Dry cell weight (g/l) Lipid yield (g/l) Lipid content (%) References

Rhodosporidium toruloides Y4 Jerusalem artichoke hydrolysates Anthrone-sulfuric acid 70 39.55 56.5 [161]
Rhodosporidium toruloides ATCC 10788 Wheat straw hydrolysate Dilute acid 9.9 2.44 24.6 [199]
Rhodosporidium toruloides 21167 Cassava starch Amylases produced by co- culture microorganism 20.1 13.04 64.9 [200]
Rhodosporidium toruloides 21167 Cassava starch Crude amylase preparation 18.5 11.69 63.2 [201]
Rhodotorula graminis Corn steep solids Dilute acid 15.14 7.9 52.18 [202]
Rhodosporidium kratochvilovae HIMPA1 Hemp seed aqueous extract Sonication 15.10 8.39 55.56 [180]
Rhodotorula glutinis ATCC 204091 Detoxified liquid wheat straw hydrolysate Dilute acid 11.8 2.44 20.7 [203]
Rhodotorula glutinis ATCC 204091 Non-detoxified liquid wheat straw hydrolysate Dilute acid 13.8 3.45 25.0 [203]
Trichosporon dermatis CH007 Corncob Enzyme 24.4 9.78 40.1 [204]
Trichosporon cutaneum ACCC 20271 corncob residues hydrolysate Acid pretreatment 38.4 12.3 32.0 [179]
Trichosporon Fermentans HWZ004 Rice straw Dilute acid pretreatment 26.4 13.78 52.2 [205]
Trichosporon coremiiforme Corncob acid hydrolysate Dilute sulfuric acid 20.4 7.7 37.8 [206]
Trichosporon fermentans Waste sweet potato vines hydrolysate Enzyme hydrolysis 26.96 9.6 35.6 [207]
T. fermentans CICC 1368 Rice straw Dilute sulfuric acid 28.6 11.5 40.1 [208]
Lipomyces starkeyi ATCC 12659 Detoxified liquid wheat straw hydrolysate Dilute acid 12.7 3.69 29.1 [164]
Lipomyces starkeyi ATCC 12659 Non-detoxified liquid wheat straw hydrolysate Dilute acid 14.7 4.58 31.2 [164]
Yarrowia lipolytica ATCC 20460 Detoxified liquid wheat straw hydrolysate Dilute acid 7.2 0.32 4.4 [164]
Yarrowia lipolytica ATCC 20460 Non-detoxified liquid wheat straw hydrolysate Dilute acid 7.8 0.36 4.6 [164]
Yarrowia lipolytica Po1g Detoxified sugarcane bagasse Hydrochloric acid 11.42 6.68 58.5 [112]
Cryptococcus curvatus ATCC 20509 Sweet sorghum bagasse Microwave 15.50 9.92 63.98 [209]
Cryptococcus curvatus ATCC 20509 Sweet sorghum bagasse Microwave with lime 10.83 7.93 73.26 [209]
Cryptococcus curvatus ATCC 20509 Detoxified liquid wheat straw hydrolysate Dilute acid 15.6 4.23 27.1 [199]
Cryptococcus curvatus ATCC 20509 Non-detoxified liquid wheat straw hydrolysate Dilute acid 17.2 6.02 33.5 [199]
Cryptococcus sp. Corncob hydrolysate Dilute sulfuric acid followed by autoclaving 10.8 6.6 61.3 [210]
Cryptococcus curvatus Wheat straw acid hydrolysate Dilute acid 17.2 5.8 33.5 [199]
 A. Patel et al. / Renewable and Sustainable Energy Reviews 62 (2016) 836–855
3.10. Biological pretreatment
Previously, pretreatment with fungi has been explored for lig-
nocellulosic pulp and paper technology. It is the eco-friendly, cost
effective, regenerative, quick and efficient approach in which lig-
nin degrading enzyme secreted by the fungus is used for enhan-
cing enzymatic saccharification [87]. There are several white-rot
fungi such as Phanerochaete chrysosporium, Cyathus stercolerus,
Pycnoporus cinnarbarinus and Ceriporiopsis subvermispora have
been used for lignin degradation of different lignocellulosic bio-
masses [77,88].
3.11. Microwave pretreatment
Pretreatment of LB with a microwave is a physicochemical
process in which biomass are soaked in dilute chemical reagent
and further treated with microwave radiation for 5–20 min [89].
The use of dilute alkali along with microwave treatment is more
suitable [90]. It has been earlier reported that microwave pre-
treatment of rice straw and bagasse soaked in water, enhance the
generation of total reducing sugars by a factor of 1.6 and 3.2 times,
respectively. Microwave pretreatment in combination with NaOH
increase the efficiency of enzymatic hydrolysis of rice straw and
obtained the maximum amount of reducing sugars [91].
4. Oleaginous yeasts: a promising feedstock for biodiesel
production
Microorganisms are more suitable candidates for biotechnolo-
gical experiment purposes as they have many advantages over
other sources. They have great diversity among them and can
grow exponentially while utilizing cheap substrates. Wild strains
can be easily genetically manipulated to perform various tasks and
cultivated at large scale. Among several microorganisms, yeasts
are more promising for biotechnological experiments as they
devoid of endotoxins which are more pronounced in the case of
bacterial species. When bacterial species are grow in the presence
of excess carbon source, it metabolizes carbon into poly-
saccharides and fatty acids in the form of polyhydroxyalkanoates
while yeast accumulates excess carbon as glycogen or lipid in the
form of Triacylglycerols (TAG). Yeasts have the unique property to
produce lipids from different carbon sources, even utilize a various
form of fatty acids present in the culture media. Even, they can
accumulate lipids with similar triglycerides composition as pro-
viding in fermenting medium [92]. It is interesting to know about
molecular aspect of lipid accumulation in cellular compartment of
oleaginous microorganisms [93]. It has been previously reported
that several oleaginous microorganisms can accumulate more than
70% lipid on their cell dry weight basis, moreover, the accumulated
lipids are similar in composition with vegetable oils [24]. Non-
oleaginous yeast such as Saccharomyces cerevisae and food yeast
(Candida ultilis) cannot accumulate lipid content more than 10% of
their total dry biomass [94]. However, when they grow in
nitrogen-limited medium with an excess carbon source, the
amount of mannans and glucans content increases, while in the
case of oleaginous yeast the excess carbon source gets converted
into lipids [23]. Oleaginous yeasts shows three phases of growth
under nutrient limitation condition (i) exponential growth phase
where cells go to rapid proliferation (ii) lipid accumulation phase
where cells show minimum growth due to limitation of nutrient
e.g. nitrogen, phosphorus, and sulfur etc. and (iii) stationary phase
or late accumulation phase where catabolic breakdown of lipid
occur with little bit accumulation of lipid. When nitrogen exhausts
843
from the culture medium, adenosine monophosphate deaminase
gets activated and catalyze the conversion of AMP into inosine 5’-
monophosphate and ammonium (Fig. 4) [95]. The adenosine
monophosphate deaminase enzyme is present in oleaginous yeast
but no such absolute dependency of this enzyme in non-
oleaginous yeast [96]. Further, the decreased concentration of
AMP responsible for the inactivation of isocitrate dehydrogenase
and isocitrate does not metabolize via tricarboxylic acid cycle
(TCA) [97]. In oleaginous yeasts, phosphatidic acid (PA) are formed
either by glycerol-3-phosphate (G-3-P) or by dihydroxyacetone
phosphate (DHAP) which is totally dependent on the availability of
carbon sources. Three phosphatidate phosphatase (PAP) iso-
enzymes are required to convert PA into Diacylglycerol (DAG)
through dephosphorylation reaction (Fig. 4). The last steps of de-
novo lipogenesis in oleaginous yeasts are varied in different strains
and show dependency on acetyl-Co-A and binds with DAG back-
bone by diacylglycerol acyltransferases that finally form TAG [98].
It has been reported that in Rhodotorula glutinis this reaction is
performed by a 35 kDa acyl–acyl carrier protein synthetase [99].
The overall reaction of TAG synthesis is known as “Kennedy
pathway” [31]. Accordingly, glucose is converted to pyruvate via
glycolysis and transported into mitochondria through the proton-
linked transporter. In mitochondria, pyruvate is converted into
acetyl-Co-A by decarboxylative dehydrogenase which further
reacts with oxaloacetate and gives citrate. Citrate forms ATP when
enters into Krebs cycle through ETC present in the mitochondrial
membrane. However, when lipid accumulation conditions are
provided to yeast cells, citrate is exchanged with intracellular
malate via citrate/malate translocase. Citrate is finally cleaved into
acetyl-Co-A and oxaloacetate by ATP–citrate lyase (ACL). Acetyl-
Co-A formed in this reaction is converted into malonyl-Co-A by
acetyl-Co-A carboxylase enzyme. In the de-novo synthesis of
lipids, both acetyl-Co-A and malonyl-Co-A are added together to
form fatty acid chains between 14 and 16 carbons long (Fig. 4).
Moreover, the fatty acid profile of oleaginous yeast is totally
dependent on provided culture conditions; it can be either envir-
onmental stress or physical stress conditions [98,99]. The fatty acid
profile of oleaginous yeasts varies in different species, however,
the main fatty acids produced by these yeasts are myristic acid
(C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid
(C18:0), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid
(C18:3). The obtained fatty acids are similar to those produced by
plants and algae. Gill et al. [26] earlier reported that oleaginous
yeasts Candida 107 accumulate more lipid in phosphate-limited
conditions (high carbon to phosphorus, C/P molar ratio) in growth
medium. Granger et al. [100] studied on the effect of various
nutrients (nitrogen; N, phosphorus; P, zinc; Zn or iron; Fe) lim-
itation on fatty acid production by Rhodotorula glutinis and con-
cluded that the P – limitation supports maximum fatty acid pro-
duction in this yeast. The same results were also observed in
Rhodosporidium toruloides Y4, that produced a high amount of
lipid under P-limited condition in growth medium [101]. In other
example, C. utilis synthesized more intracellular non-polar lipid
contents under the phosphate-limited condition, while, the polar
lipids remain relatively constant [69]. It has been suggested that
the R. glutinis showed a little bit growth under the N-limited
condition and stopped growing when N was totally exhausted
[102]. However, the condition was totally different in phosphate
exhausted condition, the culture show increased cell density and
lipid-free biomass. There are so many other factors which affect
the lipid accumulation such as temperature, pH, the presence of
trace elements, aeration rate, and dissolve oxygen [103–105].
844 A. Patel et al. / Renewable and Sustainable Energy Reviews 62 (2016) 836–855
Fig. 4. Schematic diagram of lipogenesis in oleaginous yeast and role of citrate and malate as precursors of acetyl-Co-A. Conversion of malonyl-Co-A from acetyl-Co-A by
acetyl-Co-A carboxylase enzyme and both act as precursors for the synthesis of TAG [95,96,126].
5. Comprehensive procedure for biodiesel production using
oleaginous yeasts
The idea to produce biodiesel from oleaginous yeast deals with
the screening and selection of oleaginous species. The first step
after selection of oleaginous yeast is the implementation of culti-
vation system for growth and lipid accumulation. The next step is
to harvest the biomass and extract the lipid from this oleaginous
yeast, followed by the transesterification of extracted lipid into
fatty acid methyl esters (FAME). The schematic diagram of bio-
diesel production from oleaginous yeast is presented in Fig. 1a. The
estimation of biodiesel quality is a vital step before supplying it to
the distribution unit Fig. 1b.
5.1. Screening of high triacylglycerol (TAG) accumulating oleaginous
yeasts
The first step of biodiesel production from oleaginous yeast is
the screening and selection of a suitable candidate that can grow
on a large number of low-cost substrates and accumulate high
amount of lipids in its cellular compartment. The yeast species are
cosmopolitan in nature as they are found in all natural ecosystems
such as in freshwater, marine water, in soil and from the surface to
a depth of the ocean. Some of them also belong to extremophiles
i.e. they extensively colonize at higher altitude, low temperatures,
low oxygen availabilities, and deep oceanic water. Hence, in order
to search a suitable candidate from a wide range of samples of
different niches, there is a compulsive need to develop a simple,
reliable and rapid method for screening [12,21,106,107]. Chen et al.
[108] performed experiment for the screening of oleaginous yeasts
based on their properties to grow on lignocellulose degradation
compounds. Seven typical lignocellulose degradation products
were provided to different strains. Trichosporon cutaneum 2.571
was selected as an optimal candidate to tolerate the degradation
products and screened for the utilization of lignocellulose hydro-
lysate as fermentation medium [108]. In another experiment of
screening, eight hundred and eighty-nine yeast strains were iso-
lated. The screening was performed with Sudan Black B dye
(lipophilic stain) and it was found that only 23 strains were the
best lipid producer. Kodamaea ohmeri and Trichosporonoides spa-
thulata were identified as potential candidates to grow on crude
glycerol-based medium and accumulate 53.28% and 41.50% lipid
content, respectively [109]. The screening of oleaginous yeast is
usually based on two facts, that is, fatty acids synthesized by these
yeasts should be satisfied the criteria for biodiesel feedstocks and
grown on a wide range of low-cost renewable substrates [110]. The
other factors considered for the selection of oleaginous yeasts are
mentioned below:
 The lipid productivity of oleaginous yeast in terms of g L  1 d  1
should be high.
 The TAG amount in total lipid contents should be maximum.
 Upstream and downstream processing should be easy for the
particular oleaginous yeast.
5.2. Optimization of different parameter for higher lipid yield
Before large-scale cultivation of oleaginous yeasts, it is essential
to optimize all parameters such as media components, pH of the
medium, aeration rate and temperature etc.
 A. Patel et al. / Renewable and Sustainable Energy Reviews 62 (2016) 836–855
5.2.1. Selection of carbon sources for fermentation
Yeast cells are more prone to utilize different carbon sources
present in their surrounding environment and the lipid accumu-
lation in their cellular compartments are totally dependent on the
quality and quantity of provided carbon sources. It has been earlier
reported that various oleaginous yeasts such as Y. lipolytica cannot
efficiently utilize xylose as a sole carbon source, however, bio-
conversion of glycerol is preferable than glucose by this yeast
[111]. There is a contradictory report presented by Tsigie et al.
[112], where this yeast utilized D-xylose and D-glucose simulta-
neously when both were present in same media. The consumption
rate of xylose was higher than glucose when provided separately,
while in a mixture of glucose and xylose, glucose competitively
inhibited the uptake of xylose [112]. According to Sha [113], this
oleaginous yeast produced more biomass and lipid when glucose
and xylose were provided in a mixture. In the case of non-
oleaginous yeast, Saccharomyces cerevisiae preferentially utilizes
glucose as sole carbon source, however, in the absence of this
fermentable carbon source (glucose), it is shifted to utilize variety
of other non-fermentable sugar source (glycerol) and suppresses
the transcriptional expression of genes responsible for utilization
of fermentable carbon sources. In oleaginous yeast, both pathways
of sugars utilization are activated simultaneously [114]. Recent
studies revealed that the oleaginous yeasts have a different tran-
scriptional regulation mechanism for non-fermentable carbon
sources, which get expressed 9–12 times more than that of fer-
mentable carbon sources [114]. The role of Snif1 kinase for acti-
vation of a gene responsible for utilization of non-fermentable
carbon sources indicates networking of several unexplained
mechanisms of sugar utilization. Most of the microorganisms
utilize preferably glucose over other sugars and do not shift to
other sugars till glucose is consumed, moreover, the microorgan-
ism is rarely observed which utilize glucose and other sugar
sources simultaneously [32,33].
It has been earlier reported that when non-oleaginous yeast,
Saccharomyces cerevisiae was grown in non-fermentable sugar
(glycerol or ethanol) with fermentable sugar (glucose or fructose),
triggers the extensive series of metabolic regulation at the tran-
scriptional and post-transcription level [115]. This strategy also
triggers the growth rate extensively, which is preceded by a
characteristic upshift in the synthesis of ribosomal RNA and pro-
tein. The yeast cell growing on non-fermentable sources have two
phases of glucose-induced upshift, the first initial rapid phase that
is independent of metabolism and second long term maintenance
phase that is dependent on metabolism [116]. Now, researchers
are trying to develop new strains which are capable of hexoses and
pentoses utilization simultaneously. The genetic engineering is
carried out to overcome with the problem of glucose repression by
modifying the genes involved in glucose signaling and expressing
the genes which are responsible for transporting secondary sugars
and catabolic enzymes. Recently, cellodextrin transporter and an
intracellular b-glucosidase (BGL) were introduced in host micro-
organism to accumulate the little amount of glucose in the med-
ium that enhances the overall consumption of cellodextrin and
non-glucose sugars [117]. Ethanol production by using co- fer-
mentation of mixed sugars have been intensively investigated in
recent years, however, the strategy to utilize mixed sugars for
biodiesel production from oleaginous microorganisms are in
inadequate condition [118]. In this regard different species of
Rhodosporidium, Rhodotorula and Yarrowia have been quite
important oleaginous microorganisms that have the capability of
simultaneous sugar utilization. However, each species have
divergent property to accumulate lipid depending on the nature of
provided carbon source e.g. R. toruloides CBS14 utilized glucose
(hexoses) more efficiently than xylulose and arabinose (pentoses).
It showed higher lipid accumulation when mixtures of
845
carbohydrates (hexoses and pentoses) were provided in fed-batch
cultivation [119]. In another example, R. glutinis CCMI 145 was
grown in batch culture using glucose, showed more lipid content
than on xylose [120]. R. minuta IIP-33 was supplied to sucrose,
fructose, and galactose besides the glucose. The lipid yield (w/w %)
was maximum on glucose (0.48%) than on sucrose (0.30%), fruc-
tose (0.30%) and galactose (0.11%). However, this yeast strain was
not able to utilize lactose as a substrate [121]. Easterling et al.
(2009) suggested that when R. glutinis was grown on glycerol,
dextrose and xylose separately, the lipid contents were 12.97%,
8.56% and 9.08%, respectively while it shows increased lipid con-
tent in the mixture of sugars (dextrose plus xylulose) [122]. It has
been observed that when R. glutinis was grown on dextrose plus
glycerol, the monounsaturated fatty acid contents were increased
while compared to cells grown on dextrose or glycerol, individu-
ally. The important regulatory biochemical mechanisms for lipid
accumulation is the nitrogen limitation [23]. When oleaginous
yeast is grown in nitrogen deficient condition (high carbon to
nitrogen; C/N, molar ratio), the excess carbon turned into fatty acid
via de novo pathway of fatty acid synthesis and stored in the form
of large lipid droplets (LD) in the cellular compartment
[94,97,123–125]. Evans and Ratledge suggested that not only
nitrogen limitations but also the quality of nitrogen affect the lipid
accumulation in oleaginous yeasts. They reported that Rhodos-
poridium species was synthesized more lipid when cultivated on
organic nitrogen than grown on inorganic nitrogen [126].
5.2.2. Optimization of dissolved oxygen, aeration rate and pH of the
fermentation medium
The other important factor for lipid synthesis in oleaginous
yeasts is the amount of oxygen present in the medium, however,
the aeration rate (gas volume flow per unit of liquid volume per
minute) is not entirely accountable for lipid accumulation. For
instance, some yeast species such as C. utilis and S. cerevisiae
showed increased lipid accumulation in their compartment under
aerobic condition [94]. However, Choi et al. [127] suggested that
the aeration rate determines the biomass and lipid content of
Rhodosporidium toruloides as the growth rate increases with the
constant lipid production rate (0.012 g lipid/g dry cell mass/h).
Besides the aeration rates, pH of the medium also plays an
important role in the lipid productivity of oleaginous yeasts. The
yeasts grow well in the pH range from 3.0 to 7.0 and it is strain
specific [94]. The growth of oleaginous yeast species and lipid
accumulation in their cellular compartments are two different
phenomena. It has been earlier reported that the lower pH sup-
ports the lipid accumulation in the oleaginous microorganisms
while the same pH cannot be optimal for the growth. Brown et al.
[128] reported that Trichoderma reesei (an oleaginous filamentous
fungus) accumulate highest lipid content at pH 3.2, however, the
maximum growth was observed at pH 4.0. A similar case also
reported in oleaginous yeast by Angerbauer et al. [31]. They
mentioned that Lipomyces starkeyi on sewage sludge accrued
maximum lipid content at pH 5.0 while the highest biomass was
obtained at pH 6.5 [31]. Johnson et al. [129], performed experi-
ments on Rhodotorula glutinis IIP-30 and observed the lipid pro-
duction at different pHs such as at pH 3, 4, 5 and 6 where the lipid
contents were 12%, 66%, 48% and 44%, respectively. They also
suggested a minor change in fatty acid profile with various pH.
5.3. Estimation of TAG accumulation and efficient lipid extraction
Several methods such as GC-FID, GC–MS, TLC-FID, MALDI-TOF-
MS, NMR and HPLC have been introduced to visualize, identify and
quantify the lipid classes of microbial origin. Moreover, chroma-
tographic techniques to identify and quantify the microbial lipid
classes require many tedious processes, including solvent
846 A. Patel et al. / Renewable and Sustainable Energy Reviews 62 (2016) 836–855
extraction of lipids. Fluorescence spectroscopy and microscopy are
recently introduced methods that have many advantages over
chromatographic methods for quantification and visualization of
the lipid classes, respectively. There are several types of dyes used
for the same purposes. Nile Red is the lipophilic stains that are
used to quantify the neutral lipid accumulation within the cells by
fluorescence spectroscopy [130,131]. Sudan black B is also used as
a staining dye to detect the lipid in the microorganisms but it
cannot distinguish between the lipid classes. To overcome this
problem, BODIPY493/505 is used for the staining of microbial strains
to visualize the triacylglycerols (Fig 1b). It is a lipophilic bright
green fluorescent dye, required a minimum staining time of
2 minutes with less quantity of dye. It has resistance to photo-
bleaching and can maintain its fluorescence for longer than
30 min. It is used to locate neutral lipids in a cellular compartment
of oleaginous yeasts [132]. The BODIPY493/505 has another impor-
tant characteristic that it can easily stain different sizes of lipid
droplets in yeast cells, showing bright green culture without any
pretreatment and the uptake of this dye is not affected by cell wall
composition and structure.
Biodiesel productions from oleaginous yeasts have several
limitations regarding their cell disruption and lipid extraction
procedure that are major bottlenecks for its commercial-scale
production. A number of methods for lipid extraction from olea-
ginous yeast are presently under analysis at the laboratory scale
(Bligh and Dyer method, Folch method, ultrasonication, micro-
wave, acid catalyzed hot water treatment, H2O2 with FeSO4,
osmotic shock etc.), however, not a single extraction method is
reported to be suitable for industrial scale in the case of oleaginous
yeasts [27,133]. The extraction of lipids through conventional
methods (Bligh and Dyer method, Folch method) require large
quantities of toxic organic solvents (chloroform and methanol)
including high energy input in the steps of dewatering and drying
of biomass [66,67]. At the industrial scale, lipid extraction carried
out from dry biomass; require high energy input in the drying/
dehydration process [134,135]. In solvent extraction process the
cells should contact with the organic solvent phase to extract the
lipids while in the wet condition the cells cannot mix with sol-
vents and remain in water phase due to their surface charges
[136]. Therefore, it is important to extract the lipid from the wet
biomass in order to assure process feasibility [137]. The cell dis-
ruptions through other physical or mechanical methods have also
certain limitations. Oil press or expeller press is a simplest
mechanical method that is extensively used for extraction of lipids
from algal biomass and oil seeds; however, it is not reported for
extraction of oleaginous yeast lipid. The technique of this process
is high mechanical pressure applying to the cells and the oils from
algal biomass squeezed out. High pressure generates lots of heat
and choking problems. Although it is a cost effective method but
the lipid recovery is not so good. Moreover, it works only with
low-moisture content samples and the drying of samples is
necessary that again requires high energy inputs and high cost
[138]. Bead beating is another method to extract the lipid where
the slurry of biomass spins with fine beads. Here the drying of
samples is not required as in expeller press that reduces the cost of
extraction. However, the lipid recovery from oleaginous micro-
organisms in large scale is yet again obstructing this technique
[139]. Ultrasonication is an alternative technique to extract lipid
from oleaginous microorganisms that overcome the problems
associated with the conventional methods. This technique is
simple, eco-friendly, less time consuming and can be operated in
the mild condition of temperature and pressure, it does not
require any beads or chemicals. Cavitation and acoustic streaming
are two different phenomenon that generates with the ultrasound
applying to the cells. Cavitation creates the pressure on the cells in
the form of microbubbles to disrupt the cell wall and membrane.
However, the extracted lipid quality may be detrimental to the
prolonged use of ultrasonication that produces free radicals in
working solution [140]. It has been reported that microwave
irradiation is also used to extract lipids from seeds and firstly
applied in the mid-1980s. Microwave generally applied to dielec-
tric or polar particles where friction due to inter and intramole-
cular movement produce a high amount of heat. Water molecule
present in intracellular compartment form vapor that exhorts
pressure on cell wall and the cells burst. Therefore, it behaves as
electroporator that makes membrane porous and plays an efficient
role in the extraction process. However, high amount of electricity
is utilized in microwave irradiation that further leads to high cost
mainly on a commercial scale and overall it affects the product
quality [141]. Furthermore, after all, this mechanical or physical
methods to extract the lipids from oleaginous microorganisms,
many researchers have been focused on biological method. Jin
et al. [142] used recombinant b-1,3-glucomannanase plMAN5C
enzyme on the microwave pretreated oleaginous yeast Rhodos-
poridium toruloides Y4 to degrade the cell wall [142]. High enzyme
cost per unit substrate is also an obstacle for the biological treat-
ment method.
5.4. Transesterification
The conversion of fatty acids into fatty acid methyl esters
(FAME) is usually processed through three different techniques;
microemulsions (co-solvent blending), thermal cracking (pyr-
olysis) and transesterification (alcoholysis). Among these techni-
ques, transesterification is the most suitable method to reduce the
viscosity associated with microbial lipids [17,143]. Transester-
ification reaction has various advantages over other methods such
as mild reaction condition, eco-friendly and appropriateness for
various feedstocks. This reaction can be classified into two types:
catalyzed or non-catalyzed. A catalyzed transesterification process
is usually achieved by homogeneous, heterogeneous and enzy-
matic catalysis [144,145]. Homogeneous catalytic transesterifica-
tion is most widely used reaction for biodiesel production: It can
be either acidic or basic, depending upon the composition of fatty
acids. The amount of catalyst, the molar ratio of alcohol to fat,
reaction time and temperature directly affect the final yield of
biodiesel [17]. High free fatty acid (FFA) containing feedstocks are
not suitable for homogeneous catalytic transesterification; this
problem is usually carried out by the reaction with heterogeneous
catalyst [17,146]. Moreover, it has several advantages like reusa-
bility and regeneration of catalyst, simple purification, and
downstream processing. Although enzymatic transesterification is
an alternative to heterogeneous catalytic transesterification where
the lipases isolated from different microorganisms (Penicillium
roqueforti, P. camembertii, Candida rugose, C. antactica, and C.
lypolytica) are used as a catalyst, extensively [147–149]. The mild
reaction condition (pH, temperature, and time), easy separation
and purification of enzymes and simple downstream processing of
products are main characteristics of enzymatic transesterification
reaction [147].
6. Lipid accumulation by oleaginous yeasts utilizing hydro-
lysates of various non-edible lignocellulosic biomasses
There are several literatures and review articles reported for
conversion of LB hydrolysates into oil by oleaginous yeasts
[35,94,103,150–155]. Recently, Leiva-Candia summarized non-
pretreated industrial waste and plant materials as feedstock for
microbial oil production [156]. Utilization of various pretreated
non-edible lignocellulosic biomasses by oleaginous yeasts for lipid
production is presented in Table 1. It has been earlier reported that
 A. Patel et al. / Renewable and Sustainable Energy Reviews 62 (2016) 836–855
oleaginous yeasts are utilized various lignocellulosic biomasses for
lipid accumulation such as R. gracilis was grown on wood hydro-
lysates or molasses accumulating up to 64% lipid in cellular com-
partment [157,158]. Liang et al. [159] earlier reported that Cryp-
tococcus curvatus ATCC 20509 accumulate 73.26% lipid content
while grown in sweet sorghum bagasse pretreated with micro-
wave plus lime, however, when lime was not used with microwave
for pretreatment of sweet sorghum bagasse, the lipid content was
only 63.98%. Same oleaginous yeast Cryptococcus curvatus ATCC
20509 was grown in dilute acid pretreated, non-detoxified wheat
straw hydrolysates, attained a lipid content of 33.5% with 17.2 g/l
dry cell weight while lipid content was decreased to 27.1% when
grown in detoxified liquid wheat straw hydrolysates pretreated
with dilute acid [160]. Rhodosporidium toruloides is also novel
oleaginous yeast that utilized various lignocellulosic biomass
hydrolysates and waste materials such as Jerusalem artichoke
hydrolysates, wheat straw hydrolysates, cassava starch and was
produced 56.5%, 24.6% and 64.9% lipid content with 70, 9.9 and
20.1 g/l dry cell biomass, respectively [161–163]. Yu et al. [164]
compared the lipid content of Rhodotorula graminis ATCC 204091
while grown in the dilute acid pretreated detoxified and non-
detoxified wheat straw hydrolysate, and conclude that non-
detoxified wheat straw hydrolysate was suitable for lipid produc-
tion with 25% lipid content. However, Yarrowia lipolytica ATCC
20460 was reported to accumulate less than 5% of total lipid when
grown in similar hydrolysates [164].
7. Effect of inhibitors generated during hydrolysis of LB on
growth and lipid accumulation by oleaginous yeast
Lignocellulosic biomasses are hydrolyzed to give the mono-
saccharides (C5 and C6) including certain by-products depending
on the feedstocks and type of used pretreatment methods
[165,166]. The obtained by-products such as aliphatic acids, furan
aldehydes, inorganic acids including phenolic and aromatic com-
pound act as inhibitors of microbial growth. The cellulose is
usually degraded to give hexose (glucose) while hemicellulose
gives (pentose) xylose as the main component along with man-
nose, glucose, galactose and acetic acid. At elevated temperature
and pressure hexoses are further degraded to 5-hydroxymethyl
furfural (HMF) and xylose into furfural, respectively. Both, HMF
and furfural by-products finally degraded into formic acids (levu-
linic acid usually formed by HMF). Lignin present in lignocellulosic
biomass generates phenolic compounds after degradation (Fig. 3)
[167]. Palmqvist and Hahn-Hagerdal [9] described the inhibitory
effect of by-products present in lignocellulosic hydrolysates on the
microorganisms and discussed their mechanisms of inhibition.
They divided the by-products of hydrolysates in three major
categories based on their origin: weak acids, furan derivatives, and
phenolic compounds. They also suggested the specific detoxifica-
tion methods for these compounds according to their mechanisms
of inhibition [9]. It is essence need to reduce the number of inhi-
bitors from the hydrolysates before fermentation experiments.
Several types of detoxification methods (chemical, physical and
biological methods) are used to reduce the inhibitory concentra-
tion. Neutralization with chemicals, CaOH over-liming and ion
exchange chromatographic methods are usually used for chemical
detoxification while in physical detoxification volatile inhibitor
compounds (vanillin, furfural, and acetic acid) are removed
through evaporation [168]. Enzymatic treatment of inhibitors
through peroxidase and laccase are preferred biological detox-
ification method. These enzymes are obtained from the lig-
ninolytic fungus such as Trichoderma resei and Aspergillus nidulas
[169]. In the case of biodiesel production from oleaginous yeasts,
the inhibitors tolerant species may reduce the production cost by
847
eliminating detoxification step. During past years, various research
works have been performed to see the effect of the inhibitory by-
products on ethanol fermenting microorganisms [170–176].
However, studies for the effect of these compounds on oleaginous
yeasts are limited. Hu et al. [177] performed an experiment on
Rhodosporidium toruloides to monitor the effect of lignocellulosic
biomass hydrolysis by-products. They showed that among various
inhibitors (furfural, furoic acid, furfuryl alcohol, acetate, 5-HMF,
syringaldehyde, p-hydroxybenzaldehyde and vanillin) only fur-
fural and its derivatives furfuryl alcohol and furoic acid showed
maximum % inhibition of microbial growth (45%) at very low
concentration while at high concentration of acetate, 5-
hydroxymethylfurfural and syringaldehyde showed very low
microbial growth inhibition [177]. Yu et al., 2011 used wheat straw
hydrolysate for microbial oil production with five oleaginous yeast
strains, Cryptococcus curvatus, Rhodotorula glutinis, Rhodosporidium
toruloides, Lipomyces starkeyi, and Yarrowia lipolytica and also
discussed the effect of inhibitors (acetic acid, Furfural, and HMF)
on their growth. Prior to detoxification or non-detoxified wheat
straw hydrolysate contain acetic acid (4.0 7 0.2 g/l), Furfural
(0.44 70.01 g/l) and HMF (0.05 70.00 g/l) respectively, while after
detoxification the concentration of furfural and HMF were less to
0.03 70.00 g/l and 0.02 70.00 g/l respectively. Among all above
mentioned oleaginous yeast, Cryptococcus curvatus showed more
tolerance to inhibitors [162]. Yen et al. [178] worked on oleaginous
yeast Rhodotorula glutinis in an airlift bioreactor to utilize the rice
straw hydrolysate as a carbon source. The inhibitors formic acid
(2.370.2 g/l), acetic acid (4.9 70.1 g/l), HMF (0.11 70.1 g/l), and
furfural (0.2 7 0 g/l) were present in rice straw hydrolysate,
respectively [178]. Gao et al. [179], described that the oleaginous
yeast Trichosporon cutaneum ACCC 20271 utilized corncob residues
hydrolysate for the production of lipid. Corncob residues hydro-
lysate also contained inhibitors such as HMF, furfural, formic acid,
acetic acid, levulinic acid but all of these were removed during the
xylitol production process, some inhibitor residues and the water
insoluble phenolic derivatives still existed (1.0 g/l of acetic acid
and 0.15 g/l of phenolic compounds) [179].
8. Fatty acids profile of oleaginous yeasts grown on various
non-edible lignocellulosic biomasses
Oleaginous yeast usually accumulates lipid in the form of dia-
cylglycerols (DAG) and triacylglycerols (TAG). The oleaginous yeast
species such as Rhodosporidium, Rhodotorula, Yarrowia, Crypto-
coccus, Candida, Lipomyces and Trichosporon synthesize commonly
myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic
acid (C18:1), along with linoleic acid (C18:2). Fatty acid profiles of
different oleaginous yeasts are listed in Table 2. The fatty acid
profile of a particular oleaginous yeast is totally dependent on
provided medium and cultivation conditions, e.g. Rhodosporidium
toruloides Y4 was grown in glucose synthetic medium with certain
inhibitors such as acetic acid, HMF, syringaldehyde, furfural,
vanillin, and PHB, showed different quantity of C14:0, C16:0, C18:0,
C18:1, C18:2 and C18:3. It has been reported that R. kratochvilovae
HIMPA1 grown on various substrates synthesized mainly myristic
acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid
(C18:1), along with linoleic acid (C18:2) and traces of linolenic acid
(C18:3) [180–182]. R. kratochvilovae HIMPA1, grown on Hemp seeds
aqueous extract (HSAE) the FAME profile contains mainly of C16:0,
5.90%; C18:0, 25.10%; C18:1, 37.5%; along with C20:0, 22%; C22:0, 6.5%,
and an unusual fatty acid C27:0, 3% [180]. While the fatty acids
profile was changed when this oleaginous yeast is grown in non-
edible lignocellulosic biomass of Cassia fistula L. fruit pulp, it
contains mainly C16:0, 43.06%; C18:0, 28.74%; and C18:1, 17.34% as
major fatty acids [182].
 848
Table 2
Fatty acids profiles (% fatty acids) of various oleaginous yeasts (OY) grown of non-edible lignocellulosic biomasses.

Oleaginous Yeasts Medium C 14:0 C 16:0 C 16:1 C 18:0 C 18:1 C 18:2 C18:3 C 20:0 C 22:0 References

Rhodosporidium toruloides 21167 Cassava starch 1.9 21.6 nd 5.8 51.6 17.7 nd nd nd [163]
Rhodosporidium toruloides 2F5 Inulin 0.01 22.14 nd 13.79 52.19 10.96 nd nd nd [211]
Rhodosporidium toruloides Lignocellulosic hydrolysate 1.19 29.31 0.72 9.68 49.36 9.62 2.26 0.31 0.14 [212]
Rhodosporidium toruloides 21167 Hydrolysate of cassava starch 1.66 30.51 1.5 5.59 53.34 7.4 nd nd nd [213]
Trichosporon cutaneum Corncob acid hydrolysate nd 12.5 nd 6.6 72.1 5.6 nd nd nd [179]
Rhodotorula glutinis Lignocellulosic biomass hydrolysate 1.04 13.3 0.95 5.1 55.5 20.2 6.3 nd nd [36]
Cryptococcus sp. SM5S05 Corncob hydrolysateþ Reducing sugar (2:2; w-v) 0.5 22.4 nd 7.0 58.2 6.9 0.7 2.2 0.8 [214]
Corncob hydrolysate þ Reducing sugar (2:4; w-v) 0.5 22.1 nd 7.5 57.2 7.2 0.8 2.1 0.9

A. Patel et al. / Renewable and Sustainable Energy Reviews 62 (2016) 836–855

Trichosporon cutaneum Corncob acid hydrolysate nd 28.0 nd 16.5 46.6 4.9 nd nd nd [215]
Rhodotorula graminis Corn stover hydrolysate nd 20.51 nd 7.16 42.12 17.15 2.89 nd nd [216]
Trichosporon coremiiforme Corncob acid hydrolysate on 10th day of fermentation nd 20.4 nd 22.9 39.4 12.5 Other fatty acids 4.9 [217]
Corncob acid hydrolysateþ 40 g/l glucose nd 22.5 nd 26.6 39.3 7.7 Other fatty acids 3.8
Corncob acid hydrolysateþ 40 g/l arabinose nd 22.6 nd 25.4 39.3 9.0 Other fatty acids 3.7$
Rhodosporidium toruloides Non-hydrolyzed levoglucosan 0.4 24.3 0.2 10.1 53.2 6.8 0.3 0.7 2.4 C24:0 [218]
2.4
Rhodotorula glutinis 0.2 25.1 0.8 9.2 50.8 7.2 1.1 0.4 0.7 3.5
Cryptococcus sp. SM5S05 Corncob hydrolysateþ 4 g/l glucose 0.4 21.1 nd 7.1 58.9 7.0 0.7 1.6 0.7 [219]
Corncob hydrolysateþ 6 g/l glucose 0.5 21.0 nd 7.5 57.5 7.2 0.8 2.0 0.9
Trichosporon dermatis Hydrolysates of corncobs 9th day of fermentation nd 27.1 nd 14.3 40.9 9.9 Other fatty acids 7.8 [204]
Corncob acid hydrolysateþ 70 g/l glucose nd 26.1 nd 15.0 43.8 9.4 Other fatty acids 5.7
Corncob acid hydrolysateþ 80 g/l glucose nd 26.7 nd 13.2 43.7 11.1 Other fatty acids 5.3
Corncob acid hydrolysateþ 90 g/l glucose nd 27.7 nd 13.6 43.4 10.0 Other fatty acids 5.3
Trichosporon fermentans 100% molasses nd 25.35 nd 18.04 2.87 nd 31.37 Other fatty acids 22.38 [220]
100% Molassesþ 10% Sweet potato vines hydrolysate (SVH) nd 32.86 nd 17.06 4.25 nd 25.27 Other fatty acids 20.74
Glycerol minimal media þ SVH nd 33.1 nd 31.95 23.12 nd nd Other fatty acids 11.83
Y. lipolytica Non-detoxified liquid wheat straw hydrolysate (NDLH) nd 6 nd 2 56 19.9 nd nd nd [199]
Detoxified liquid wheat straw hydrolysate (DLH) nd 5.7 nd 0.8 55.3 20.9 nd nd nd
C. curvatus NDLH nd 25.9 nd 15.2 47.7 6.42 nd nd nd
DLH nd 27 nd 15.3 45 7.3 nd nd nd
R. glutinis NDLH nd 23.5 nd 9 43.4 15.4 nd nd nd
DLH nd 22.4 nd 9.3 42.7 17 nd nd nd
R. toruloides NDLH nd nd nd nd nd nd nd nd nd
DLH nd 19.8 nd 5.9 53.4 13.5 nd nd nd
L. starkeyi NDLH nd 36.2 nd 4.5 46.3 3.4 nd nd nd
DLH nd 37.1 nd 5.5 45.1 4.9 nd nd nd
Y. lipolytica Jerusalem artichoke tubers 1.68 28.32 2.12 4.65 58.48 2.14 nd nd nd [221]
Rhodosporidium toruloides 21167 Cassava starch 1.9 21.6 nd 5.8 51.6 17.7 nd nd nd [41]
R. toruloides Carob pulp syrup with 75 g/l of total sugars 1.17 23.2 0.83 5.69 48.86 16.1 2.7 0.18 nd [222]
Carob pulp syrup with 100 g/l of total sugars 1.28 23 1.03 4.93 47 18.4 3.2 nd nd
Sugarcane molasses with 75 g/l of total sugars 1.36 25.6 0.97 5.93 41.9 19.9 4.1 nd nd
Sugarcane molasses with 100 g/l of total sugars 1.58 26.3 1.28 4.31 38.3 22.3 5.7 nd nd
R. toruloides Y4 Corn stalk hydrolysates with closed recycling 1 1.7 27.5 0.7 9.6 54 4.6 nd nd nd [223]
Corn stalk hydrolysates with closed recycling 2 1.3 25.6 0.3 15.4 50.3 6.7 nd nd nd
Corn stalk hydrolysates with closed recycling 3 1.6 27.6 0.5 12 53.5 4.7 nd nd nd
Corn stalk hydrolysates with semi-closed recycling 1 1.3 28.2 0.6 10 51.2 6.8 nd nd nd
Corn stalk hydrolysates with semi-closed recycling 2 1.5 30 0.6 10.6 51 6.3 nd nd nd
Corn stalk hydrolysates with semi-closed recycling 3 1.5 29 0.8 10.2 51.1 5.9 nd nd nd

nd - not determined
 A. Patel et al. / Renewable and Sustainable Energy Reviews 62 (2016) 836–855
9. Estimation of biodiesel properties on the basis of fatty acids
profile
In order to use biodiesel for diesel engines it has to comply
with quality standards given by ASTM D6751 (United States) and
EN 14214 (European Union) for automotive fuel [183,184]. Various
important physical properties which have to be evaluated for
vehicular quality include ester content (% mol/mol), density (kg/
m3), kinematic viscosity (mm2/s), flash point (°C), sulfur content
(mg/kg), carbon residue (%), and cetane number. The other prop-
erties also determine the quality of fuel for efficient engine per-
formance such as oxidative stability (h), saponification value
(mg KOH/g), iodine value (gI2/100 g), linolenic acid content (% mol/
mol), degree of unsaturation (%), cold filter plugging properties
(°C), pour point (°C), cloud point (°C), and high heating value/
calorific value (MJ/kg). After biodiesel meets all the above listed
quality parameters, it can directly be used in unmodified diesel
engines [185,186]. FAME composition affects the above listed
physical properties. High saturated fatty acids (SFA) present in
FAME mitigate the biodiesel to auto-oxidation and thereby
increasing its self-life while unsaturated fatty acids (UFA) quan-
tities determine its cold flow plugging properties (CFPP). Therefore
optimum ratio of SFA to UFA is necessary for better cold flow,
oxidative stability, kinematic viscosity and cetane number [187]. In
this study empirical formulas were derived for cetane number
(CN), iodine value (IV), saponification value (SV), degree of unsa-
turation (DU), long chain saturation factor (LCSF), cold filter
plugging point (CFPP), kinematic viscosity (KV), density, oxidative
stability (OS) and high heating value (HHV)/calorific value
[146,184,188–190] and were used to evaluate biodiesel quality
using FAME profile of several oleaginous yeasts as listed in Table 3.
Empirical formulas for estimation of biodiesel properties
[188,191–193]
P
SV ¼ 560ð%FC Þ=M
P
IV ¼ 254 DB  %FC=M
CN ¼ 46:3 þ 5458=SV–ð0:255  IVÞ
DU ð%Þ ¼ MUFA þ ð2  PUFAÞ
LCSF ¼ ð0:1  C16Þ þ ð0:5  C18Þ
CFPP ¼ ð3:417  LCSFÞ–16:477
HHV ¼ 49:43–0:041ðSV Þ–0:015 ðIV Þ 
P
P
lnðKV Þ ¼  12:503 þ 2:496  ln M –0:178  DB
P P
Density ¼ 0:8463 þ 4:9= M þ 0:0118  DB
OS ¼ 117:9295=ðwt %C18 : 2 þ wt%C18 : 3Þ þ 2:5905 where M is
the molecular mass of each fatty acid component, DB the number
of double bonds, FC the % of each fatty acid component, MUFA the
weight % of monounsaturated fatty acids, and PUFA the weight % of
poly unsaturated fatty acids.
Fatty acid unsaturation can be estimated by iodine value (IV).
EU has set a limit of 120 g I2/100 g which excludes sunflower, soya
bean, grape seed oil as potential feedstocks for biodiesel produc-
tion. High IV results in polymerization of glycerides and on heating
causing gum formation [146,186]. The IV values recorded were
within the threshold limit ranging from 19.83 g I2/100 g obtained
in Trichosporon fermentans cultivated in glycerol minimal med-
iaþ SVH to 99.86 g I2/100 g attained in Rhodotorula glutinis grown
in lignocellulosic biomass hydrolysate as shown in Table 2. Higher
CN imparts the better ignition of biodiesel than the conventional
diesel fuel ensuring better cold start behavior, smooth engine run
and complete combustion leading to reduced gaseous and parti-
culate emissions [194,195]. ASTM has set a minimum limit of 47
while EU has a minimum limit of 51. The Degree of unsaturation
(DU) depicts the oxidation stability influencing the feasibility of
prolonged storage [188]. Low DU is desirable making biodiesel
more stable. The highest CN (72.89) and lowest DU (23.12%) was
recorded in Trichosporon fermentans cultivated in glycerol minimal
media þ SVH (Table 2). Higher heating value (HHV) is the amount
849
of heat produced by a unit of diesel after its complete combustion
including the energy in the water vapor formed during the reac-
tion. Biodiesel has approximately 12% less HHV than that of diesel
(39.57–41.33 MJ/kg), thus lower in energy content. This leads to
higher consumption of biodiesel in order to attain similar yield as
that of conventional diesel [196]. The HHV increases with more
carbon in FAME and with an increase in the ratio of carbon and
hydrogen to oxygen and nitrogen [194]. Obtained HHV had ranged
between 39.85 and 42.06 MJ/kg (Rhodotorula glutinis grown in
lignocellulosic biomass hydrolysate and Trichosporon fermentans
cultivated in glycerol minimal media þSVH respectively) as shown
in Table 2. Low-temperature operability of biodiesel can be ana-
lyzed by cold flow plugging properties (CFPP). CFPP is the lower-
most temperature at which 20 ml of biodiesel totally flows under
vacuum through a wire mesh filter screen within 60 s. Once CFPP
is reached, the fuel forms solids which block the engine and make
it inoperable [197]. CFPP is directly dependent on SFA mainly C16:0
and C18:0 as these two fatty acids precipitate faster under lower
temperatures [184,188]. Most of the oleaginous yeast listed in
Table 2 showed CFPP within the limit set by EU ( r5/ r  20), such
as Y. lipolytica ( 13.16,  11, 1.14), L. starkeyi (3.58), R. toruloides
( 7.93, 0.19,  0.13, 0.36, 2.40), R. toruloides 21167 (3.49), Rho-
dotorula glutinis (  3.21), and Trichosporon cutaneum ( 0.92),
respectively. Kinematic viscosity measures the resistance to liquid
fuel flow affecting atomization. High viscosity is responsible for
large droplet size, poor vaporization, narrow injection spray angle
leading to increased emissions, oil dilutions and poor combustion
[195]. High KV is observed at low ambient temperatures, higher
fatty acid carbon number, low density, and trans-configuration of
double bonds. KV limits are set to 1.9–6.0 mm2/s and 3.5–5.0 mm2/
s as per UN and EU standards, respectively [184]. CN and high
heating value are related to the density of biodiesel [186]. Fuel
injection point impels the diesel by volume which depends on its
density. Density also affects the air to fuel ratio and energy con-
tent. KV is directly proportional to the degree of saturation while
density directly correlates with the degree of unsaturation. High
KV along with high-density of biofuel decrease the overall per-
formance of the diesel engine [195]. ASTM D6751-02 limit for KV is
1.9–6.0 mm2/s while for EN 14214 is 3.5–5.0 mm2/s and density
limit is 0.86-–.90 g/cm3. Biodiesel KV and density derived from
analyzed yeasts were within the set limit. Oxidative stability (OS)
reflects the time (h) for which biodiesel can be stored without
undergoing auto-oxidation. The higher the OS, the shelf life of
biodiesel will be long. With the increase in a number of double
bonds, the oxidation rate increases in a nonlinear manner. FAME
with one double bond has oxidation rate of 1 while for double and
triple bonds the rate increases by a factor of 41 and 98, respec-
tively [195,198]. OS is directly correlated to the length of fatty acid
chain and inversely to double bonds in the cis configuration. The
highest OS (57.69 h) was observed in Y. lipolytica grown in Jer-
usalem artichoke tubers. Linolenic acid (C18:3) contains two bis-
allylic groups making it more prone to auto-oxidation. Hence, EU
B100 standards have a limit of 12% linolenic acid content. All the
listed biodiesel have less than 12% of linolenic acid content in its
FAME profile. After estimation of physical properties of all the
yeasts listed in Table 3, four yeasts showing potential for vehicular
quality biodiesel were Y. lipolytica (Jerusalem artichoke tubers)
followed by Trichosporon cutaneum (corncob acid hydrolysate),
Rhodosporidium torulodies 21167 (hydrolysate of cassava starch )
and finally L. starkeyi (NDLH) respectively.
10. Conclusion
According to the survey of National biodiesel board USA, pre-
sent biodiesel production cannot fulfill the increasing demand of
850 A. Patel et al. / Renewable and Sustainable Energy Reviews 62 (2016) 836–855

Table 3
Estimation of biodiesel properties on the basis of the fatty acid profile of oleaginous yeasts listed in Table 2.

Oleaginous Yeasts Medium IV SV CN DU LCSF HHV CFPP KV D OS 18:3

Rhodosporidium toruloides Cassava starch 74.86 191.43 55.72 87 5.06 40.46 0.81 4.24 0.87 9.25 0
21167
Rhodosporidium toruloides Inulin 63.72 191.47 58.55 74.11 9.10 40.62 14.64 4.25 0.87 13.05 0
2F5
Rhodosporidium toruloides Hydrolyzate 65.55 200 56.87 73.84 8.29 40.24 11.85 4.59 0.87 12.51 2.26
Rhodosporidium toruloides Hydrolysate of cassava starch 59.97 195.76 58.88 69.94 5.84 40.50 3.49 4.13 0.87 18.53 0
21167
Trichosporon cutaneum Corncob acid hydrolysate 71.54 18.40 57.49 83.3 4.55 40.75 -0.92 4.53 0.87 23.64 0
Rhodotorula glutinis Lignocellulosic biomass hydrolysate 99.86 197.12 48.52 109.45 3.88 39.85 -3.21 3.96 0.88 7.04 6.3
Cryptococcus sp. SM5S05 Corncob hydrolysate þReducing sugar (2:2; w-v) 63.69 190.41 58.72 73.4 9.14 40.67 14.75 4.74 0.87 18.10 0.7
Corncob hydrolysate þReducing sugar (2:4; w-v) 65.22 189.48 58.48 75.1 8.06 40.68 11.06 4.56 0.88 17.33 0.8
Trichosporon cutaneum Corncob acid hydrolysate 48.45 186.57 63.19 56.4 11.05 41.05 21.28 4.52 0.87 26.66 0
Rhodotorula graminis Corn stover hydrolysate 73.32 173.89 58.99 82.2 5.63 41.20 2.76 4.19 0.87 8.47 2.89
Trichosporon coremiiforme Corncob acid hydrolysate on 10th day of 55.41 183.69 61.88 64.4 13.49 41.06 29.61 4.52 0.87 12.02 0
fermentation
Corncob acid hydrolysateþ 40 g/l glucose 47.03 185.61 63.71 54.7 15.55 41.11 36.66 4.53 0.87 17.90 0
Corncob acid hydrolysateþ 40 g/l arabinose 49.27 186.1 63.03 57.3 14.96 41.06 34.64 4.52 0.87 15.69 0
Rhodosporidium toruloides Non-hydrolyzed levoglucosan 58.37 193.42 59.63 67.6 16.58 40.62 40.17 4.98 0.87 19.20 0.3
Rhodotorula glutinis 59.66 190.34 59.76 68.2 15.56 40.73 36.69 4.98 0.87 16.79 1.1
Cryptococcus sp. SM5S05 Corncob hydrolysateþ4 g/l glucose 64.46 187.99 58.89 74.3 8.31 40.75 11.92 4.74 0.87 17.09 0.7
Corncob hydrolysateþ6 g/l glucose 63.86 187.68 59.09 73.5 9.2 40.77 14.95 4.75 0.87 17.33 0.8
Trichosporon dermatis Hydrolysates of corncobs 9th day of fermentation 52.02 179.31 63.42 60.7 9.86 41.29 17.21 4.52 0.87 14.50 0
Corncob acid hydrolysateþ 70 g/l glucose 53.82 183.09 62.38 62.6 10.11 41.12 18.06 4.52 0.87 15.14 0
Corncob acid hydrolysateþ 80 g/l glucose 56.68 184 61.06 65.9 9.27 41.05 15.19 4.53 0.87 13.21 0
Corncob acid hydrolysateþ 90 g/l glucose 54.52 184.16 62.03 63.4 9.57 41.06 16.22 4.53 0.87 14.38 0
Trichosporon fermentans 100% molasses 84.32 152.07 60.68 65.61 11.55 41.93 23.09 4.31 0.88 6.34 31.37
100% Molassesþ 10% Sweetpotato vines hydro- 69.59 156.17 63.38 54.79 11.82 41.96 23.89 4.30 0.88 7.52 25.27
lysate (SVH)
Glycerol minimal media þ SVH 19.83 172.43 72.89 23.12 19.28 42.06 49.91 4.08 0.87 – 0
Y. lipolytica Non detoxified liquid wheat straw hydrolysate 82.43 160.05 59.37 95.8 1.6 41.63 -11 4.52 0.87 8.51 0
(NDLH)
Detoxified liquid wheat straw hydrolysate(DLH) 82.43 153.17 60 95.8 0.97 41.95 -13.16 4.52 0.87 8.52 0
C. curvatus NDLH 52.02 184.75 62.57 60.54 10.19 41.07 18.34 4.53 0.87 20.95 0
DLH 51.23 183.79 62.93 59.6 10.35 41.13 18.89 4.52 0.87 18.74 0
R. glutinis NDLH 63.85 177.09 60.83 74.2 6.85 41.21 6.92 4.52 0.87 10.24 0
DLH 66.02 177.10 60.28 76.7 6.89 41.18 7.06 4.52 0.87 9.53 0
R. toruloides NDLH
DLH 69.15 178.89 59.17 80.4 4.93 41.05 0.36 4,52 0.87 11.32 0
L. starkeyi NDLH 45.60 177.61 65.40 53.1 5.87 41.46 3.58 4.52 0.87 37.27 0
DLH 47.16 181.94 64.27 54.9 6.46 41.26 5.59 4.53 0.87 26.65 0
Y. lipolytica Jerusalem artichoke tubers 55.89 190.05 60.69 64.88 5.16 40.78 1.14 4.12 0.87 57.69 0
Rhodosporidium toruloides Cassava starch 74.86 191.43 55.72 87 5.06 40.46 0.81 4.24 0.88 9.25 0
21167
R. toruloides Carob pulp syrup with 75 g/ L of total sugars 72.53 192.29 56.18 98.67 2.5 40.46 -7.93 4.29 0.87 8.86 2.7
Carob pulp syrup with 100 g/ L of total sugars 81.45 192.18 53.92 91.23 4.76 40.32 -0.19 3.96 0.88 8.05 3.2
Sugarcane molasses with 75 g/ L of total sugars 81.95 194.45 53.46 90.87 5.52 40.22 2.40 3.96 0.88 7.51 4.1
Sugarcane molasses with 100 g/ L of total sugars 87.48 194.84 52.00 92.58 4.78 40.13 -0.13 3.96 0.88 6.80 5.7
R. toruloides Y4 Corn stalk hydrolysates with closed recycling 1 54.94 191.39 60.80 63.9 7.55 40.75 9.32 4.13 0.88 28.22 0
Corn stalk hydrolysates with closed recycling 2 55.02 193.59 60.46 64 10.26 40.66 18.58 4.12 0.87 20.19 0
Corn stalk hydrolysates with closed recycling 3 54.50 194.71 60.43 63.4 8.76 40.62 13.45 4.13 0.87 27.68 0
Corn stalk hydrolysates with semi-closed recy- 56.25 191.36 60.47 65.4 7.82 40.74 10.24 4.13 0.87 19.93 0
cling 1
Corn stalk hydrolysates with semi-closed recy- 55.21 195.35 60.15 64.2 8.31 40.59 11.88 4.13 0.87 21.30 0
cling 2
Corn stalk hydrolysates with semi-closed recy- 54.80 192.37 60.69 63.7 8 40.72 10.85 4.13 0.88 22.57 0
cling 3

IV—iodine value; SV—saponification value; CN—cetane number; DU—degree of unsaturation; LCSF—long chain saturation factor ; HHV—high heating value; CFPP—cold
filter plugging point ; KV—kinematic viscosity; D—density ; OS—oxidative stability; 18:3—linolenic acid content.

energy in near future. It has been reported that only US consumers
utilized a record of approximately 2.1 billion gallons of biodiesel in
2015. The data was almost 25% higher than consumed in 2014
(1.97 billion gallons) and in 2013 (1.50 billion gallons), respec-
tively. Apart from this, an important fact is the reduction of
America's carbon emissions by at least 18.2 million metric tons. It
is assessed that international biodiesel market continues to grow
in the future. Current biodiesel production is based on high-cost
feedstocks such as palm oil, soybean oil, and other vegetable oils.
In terms of obtainability and sustainability, compared to virgin
vegetable oils, lipids obtained from oleaginous yeasts have been
confirmed to be precise noble feedstocks for high-quality biodiesel
production. The use of oily yeast as biodiesel feedstock represents
one of the promising ways to combat the present scenario of food
security and energy crisis. The utilization of non-edible lig-
nocellulosic biomass as a feedstock for biodiesel production from
oleaginous yeast is more convenient than its production with
vegetable oil or some energy crops. Non-edible lignocellulosic
biomasses are abundant resources that are freely available for
biofuels production without affecting environment sustainability.
However, due to its recalcitrant nature, various pre-treatments are
required to make it suitable for saccharification. Generation of
 A. Patel et al. / Renewable and Sustainable Energy Reviews 62 (2016) 836–855
inhibitors during hydrolysis of lignocellulosic biomasses are a
major obstacle to utilizing it as fermenting media for lipid pro-
duction. The screening and selection of suitable inhibitors tolerant
oleaginous yeasts and proper detoxification methods can over-
whelm the associated problems.
Acknowledgment
Authors are thankful for financial support from the Department of
Biotechnology, Govt. of India, Bio Care Programme, DBT Sanction no.:
102/IFD/SAN/3539/2011-2012 (Grant no.: DBT-608-BIO) and SRF to
Alok Kumar Patel from UGC, India (Grant no.: 6405-35-044).
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