This document provides a critical review of the potential for direct biodiesel synthesis from microalgae biomass. It discusses microalgae species commonly used for biodiesel production and factors that influence lipid accumulation, such as nitrogen starvation. Direct biodiesel synthesis combines lipid extraction and transesterification into a single step, avoiding costly extraction methods. The review analyzes technologies for direct conversion of microalgae biomass to biodiesel and parameters that affect the process. It also evaluates methods to enhance biomass productivity and lipid content in microalgae. Finally, it covers challenges and the economic outlook for microalgae biodiesel production.

1. Contents lists available at ScienceDirect
Renewable and Sustainable Energy Reviews
journal homepage: www.elsevier.com/locate/rser
Sustainability of direct biodiesel synthesis from microalgae biomass: A
critical review
Brandon Han Hoe Goha
, Hwai Chyuan Onga,⁎
, Mei Yee Cheaha
, Wei-Hsin Chenb
, Kai Ling Yuc
,
Teuku Meurah Indra Mahliad
a
Department of Mechanical Engineering, Faculty of Engineering, University of Malaya, Kuala Lumpur, Malaysia
b
Department of Aeronautics and Astronautics, National Cheng Kung University, Tainan 701, Taiwan
c
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
d
School of lnformation, Systems and Modelling, Faculty of Engineering and Information Technology, University of Technology Sydney, NSW 2007, Australia
A R T I C L E I N F O
Keywords:
Biofuel
Microalgae
Oil extraction
Biodiesel conversion
Alternative energy
A B S T R A C T
Microalgae has been identified as a potential feedstock for biodiesel production since its cultivation requires less
cropland compared to conventional oil crops and the high growth rate of microalgae. Research on microalgae
oils often are focused on microalgae oil extraction and biomass harvesting techniques. However, energy in-
tensive and costly lipid extraction methods are the major obstacles hampering microalgae biodiesel commer-
cialisation. Direct biodiesel synthesis avoids such problems as it combines lipid extraction techniques and
transesterification into a single step. In this review, the potential of direct biodiesel synthesis from microalgae
biomass was comprehensively analysed. The various species of microalgae commonly used as biodiesel feedstock
was critically assessed, particularly on high lipid content species. The production of microalgae biodiesel via
direct conversion from biomass was systematically discussed, covering major enhancements such as hetero-
geneous catalysts, the use of ultrasonic and microwave- techniques and supercritical alcohols that focus on the
overall improvement of biodiesel production. In addition, this review illustrates the cultivation conditions for
biomass growth and lipid productivity improvement, the available harvesting and lipid extraction technologies,
as well as the key challenges and future prospect of microalgae biodiesel production. This review serves as a
basis for future research on direct biodiesel synthesis from modified microalgae biomass to improve profitability
of microalgae biodiesel.
1. Introduction
Biofuel has commanded a growing interest globally due to the
limited fossil deposits and the volatile fuel prices. Energy support ci-
vilization, for development and mobility. But the continuous usage of
fossil fuel, especially for transportation, has long being identified major
contributor of air pollutants. The emissions of unburned hydrocarbons,
carbon monoxide and nitrogen oxides [1] are found to take years off
people's lives. This has prompted the needs to find an alternative re-
newable fuel source for transportation and, biofuel being one of the
alternatives. Biofuel can be defined as fuel that is produced directly or
indirectly from biomass sources [2]. Major issues like energy security,
rural development and climate change [3] have catalysed the rapid
development of biofuel.
One of the most extensively studied fuel types is biodiesel, a sub-
stitute for fossil diesel. The advantages biodiesel possess over fossil like
renewability, toxic free, sulphur free and offer better lubricity, have
been well documented [4]. In the last two decades, different genera-
tions of biodiesels are being developed [5,6] and each generation of
biodiesel differs by the feedstock used. Table 1 summarises the differ-
ences between each generation of biodiesels.
In 2015, 69% of the global biodiesel output was produced from
edible vegetable oils such as soybean oil, rapeseed oil and palm oil [12].
Such high percentage of edible sources usage causes a dilemma due to
its implications on food security and commodity prices. Despite the fact
that, oil crops cultivation helps boosted rural areas’ economies but the
large areas needed for fuel's crops cultivation is not sustainable in the
long run.
All of these have led researchers to microalgae, deemed as the third
generation biodiesel. This feedstock has less competition with oil crops
for land availability [2]. Microalgae contain neutral lipids, that can be
used for biodiesel production, as well as, it also contains other high
https://doi.org/10.1016/j.rser.2019.02.012
Received 16 May 2018; Received in revised form 29 January 2019; Accepted 13 February 2019
⁎
Corresponding author.
E-mail address: onghc@um.edu.my (H.C. Ong).
Renewable and Sustainable Energy Reviews 107 (2019) 59–74
1364-0321/ © 2019 Elsevier Ltd. All rights reserved.
T

2. value compounds such as carotenoids (used in the food and feed in-
dustries) and long-chain polyunsaturated fatty acids like DHA and EPA
that are beneficial for human health [13].
Microalgae only produce small amounts of lipid under optimum
growth conditions, but when the organism is subjected to environ-
mental stress, it accumulates lipids as the carbon and energy storage
[14]. The lipid accumulation of the current microalgae mass culture
system is lower lipid than its theoretical maximum [15]. Hence, many
studies have been conducted to enhance the microalgae biomass, lipid
productivity, and production efficiency, concurrently keeping the pro-
duction cost at a minimum [16].
The advantages of microalgae as biodiesel feedstock, parameters
affecting the lipid extraction-transesterification process and the eco-
nomic potential of microalgae biodiesel have been extensively reviewed
[17–19]. Yet, not many reviews were found on direct biodiesel synth-
esis from microalgae biomass. Direct biodiesel synthesis or in-situ
transesterification has omitted the oil extraction process where lipid is
directly transesterified from biomass. This biodiesel synthesis process
has been tested on feedstocks such as Jatropha oilseeds, rapeseed and
rice bran [20–22]. In the conventional method, oil is first extracted out
before the transesterification reaction; hence, various solvents are
needed for these processes. However, in in-situ transesterification only
a single solvent is used for the entire reaction [23]. This process in-
tensification step eliminates the need for excessive solvent and oil re-
fining processes [22]. Additionally, direct biodiesel synthesis helps
prevents lipid loss during processing that maximises the conversion
rate, thus improves the profitability [23].
This paper aims to provide a critical review on the potential of in-
stantaneous biodiesel synthesis from microalgae biomass. The potential
harvesting and extraction methods to increase biomass and lipid yields
are deliberated. Discussions on the different technologies applied to
produce biodiesel from microalgae biomass are included. Besides, the
parameters affecting direct biodiesel synthesis from biomass are de-
liberated from the fatty acid methyl ester (FAME) yield. Furthermore,
the methods to enhance lipid accumulation and biomass productivity
are evaluated. Finally, the challenges and future economic outlook of
microalgae biodiesel will be duly covered as well.
2. Microalgae cultivation conditions
Microalgae can grow anywhere with sufficient sunlight and CO2
[24]. In order for microalgae to be a sustainable and feasible biodiesel
feedstock, the cultivation and production costs must be kept low. High
value compounds like carotenoid, phytosterol, polyunsaturated fatty
acid, etc. (nutraceuticals) can be co-produced from microalgae to di-
versify revenue.
Various microalgae species have been identified as potential bio-
diesel feedstock owing to their high lipid accumulation abilities (2–40%
of their weight) [24]. Table 2 shows the potential microalgae species for
biodiesel production and their lipid content under normal cultivation
conditions.
There are numerous studies on the optimum cultivation methods for
high-lipid-content microalgae (for biodiesel production) and some
common microalgae genus like Nannochloropsis, Chlorella and Spirulina
have been identified as potential feedstock [32–34]. Past works also
concluded that lipid accumulation in microalgae are species dependent
and can be affected by the culture conditions [35]. Despite of that, the
high amounts of lipid often come at the cost of lower biomass pro-
ductivity [36] and the relative amount of each lipid class can also vary
under different culture conditions [37]. Therefore, lipid productivity is
an important factor in determining the feasibility of using microalgae as
biodiesel feedstock.
Environmental stress such as pH, nitrogen, salinity and light are
common methods use to induce lipids, carbohydrates and other valu-
able compounds [38]. Nitrogen and phosphorus are crucial nutrients
for microalgae growth and lipid production. Nutrient starvation is
found to increase lipid content in microalgae, but at the same time
compromises cell growth. In order to circumvent the growth restriction,
the cultivation of microalgae can be divided into two stages. Sufficient
nitrogen is provided for biomass production in the first stage. In the
second stage, nitrogen starvation is introduced to induce lipid accu-
mulation [38]. In a nutrient-deplete environment, the need for mem-
brane lipids will cease and more fatty acids are converted into neutral
lipids [39]. Cellular growth will be disrupted and all available carbon
will be converted into lipids instead of protein [40].
2.1. Nitrogen starvation condition
Nitrogen starvation causes the microalgae cells to synthesize lipid in
the form of triglyceride fatty acids (TG-FA) that are used as carbon and
energy storage [41]. For biodiesel production, TG-FA is converted to
methyl or ethyl esters via the transesterification process [42]. There-
fore, one of the main criteria for biodiesel-feedstock species selection is
the ability to accumulate high amount of lipid. Table 3 shows several
microalgae species and their lipid content under nitrogen-starved con-
ditions.
Nutrient limitations will affect the photosynthetic capacity of mi-
croalgae but how severe such limitation has on cell division and TG
lipid synthesis is species-specific. Some species are better adaptor than
other species under nutrient limiting conditions. For example,
Nannochloropsis gaditana are able to retain its chloroplast structure and
photosynthetic capacity for a longer period of nitrogen starvation than
Neochloris oleoabundans. The latter species is found to dissipate energy
Table 1
Differences between generations of biodiesel.
Biodiesel Feedstock Processing technology References
First Generation Edible Vegetable Oils Esterification and Transesterification of oils [7,8]
Second Generation Non-edible vegetable oils, waste cooking oil, lignocellulosic feedstock
materials, Animal Fats
Esterification and transesterification of oils/ seeds (utilises organic
catalyst/additives)
[9,10]
Third Generation Aquatic cultivated feedstock (microalgae) Algae cultivation, harvesting, oil extraction, transesterification [2,11]
Table 2
Lipid content of microalgae species under normal cultivation conditions.
Microalgae species Lipid content by weight (%) References
Anabaena cylindrica 4–7 [24]
Chlamydomonas reinhardtii 6 [25]
Chlorella vulgaris 49–52 [26]
Chlorella pyrenoidosa 38 [27]
Chlorella sorokiniana 22–24 [28]
Dunaliella bioculata 8 [28]
Dunaliella salina 6–25 [28]
Nannochloropsis sp. 30 [29]
Nannochloropsis. granulata 28.5 [30]
Nannochloropsis oculata 45 [30]
Neochloris oleoabundans 35–54 [24]
Porphyridium cruentum 9–14 [24]
Prymnesium parvum 22–38 [24]
Scenedesmus dimorphus 10 [31]
Scenedesmus obliquus 30–50 [28]
Scenedesmus quadricauda 1.9 [28]
Tetraselmis sp. 20–50 [29]
B.H.H. Goh, et al. Renewable and Sustainable Energy Reviews 107 (2019) 59–74
60

3. under nitrogen-limited conditions and disintegrate its plastid to reduce
its energy intake [56]. Species with high biomass productivity under
unfavourable conditions is beneficial for lipid production [57].
Roopnarain et.al [58] found that nitrogen concentration is directly
proportional to biomass productivity. Due to this, the overall lipid
productivity in nitrogen-starved conditions may actually be lower than
normal growth conditions [59] because the overall biomass has failed
to reach a desirable amount as cell divisions were disrupted by the lack
of nitrogen. Limited cells are available for lipid accumulation, thus
reducing the overall lipid productivity.
Mendoza et. al [60] has reported that certain species of algae that
lacked cell wall had a higher extractable lipid than those with cell wall.
In addition, some species demonstrated sequential accumulation of
starch and lipids when subjected to nitrogen-deplete condition for an
extended duration, during which all available carbon will be converted
into lipid and starch from chlorophyll will too be converted into lipid.
When this occurs, the space previously occupied by plastid and chlor-
ophyll will be used to store the lipid bodies [45]. This exchange of
starch for lipid via nutrient manipulation is another option for effective
lipid accumulation enhancement. This is an almost similar approach as
bio-engineered-strains, where starch synthesis pathway is blocked to
increase lipid production, but has failed to achieve its objective, largely
due to decreased growth.
2.2. Phosphorus starvation condition
Besides nitrogen, phosphorus is one of the essential nutrients for
biomass growth needed for the synthesis of key molecules such as nu-
cleic acids, adenosine triphosphate (ATP) and phospholipids [61]. ATP
is the primary product of photosynthesis and is crucial for microalgae
growth. Microalgae utilise phosphorus in two steps, the first, through
the construction of organic cellular components such as phospholipids,
and in the second step, the remaining excess is used to produce in-
organic polyphosphate granules [62]. Currently, the agriculture in-
dustry depends on phosphate rocks as phosphorus source, however the
deposits are expected to be exhausted by the end of this century [63].
Owing to that, it is important to practise sustainable use of materials.
Wastewater contains nitrogen and phosphorus that can be
converted into biomass and other bio-products via microalgae.
Delgadillo-Mirquez [64] found that mixed microalgae and bacteria are
able to completely remove phosphate ions at 25 °C in a high rate algal
pond. Hence, the use of wastewater for microalgae cultivation can
significantly reduce biodiesel production cost. However, it should be
noted that some wastewater may contain possible inhibitors that could
impede microalgae growth [65].
Phosphorus starvation has lesser effects on microalgae photo-
synthesis in comparison to nitrogen starvation [66]. In a study of
Chlamydomonas reinhardtii under phosphorus-limiting condition, it is
found that the number of ribosomes was reduced to maintain protein
synthesis and polyphosphate storage [38]. In another example using
Monodus subterraneus, phosphorus starvation is found to induce a six
fold increase in lipid production [67]. Meanwhile, Chu [59] has de-
monstrated that the biomass production of Chlorella vulgaris under
sufficient phosphorus supply in a nitrogen-starved condition was si-
milar to that of nitrogen-sufficient condition, where the maximum lipid
content attained was 58.39 mg/L/day. Therefore, it can be deduced
from these studies that the impact from phosphorus starvation is less
than that of nitrogen starvation.
2.3. Calcium starvation and magnesium supplement condition
Other important nutrients for microalgae growth but rarely studied
are calcium and magnesium [68]. Magnesium is used for plant protein
synthesis and is also part of the enzymes involved in photosynthetic
carbon fixation and metabolism [69]. Meanwhile, calcium is used in
cell wall and membrane structure and serves as an intracellular mes-
senger that coordinates responses to environmental changes [68]. There
are limited studies on microalgae responses during magnesium and
calcium starvation in terms of biomass growth and lipid accumulation.
Esakkimuthu [68] found that a supplement of magnesium induced
54.6% lipid accumulation in Scenedesmus obliquus without affecting its
biomass growth. On the other hand, lipid accumulations due to calcium
and phosphorus starvation caused 52.9% and 47.6% lipid accumulation
respectively. These results suggested that culturing media that contain
high concentration of magnesium but low amount of calcium might
provide a better alternative than conventional nutrient starvation
Table 3
Lipid content of algae species under nitrogen-starved conditions.
Algae species Cultivation conditions Lipid content of cell dry weight (%) References
Chlamydomonas reinhardtii,
CC1010
Constant illumination (2000 lx) at a distance of 50 cm for alternate photoperiod
(light: dark – 12:12 h cycle) at 25 °C under shaking (90 rpm).
61 [43]
Chlorella regularis Alternate photoperiod of 14 h light and 10 h dark. The temperature was
maintained at 25 ± 1 °C with 160 rpm agitation.
42.3 (excess phosphorus) [44]
Chlorella sorokiniana Mixotrophic culture, cool white light intensity 100 μmol m−2
s−1
, 25 °C on an
orbital shaker at 150 rpm
44 [45]
Chlorella protothecoides 180 h in fed-batch culture at 30 °C 39.2 (hyperosmotic stress) [46]
Chlorella pyrenoidosa 120 rpm shaking incubator under continuous cool white light illumination 58 in mixotropix-heterotrophic two phase
mode and 52 in mixotropic mode
[16]
Chlorella vulgaris ESP-31 Mixotrophic cultivation on Modified Bristol's medium 40–53 [47]
Isochrysis zhangjiangensis
(Chrysophyta)
Addition of nitrogen at 24 h interval (High lipid yields under nitrogen rich
conditions)
53 [48]
Nannochloropsis oceanica DUT01 Alternate photoperiod of 14 h light and 10 h dark. Light intensity 60 μmol
m−2
s−1
. F/2 medium containing 37.5 mg/L NaNO3 combined with 1/5 fresh
medium replacement.
64 [49]
Nannochloropsis oculata Batch mode, sudden starvation of nitrogen on low initial biomass 50 (43% triglyceride fatty acids) [41]
Neochloris oleoabundans 25 °C, 300 rpm, Exponentially fed-batch cultures 0.042 h−1
growth rate 53.8 [50]
Pseudochlorococcum sp low light intensity of 20 μmol photons m−2
s−1
, orthophosphate (Pi) used as
AGPase inhibitor and 6-methoxy-2-benzoxazolinone (MBOA) used as α-amylase
inhibitor
52.1 (low light conditions) [51]
Scenedesmus obliquus NIES− 2280 6 day cultivation, maintained in the dark, stirred at 100 rpm and maintained at
24 ± 2 °C
38–48 (sufficient phosphorus) [52]
Scenedesmus sp. CCNM 1077 25 ± 2 °C with 12:12 h light dark period after 15 days cultivation 27.93 [53]
Monoraphidium sp. T4X Nitrate concentration of 0.036 mg/L. Temperature set at 25 °C ± 2. 18.42 [54]
Mixed microalgae Ambient temperature 24 ± 1 °C, constant agitation rate of 150 rpm, and
continuously illuminated with a light intensity of 30 μmol m−2
s−1
. Starvation
after 13 days
33.9 [55]
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61

4. method for microalgae biomass and lipid productivity.
2.4. Environmental stress condition
Microalgae biomass growth and lipid accumulation are also affected
by environmental stresses such as light intensity, pH and salinity.
Although the starvation of certain nutrients can induced lipid accu-
mulation in microalgae, the biomass productivity is likely compro-
mised. Light is an important factor for microalgae growth. It provides
energy to induce the conversion of dissolved inorganic matter in the
medium during photosynthesis [70]. Light wavelength and intensity are
important for biomass growth but microalgae can only absorb certain
wavelengths of the natural light spectrum (400–700 nm) [28].
When microalgae is grown in wastewater, light intensity is the
limiting factor rather than nutrient availability [71]. Microalgae under
light limiting condition increase the amount of chlorophyll linearly
with light intensity. However, under high light intensity, microalgae
will develop an acclimation mechanism known as photo inhibition to
prevent excessive energy harvesting [72] which is attained by reducing
the chlorophyll level.
Despite the fact that high light intensity induces lipid synthesis, but
excessive exposure would lead to the generation of reactive oxygen
species and acidification that cause irreversible damage to the chlor-
ophyll [73]. For microalgae grown in photo-bioreactors, light dis-
tribution in the culture is crucial due to shading where the light in-
tensity received by every cell differs [74]. Kurpan Nogueira [14] found
that high light intensity of 300 μmol photons m−2
s−1
and low tem-
perature of 20 °C induced a 70% increase in oil content in microalgae.
On the other hand, Wang et al. [75] found that red light diodes promote
better biomass growth because the adsorption bands of chlorophyll
were present in the red light wavelength. LEDs provide several ad-
vantages over the use of fluorescent lamps for microalgae growth be-
cause of their narrow band spectrum, low heat generation and high
efficiency [76]. Fluorescent dyes are another innovation employed to
alter high-energy protons to low-energy light wavelength for micro-
algae growth. Seo [77] found that maximum biomass growth is ob-
tained when using red light, whereas maximum lipid content is
achieved with blue paint. The selection of suitable microalgae strain
should include the optimisation of culture and lighting conditions for
maximum production efficiency.
Carbon dioxide (CO2) is reduced via photosynthesis where the
captured carbon is converted into carbohydrates, proteins, nuclei acids
and lipids [78]. On the other hand, dissolved CO2 in the marine en-
vironment reacts with the water to form carbonic acid (H2CO3), which
will dissociate into bicarbonates −
(HCO )
3 , and bicarbonate will dis-
sociate into carbonate ion −
(CO )
3 and hydrogen ions (H+
), lowering the
ambient pH that inhibit microalgae growth [79]. In an alkaline en-
vironment, −
HCO3 is the dominant dissolved inorganic carbon that can
be found in wastewater [80]. −
HCO3 is consumed by microalgae via
cation exchange, active transport and/or through catalytic conversion,
in the forms of CO2 and OH−
[81].
pH is important for microalgae biomass and lipid accumulation
because it affects microalgae metabolism [82]. Common microalgae
species such as Chroococcus turgidus, Lyngbya confervoides, Nostoc com-
mune, Chlorella sp., Chaetoceros calcitrans and Skeletonema costatum
showed best growth rates at neutral pH, while other species such as
Chlorella sorokiniana (pH 8) and N. oculata (pH 8.5) prefer slightly alkali
pH values [83]. Breuer [84] reported that the neutral pH and tem-
perature of 27.5 °C were the optimum conditions for maximum lipid
accumulation in Scenedesmus obliquus. However, for Chlorella sor-
okiniana DOE 1412, the optimum growth and lipid accumulation pH
were at pH 6 due to the microalgae preference of acidic CO2 over al-
kaline −
HCO3 [82]. Liao et al. [73] observed that while low pH condi-
tions leads to lower biomass growth rates, the effect is reversible and
gentler than high intensity light conditions. This suggests that pH,
which is species specific, should be optimised prior to light intensity in
a culture to avoid photoinhibition that causes permanent damage to
essential proteins [85].
Similar to other environmental stresses, salinity also affects the
biomass growth and lipid accumulation of microalgae. High salinity
creates high osmotic pressure of the external environment, which
generates pressure within the microalgae cell that affects membrane
fluidity and permeability [28]. In high salinity environment, the cel-
lular osmoregulation mechanism is activated to equilibrate the in-
tracellular osmotic pressure with that of the external. This mechanism
will induce the accumulation of lipid due to changes in the fatty acid
metabolism [86]. Sodium chloride (NaCl) is commonly used as salinity
modifier to induce lipid accumulation in microalgae, while the studies
on usage of other types of salt such as potassium chloride (KCl), mag-
nesium chloride (MgCl2) and calcium chloride (CaCl2) are limited [87].
An increase of 2.52 fold in lipid content was obtained for Scenedesmus
obliquus XJ002 when the cells were treated with 0.20 M of NaCl [88]
but in another study, 47% cell death was also reported when Chlamy-
domonas reinhardtii CC124 was exposed to high concentration of NaCl
after 3 days [89] though the lipid content has increased. While in
comparison study of using different type of salt, Wang et al. [90] re-
ported that no significant differences were found on the lipid accu-
mulation of Chlorella protothecoides between NaCl and KCl. On the other
hand, Srivastava et al. [87] observed that CaCl2 intensified lipid accu-
mulation in Chlorella CG12 and Desmodesmus GS12 the most, compared
with other types of salt.
As most of the factors (i.e. light intensity, pH and salinity) affecting
microalgae biomass and lipid productivity are species-specific, specially
tailored cultivation conditions are needed to ensure maximum pro-
ductivity, and at the same time, keeping the production cost low. The
two stage cultivation process where microalgae is first grown to achieve
maximum biomass growth before the stress-induced lipid accumulation
step is preferred, to prevent loss of biomass [91].
3. Harvesting and extraction technologies
After microalgae species selection, the next important step is the
harvesting technique. Effective harvesting technique will minimize
energy wastage and prevent loss of biomass yield. The next process is
lipid extraction. The combination of effective harvesting and lipid ex-
traction methods will enhance production efficiency. There is also op-
portunity for the coproduction of other high value products like nu-
traceuticals, pigments, food and etc [92]. Fig. 1 illustrates the
conventional microalgae biodiesel production process.
Selection of microalgae species
Optimum nutrient conditions for biomass growth
Optimisation of culture conditions to enhance lipid
production
Biomass harvesting techniques and extraction of lipids
Biomass further processing
High Valued Bio-products
Pre-treatment if free fatty
acid content is too high
Transesterification to
produce high quality
biodiesel
Fig. 1. Microalgae biodiesel and bio-products production process.
B.H.H. Goh, et al. Renewable and Sustainable Energy Reviews 107 (2019) 59–74
62

5. 3.1. Harvesting techniques
Since microalgae are cultivated in suspension in culturing media,
biomass harvesting requires huge amount of energy, and accounted for
almost one third of the total cost of production [5]. Many different
techniques have been applied, and the major ones include centrifuga-
tion, flocculation, filtration and screening, gravity sedimentation,
floatation and electricity assisted techniques [36]. The advantages and
disadvantages of each technique are discussed in the following sub-
sections. Generally, common disadvantages shared by these techniques
are high capital cost, high energy consumption and long extraction
period [93].
3.1.1. Centrifugation
Centrifugation is a popular option for microalgae bulk harvesting.
Centrifugal recovery is rapid and this technique avoids the use of che-
mical solvents for separation, which eliminates the risk of chemical
contamination [92]. However, the maintenance of freely moving parts
and the high energy consumption needed to spin cells out of suspen-
sion, has made the process costly [5]. Cell damage may also occur due
to the generated heat, high sheer and gravitational forces applied [94].
Furthermore, long operation time will be required for efficient se-
paration of biomass in large volume suspension [95].
3.1.2. Flocculation
Flocculation is considered among the most cost effective methods
for microalgae biomass recovery and it is able to accommodate large
volumes of cultures [96]. Flocculation and coagulation can be carried
out chemically, biologically or magnetically. Metal salts such as alum
and ferric chloride can be used as coagulants where the metal ions will
hydrolyse in water and precipitate [96]. One major disadvantage of
chemical flocculation is that the metal will remain in the residue after
lipid extraction, thus affecting the subsequent use of residue as animal
feed [94].
Bio-flocculation refers to flocculation that is achieved though bio-
logical means. Flocculation can occurs naturally and spontaneously
during microalgae blooms in lakes and ponds, and it is believed to be
caused by the presence of extracellular polymer substances in the
water. Flocculation can also be induced using bacteria, fungi or other
naturally flocculating microalgae, but the cultivation of bacteria, fungi
or in combination with other microalgae requires carbon source, and
wastewater is rich in carbon source. The bio-flocculation technique has
been implemented successfully in wastewater treatment plants, how-
ever, the underlying mechanism is still not very clear [96].
Magnetic adsorbents like magnetite (Fe2O3) nanoparticles can be
added to microalgae suspension to induce magnetic flocculation.
Separation of microalgae from medium is achieved when a magnetic
field is applied [93]. This approach is simple and reusable, where it has
been adopt in other industries as well [97]. However, the separation of
the magnetic adsorbents from the residue for reuse purposes can be
costly [93].
3.1.3. Filtration and screening
Dewatering is usually achieved by filtration and screening, and is
normally carried out after flocculation to improve harvesting efficiency.
A filtration membrane is used to collect microalgae deposits as the
system forces the suspension fluid to flow through it [98]. However, as
the membrane thickens, the flow resistance also increases due to
clogging, and membrane replacement is needed to maintain efficiency,
that will add to the operational cost [99]. Cross flow filtration system,
which uses tangential flow to remove thick filter cakes is introduced to
circumvent this problem, but this approach increases the operation time
[100]. Low density culture can be harvested using this approach, but it
may not be feasible for large scale setup [98].
3.1.4. Gravity sedimentation
Microalgae settling rate is crucial for the gravitational sedimenta-
tion method. Christenson & Sims [99] concluded that the settling rates
from 0.1 to 2.6 cmh-1
are too slow and will lead to the deterioration of
biomass during settling. Agar gel is found to promote microalgae cluster
growth that will increase the settling velocity [101]. Gravitational se-
dimentation may be low in cost and require minimal use of energy, but
is too unreliable for industrial settings. The combination of gravita-
tional sedimentation and flocculation has been suggested to improve
harvesting efficiency.
3.1.5. Flotation
Flotation can be described as inverted sedimentation where air
bubbles provide lifting force for particle separation [98]. Surface-active
components are absorbed on the surface of air bubbles that allows re-
covery of biological component at a higher concentration coefficient in
a single step [102]. The addition of surfactant will cause the micro-
algae's surface to be more hydrophobic, which will allow for easier
biomass removal from the culture [103]. Bubble size, surfactant con-
centration and pH are important parameters affecting the efficiency of
this approach [98].
3.1.6. Electricity assisted techniques
Electricity has been applied to improve the efficiency of microalgae
harvesting techniques. This approach can be deemed as en-
vironmentally friendly as they do not require the use of chemicals.
Since microalgae cells are negatively charged, when the culture is
subjected to an electric field, the microalgae particles will concentrate
at the anode [98]; this process is known as electrophoresis. Moreover,
electricity is also used to enhance flocculation and floatation, these
techniques are known as electro-flocculation and electro-flotation.
Electro-flocculation utilises charge neutralisation which creates sorp-
tion affinity for negatively charged particles [104]. On the other hand,
electro-flotation is achieved by formation of microbubbles at the
cathode that will capture floating particles and allows for better mi-
croalgae separation [105].
3.2. Lipid Extraction Techniques
There are currently no established methods for microalgae lipid
extraction. However, much research has been done to reduce solvent
consumption, to enhance extraction yield, to reduce extraction time, to
improve end product properties, among others [106]. A few factors
have been identified as crucial for large scale lipid extraction, they are
extraction efficiency, process duration, reactivity with lipids, capital
and operational cost, process safety and waste generated [107]. Cell
wall disruption is an important step to improve lipid extraction effi-
ciency since passive diffusion across cell wall is slow. Cell wall and
membrane disruptions will allow direct access of solvent to the in-
tracellular lipid [108], which increases the lipid recovery efficiency.
There are many different types of cell disruption method available,
which can be generally categorized into mechanical and non-mechan-
ical methods. Fig. 2 shows the various types of cell disruption methods.
3.2.1. Mechanical Methods
Mechanical methods of lipid extraction include mechanical
pressing, ultrasonic assisted oil extraction, microwave assisted oil ex-
traction, electric pulse modes, and cell homogenization. Generally,
mechanical methods result in high biomass losses and low selectivity
towards the lipids [109]. However, certain mechanical processes reduce
harmful solvent utilisation as well as decrease processing duration
[110]. Unicellular microalgae strains often have thick cell wall that
block the release of intracellular lipid, which is unsuitable for me-
chanical pressing [111]. Hence, for industrial scale lipid extraction, the
conventional mechanical pressing is not a feasible method to utilise.
Simple mechanical pressing use equipment such as screw press, bead
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6. milling, piston, extruder or pulverisation in mortar to separate biomass
cake from the oil component [112].
Abbassi [113] has reported that the utilisation of hydraulic pressing
alone for N. oculata microalgae can only achieved 51.05 ± 3.23%
disruption fraction for lipids, whereas the addition of liquid nitrogen
increased the disruption fraction to 94.77 ± 0.72%. This significant
increase is due to the sudden freezing of microalgae cells, rendering the
cell wall brittle, thus aiding the release of intracellular lipid. On the
other hand, Meullemiestre [114] found that bead milling showed better
extracted lipid quality as compared to ultrasonic and microwave
methods.
Ultrasonication is a promising technique as it facilitates the mixing
of solutions and does not require high temperatures to disrupt cell walls
[115]. This technique utilises sound waves to propagate pressure fluc-
tuations that induces cavitation [116]. X. Zhang [117] studied the use
of ultrasonic assistance for biodiesel production of Trichosporon oleagi-
nosus sludge, and found that the duration was shortened by 23 h with a
yield of 95%. Gerde [115] found that by increasing the sonication
power, the extraction of intracellular products also increases, but may
also led to poorer quality of lipid because of lipid oxidation. Ad-
ditionally, Natarajan [118] concluded that ultrasonic lipid extraction
may be efficient for rigid walled microalgae such as Chlorella sp., but
species with flexible walls such as T. suecica and Nannochloropsis sp., the
cell walls tend to coil up and retain membrane lipids. Therefore, ul-
trasonic assisted methods are only suitable for microalgae species with
rigid cell walls.
Microwave assisted techniques offers rapid and even heating, which
uses minimal amount of solvents, and requires a shorter heating dura-
tion [119]. It is comparable to ultrasonic methods as both methods
increase lipid yields and reduce extraction time. Microwave energy
offers a more effective heating due to faster energy transfer and reduces
thermal gradients. Additionally, it also demonstrates selective heating,
uses smaller equipment size, and most importantly, microwave assisted
technique increases production and eliminates process steps [120].
Guldhe [121] compared both ultrasonic and microwave lipid ex-
tractions on sun dried Scenedesmus sp. and found that the microwave
method had higher lipid yields (28.33% g-1
of dry cell weight) com-
pared to ultrasonic method. Dai [119] also arrived at the same con-
clusion that microwave method offers better lipid extraction than
ultrasonic method, in their experiment, the highest oil yield of 30 wt%
was extracted from microalgae frond using microwave method. Al-
though ultrasonication and microwave have commendable advantages
to conventional extraction, both have difficulties in extracting from
dense medium, which will affect scalability of these processes [122].
Chemical or physical treatments are found to induce excessive heat
and stress on cells, which may decrease cell viability [123]. Electric
pulse treatment produces pulsed electric fields to affect the membrane
properties of a biological cell. Under application of an electric field, the
external cell membranes will receive an increase of transmembrane
voltage that increases the membrane's permeability and conductivity
[124]. Electric pulse treatment is also known as electroporation. Elec-
troporation is highly selective and allows release of intracellular matter,
while the extraction of lipid will require use of solvent [125]. Jaeschke
[126] achieved 83% lipid yield from Heterochlorella luteoviridis under
moderate electric field and ethanol pre-treatment. Meanwhile, Garoma
& Janda [127] reported that lipid extraction for Chlorella vulgaris using
electroporation exhibited low lipid yields (5.3%). However, this
method obtained the highest energy gain per energy input compared to
microwave and ultrasonic methods with n-hexane/methanol/water
solvent solution.
Cell homogenization eliminates the need for cost intensive drying
and can be scaled to be used in large volumes [128]. Halim [129] found
that Chlorococcum sp., 73.8% of cell disruption were achieved using cell
homogenization, which was higher than acid treatment, bead beating
and ultrasonic methods. On the contrary, for Chlorella vulgaris, dos
Santos [130] found that homogenization (16%) showed poorer lipid
yields than ultrasonic methods (19%) under similar solvent conditions.
Sudden depressurization technique is also a common cell disruption
method, used to obtain intracellular compounds. During static com-
pression, a diffusible gas such as supercritical carbon dioxide is allowed
to penetrate the cell wall until saturation is achieved before a sudden
depressurization is initiated. A sharp pressure gradient is formed along
the cell wall where the gas expanded that result in high disruption ef-
ficiency [131]. The main advantage of sudden depressurization is that
the process is free from toxic solvents [132]. When the decompression
rate is increased, the efficiency of disruption will also increase, since it
induces a higher pressure drop along the cell wall [133]. Halim et al.
[134] found that the use of supercritical carbon dioxide was able to
Cell
Disruption
Techniques
Mechanical
Conventional mechanical
pressing
Ultrasonic assistance
Microwave assistance
Electric pulse
Cell homogenization
Sudden depressurization
Non-mechanical
Conventional chemical
solvents
Acids
Nanoparticles
Supercritical fluids
Ionic liquid
Biological enzymes
Fig. 2. Types of cell disruption techniques.
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64

7. shorten the Chlorococcum sp. lipid extraction time by 5.6 times in
comparison to conventional Soxhlet extraction.
Despite numerous studies on the mechanical modes of lipid ex-
traction, all methods have its limitations. The thick cell walls of mi-
croalgae make conventional mechanical pressing inefficient.
Ultrasonication and microwave produce impressive lipid yields, but
both encounter difficulties from dense mediums extraction, which
might not be suitable for industrial setting. In addition, electric pulse
treatment, cell homogenization and sudden depressurization are energy
intensive methods, which increase the cost of production of microalgae
biodiesel.
3.2.2. Non-mechanical methods
Besides mechanical methods of lipid extraction, other methods of
lipid extraction include the use of chemicals or enzymes to disrupt the
cell wall. Chemical or biological materials interact with the cell mem-
branes to allow direct passage of intercellular components to the sur-
rounding [135]. Lipid extraction efficiency depends on the use of non-
polar and polar solvents [136]. For microalgae lipid extraction, polar
solvents are paired with non-polar solvents to ensure total extraction of
all neutral lipids, which include free-standing globules and membrane
associated complexes [137].
Commercially, lipid extraction has been conducted with solvents
such as ethanol, methanol, hexane and chloroform. The Bligh and Dyer
method is one of the common methods used for lipid extraction, where
a two-phase system is created and lipid will be fractioned in the
chloroform phase [138]. However, not all solvents are safe for the en-
vironment. One major drawback of conventional solvents such as
hexane and chloroform is their toxicity and adverse effect to the en-
vironment [116]. In addition, these solvents also affect the quality of
product by dissolving unwanted products such as chlorophyll [139].
Table 4 shows the common types of solvent used for lipid extraction of
various feedstocks.
Ideally, extraction solvent should be highly specific to lipid but
volatile enough to ensure low energy distillation and easy separation
from lipid [116]. Considering the health and environmental issues as-
sociated with conventional solvents, new environmentally friendly and
sustainable solvents have been introduced for microalgae lipid extrac-
tion, such as acid, nanoparticle, supercritical fluid, ionic liquid and
biological enzyme.
Acid mediated cell disruption is often accompanied with heat. The
strong acid will catalyse the hydrolysis of bio-component at elevated
temperatures [151]. J.-Y. Park et. al [152] found that 1% sulphuric acid
heated at 120 °C for 60 min increased lipid yields of Chlorella vulgaris by
approximately 4 times. On the other hand, I. Lee et. al [151] found that
increasing nitric acid concentration lead to decrease in lipid yields of
Nannochloropsis salina. The use of acids at accurate concentrations
should be studied along with microalgae strains for efficient lipid ex-
traction.
Nanoparticles can easily penetrate and interact with biomolecules
due to their size. Abdul Razack [153] found that silver nanoparticles
cause cell wall damage to Chlorella vulgaris and are suitable for lipid
extraction. Zinc oxide nanoparticles are also able to increase the per-
meability of cellular membranes and depolarise cells [154]. W.-C.
Huang & Kim [155] studied the use of nickel oxide nanoparticles for
Chlorella vulgaris lipid extraction and found a 208% increase in ex-
traction efficiency. However, the synthesis cost, environmental con-
cerns and reusability of nanoparticles have yet to be fully addressed for
commercial applications of these technologies [156].
Supercritical fluids have emerged as an interesting alternative to
conventional solvents due to their low viscosity, high diffusivity, easy
separation, high dissolving power and low surface tension [157]. Su-
percritical CO2 is the most widely used supercritical fluid for extraction
of bio-compounds and is recyclable [158]. S. Tang [159] achieved
33.9% lipid yield from Schizochytrium limacinum powder with ethanol
(95%) and supercritical CO2 (5%) as extraction solvents. Millao &
Uquiche [160] studied the effects of supercritical CO2 on Nanno-
chloropsis gaditana lipid extraction and found that temperature and CO2
density increase lead to higher lipid and carotenoid yields. In order to
make supercritical CO2 lipid extraction process more economical, si-
multaneous lipid and carotenoid extraction have been explored.
Ionic liquids are green organic solvents that are non-volatile and
possess good thermal stability [161]. Ionic liquid has been applied to
not only extract lipid, but also to recover other valuable compounds
such as proteins and polysaccharides from wet biomass [162]. Choi
[163] compared the lipid yields of Chlorella vulgaris with organic sol-
vents and ionic liquids, and found that 1-ethyl-3-methyl imidazolium
acetate, 1-ethyl-3-methyl imidazolium diethylphosphate, 1-ethyl-3-
methyl imidazolium tetrafluoroborate, and 1-ethyl-3-methyl imidazo-
lium chloride showed high lipid yields of more than 200 mg/g cell
compared to 185.4 mg/g cell achieved by the conventional hexane-
methanol solvent. However, there are cases where certain ionic liquids
resulted in lower lipid yield than conventional organic solvents. In
addition, Olkiewicz [162] found that ionic liquids showed better ex-
traction yields from raw sludge than dried sludge, which would elim-
inate the need for costly drying process.
Enzyme can facilitates the recovery of lipid by selective degradation
of cell wall and membrane while preserving most labile compounds
[164]. Published studies have concluded that proper enzyme selection
and optimal process conditions determination are essential for effective
enzymatic treatment [165]. Sierra [108] found that lipid yield of
Chlamydomonas reinhardtii incubated with autolysin was found to be
Table 4
Conventional solvents for lipid extraction.
Solvent used Feedstock Lipid yield Operating conditions Reference
Ethanol-hexane (1:2) Acutodesmus obliquus 92% (Soxhlet) 60 °C, at 12 h (Soxhlet) and 2 h (ultrasonic) [140]
59% (Ultrasonic)
Ethanol Heterochlorella luteoviridis 83% 75 ml/100 ml ethanol solution, moderate electric field (90 V) [126]
Chloroform-methanol mixture (75% v/v
to methanol)
Botryococcus braunii 98.9 wt% 5 h (Butt tube systems) [141]
Hexane Nannochloropsis gaditana 69.1 wt% Homogenization at 1700 bar and low temperature (20–22 °C) [142]
Chloroform: methanol (2:1 v/v) Chlorella pyrenoidosa 19.74% (magnetic stirring) Stirring at 700 rpm at 20 °C [143]
19.43% (ultrasonic)
Methanol: Chloroform: water (25:12.5:5) Chlorella vulgaris 52.5% Sonicated for 40 min [144]
Methanol Chlorella vulgaris 20.7% Sohxlet at 373 K for 96 h [145]
Methanol-ethyl acetate (2:1) Chlorella sp. 18.1% 60 °C, 2 h [146]
Hexane Scenedesmus sp. 16.3 ± 0.2 wt% Hot compressed hexane (235 °C, 31 bar) [147]
CO2 expanded ethanol Schizochytrium sp. 87 wt% Pressure 6.9 MPa, Temperature 313 K, ethanol flowrate 1.0 ml/
min, CO2 flowrate 6.0 ml/min, 30 min
[148]
CO2 expanded methanol Botryococcus braunii 24 wt% 35 °C and 7.2 MPa [149]
Dipotassium hydrogen phosphate mix
with ethanol
Chlorella sp. 69 ± 2% Three phase partitioning for 2 h [150]
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8. 30% higher than without biomass treatment. High lipid extraction
(88.3%) was achieved from slurry of Nannochloropsis oceanica with the
use of thermal lysin, Aspergillus niger cellulose and surfactants [166].
The combination of several cell disruption methods may help increase
lipid yields from microalgae. However, it should be noted that enzymes
should not be exposed to mechanical, thermal or chemical stress to
ensure reusability.
Lipid extraction using solvents can be done on either dry or wet
biomass. However, extraction of lipid from dry biomass is usually more
efficient [135]. The drying process is energy and cost intensive but wet
extraction usually resulted in lower yield due to the tendency of mi-
croalgae cells to remain in the water phase and not interact with the
organic solvents used for lipid extraction [167]. Considering the scale
up difficulty faced by mechanical modes of lipid extraction, the solvent
method is usually more suitable for commercial use as it is less energy
intensive and has shown to produce higher yields of lipid extraction
[167]. Balasubramanian et al. [168] studied factors affecting lipid ex-
traction from marine microalgae such as biomass drying method,
moisture content and solvent extraction system. They discovered that
drying method had no significant effect on lipid yield, but it would
affect the total free fatty acid produced. Therefore, choosing the right
kind of solvent for microalgae cell wall disruption is important to en-
sure optimum lipid yield. Another technique that avoids the use of
harmful solvents for lipid extraction and utilises less energy is in-situ
transesterification, where lipid is transesterified directly from the mi-
croalgae biomass
4. Direct biodiesel synthesis from microalgae biomass
Conventionally, biodiesel is produced from extracted lipids. Lipid
extraction is costly as it requires the breaking of cell wall and normally
this includes the dewatering process, which is also energy intensive
[169]. A life cycle analysis found that drying and extraction processes
accumulated to 90% of the total biodiesel production costs [170]. The
transesterification process, developed several decades ago, is con-
sidered to be highly efficient [171]. It has been successfully applied in
commercial biodiesel production, and has a stable conversion efficiency
of above 95% [17]. Conventionally, the transesterification reaction
involves blending of alcohol with vegetable oil in the presence of a
catalyst. The reaction will convert the oil to a methyl or ethyl ester
(biodiesel) and glycerol [171]. The choice of alcohol and catalyst is
dependent on nature and type of feedstock used [172]. The combina-
tion of lipid extraction, solvent recovery and transesterification into a
single step provides a more economical alternative for biodiesel pro-
duction. In recent years, many researchers have work on the direct
biodiesel synthesis of wet or dry microalgae biomass and some studies
has found that higher moisture content reduces the biodiesel yield
[173]. In the direct biodiesel synthesis from microalgae biomass, al-
cohol acts as the extraction solvent as well as the transesterification
reactant [174]. The use of co-solvent may help improve the process
efficiency by acting as an extraction agent and forming homogenous
system between the microalgae oil, alcohol and catalyst [175]. Direct
biodiesel synthesis eliminates the loss of lipids where all lipids are
converted to biodiesel and offers the concurrent production of valuable
co-products such as ethyl levulinate, ethyl formate, diethyl ether and
glycerolcarbonate [23].
Common species of microalgae being studied for direct biodiesel
synthesis include Chlorella spp. and Nannochloropsis spp.. Lemoes et. al
[176] found that direct biodiesel synthesis had higher ethyl and methyl
ester yields than the conventional extraction-transesterification process.
Ghosh [177] also found that the combination of extraction and trans-
esterification using acid catalyst for Chlorella sp. MJ11/11 yielded more
biodiesel than a two-step process. However, Chen [178] observed that
direct transesterification from biomass would require a higher catalyst
loading when compared to the transesterification of extracted oils.
There are several parameters which affect the biodiesel yield from
single step transesterification which include application of catalyst,
ultrasonic or microwave technologies and use of supercritical alcohols.
Table 5 shows the direct biodiesel synthesis from microalgae biomass
using conventional methods.
4.1. Catalyst
Selection of catalyst is an important step for high biodiesel yields.
Conventional catalysts for biodiesel production include base or acid
catalyst, which depends on the fatty acid content of the oil. A base
catalysed reaction occurs by creating a nucleophilic alkoxide from the
alcohol to attack the electrophilic part of the carbonyl group of trigly-
cerides, whereas in an acid catalysed reaction, the carbonyl group of
triglycerides is protonated and the alcohol will attack the protonated
carbon to create a tetrahedral intermediate [188]. The drawback of acid
catalysed transesterification is the low yield compared to conventional
base catalysed [189]. Base catalyst demonstrates faster reaction kinetics
than acid catalysed but are only suitable in the biodiesel synthesis of
lower free fatty acid content lipids (< 0.5%) [190]. Microalgae lipids
have been known to contain high levels of free fatty acid (up to 70 wt%
depending on storage conditions) and require the use of acid catalyst
during transesterification [191]. Homogenous catalysts are difficult to
separate from product due to their homogeneity, incurring additional
cost for product purification and at the same time creating extra waste
Table 5
Direct biodiesel synthesis from microalgae biomass using conventional methods.
Algae species Transesterification conditions Biodiesel yield (%) Reference
Chaetoceros gracilis (wet) 100 mg biomass, 2 ml Methanol, add chloroform to form single phase solution, 1.8% sulphuric acid
catalyst, 80 °C for 20 min
84 (FAME) [179]
Chlorella sp. Ultrasonic power 137 W, reaction time 100 min, molar ratios of methanol to oil of 83 and chloroform
to oil of 30, 0.08 mol sulphuric acid concentration
81.2 [180]
Chlorella pyrenoidosa (wet) 1 g biomass, 4 ml chloroform, 4 ml methanol, 0.2 ml sulphuric acid catalyst, microwave assisted for
30 min
10.51 [181]
Nannochloropsis gaditana 4 kg biomass in 16 L of water, 36.4 L hexane, 36.4 L methanol, 18.2 L 98% sulphuric acid catalyst,
2 h, vacuum distillation refining
85.5 ± 2.6 (FAME) [182]
Nannochloropsis gaditana 0.75 g biomass, 4.06 M sulphuric acid catalyst with 6.67 ml (ethyl acetate) /g dried algae, heated at
113.6 °C for 2 h
97.8 wt% (FAEE) [183]
Nannochloropsis oceanica (wet) 0.2 g biomass, 1 ml methanol, 2 ml chloroform, 0.4 ml sulphuric acid catalyst, 95 °C for 120 min 91 (FAME) [173]
Nannochloropsis sp. (wet) Mixture of biomass (20% water), methanol and sodium hydroxide catalyst, microwave assisted at 50°
for 10 min
75 [184]
Nannochloropsis sp 1:400 M ratio of lipid to methanol1:1 vol ratio of methanol to n-hexane, heatedat 60 °C for 4 h 90.9 [185]
Nannochloropsis gaditana 5 g biomass, 1.98 ml of 1, 2-dichloroethane/ g biomass, 4.69 ml ethanol, heated at 185.08 °C for 3 h 92 (FAEE compared to
biocrude)
[186]
Nannochloropsis gaditana 0.15 g dry biomass saturated to 80 wt% moisture, 1.5 ml methanol, 0.1 ml chloroform, 0.3 ml
hydrochloric acid catalyst, heated at 95 °C for 2 h
90 [187]
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9. [192]. Therefore, heterogeneous catalyst is the better choice as it
eliminates the need for catalyst recovery, reducing the cost of biodiesel
production [169]. Table 6 shows the direct biodiesel synthesis using
various heterogeneous catalysts.
The use of calcium oxide (CaO) catalyst, which can be synthesized
from egg shells, mollusc shells, chicken bone, oyster and mud crab shell
is gaining much attention for biodiesel production [199]. Calcium oxide
catalyst recycles shell waste from being disposed and its usage also
increases the commercial value of seafood production [200]. The cat-
alytic capacity of CaO in transesterification is dependent on the ex-
istence of basic sites and their spatial dispersion [201]. Nevertheless,
because CaO is a basic catalyst and it can only be used for transester-
ification of microalgae with low free fatty acid content. Microalgae
biomass that contains high free fatty acid content can be transesterified
by acid or enzymatic catalyst. Research has found that lipases work at
lower temperature (25–50 °C) and the subsequent separation of bio-
diesel and glycerol is relatively easier [202]. Lipase catalyst is applied
in the transesterification of vegetable oils but rarely on microalgae
biomass.
There is also a favourable trend of using lipase in biodiesel pro-
duction due to its high production rate and low cost [203]. Guldhe
[204] optimised the transesterification of lipid from Scenedesmus ob-
liquus using immobilized Aspergillus niger as catalyst and obtained a
90.82% conversion yield. However, the use of extracellular lipases re-
quires an immobilisation process, and the subsequent recovery and
purification are also costly and difficult [202]. The use of hetero-
geneous catalyst allows for easy catalyst recovery as alcohols do not
mix with the solid catalyst [205]. Therefore, heterogeneous catalyst
looks like a promising approach to microalgae biodiesel commerciali-
sation.
4.2. Process intensification
Besides the use of catalyst, microwave or ultrasonic assistance
during transesterification also improves biodiesel synthesis efficiency.
The use of ultrasonic assisted acid catalysed transesterification on mi-
croalgae feedstock is rarely studied. Most microalgae biodiesel direct
transesterification utilises microwave assistance. Teo [206] reported
that microalgae biodiesel produced with microwave assistance showed
higher lubricating property, good cetane number and shorter carbon
chain FAME compared to biodiesel produced using the conventional
method. Similar to lipid extraction methods, sonication encourages
better lipid solubilisation. Martinez [175] found that ultrasonic assis-
tance improved yield of biodiesel from transesterification of Spirulina
sp. and a higher lipid recovery. On the other hand, microwave allows
selective heating, faster energy transfer and thus more efficient heating
[207]. Table 7 shows the major differences with microwave and
ultrasonic technologies.
Koberg [197] compared the ultrasonic and microwave assistance on
Nannochloropsis microalgae and obtained better yields from the mi-
crowave assisted process. Cheng [181] found that the kinetic rate of
direct biodiesel synthesis with microwave assistance was 6 times faster
than the conventional extraction-transesterification of Chlorella pyr-
enoidosa oil. Sharma [214] optimised biodiesel production from
Chlorella vulgaris under microwave irradiation and achieved an 84.01%
yield. Meanwhile, Ma et. al [215] utilised heterogeneous catalyst KF/
CaO in an ultrasonic-microwave synergistic extraction apparatus and
obtained a maximum biodiesel yield of 93.07 ± 2.39%. The use of a
synergized setup as such may be a viable option as it allows testing of
both technologies for any selected microalgae to achieve maximum
biodiesel yields.
4.3. Supercritical conditions
The transesterification process can also be conducted under super-
critical conditions, which eliminate the use of catalyst, thus avoid the
production of pollutants [216]. Non-catalytic transesterification re-
quires elevated temperature to beyond the critical temperature of the
alcohol to form a homogenous reaction phase, but does not need to be
in the supercritical state [217]. Supercritical methanol has been widely
used in the transesterification of several feedstock such as rapeseed oil
[218], palm oil [219], Jatropha oil [220] and waste vegetable oil [221].
Although this process produces high yield of biodiesel, the stringent
reaction conditions often pose a challenge towards the efficient scale up
of the production [222]. Methanol in supercritical conditions breaks the
rigid microalgae cell walls and allows for solvent diffusion into lipid
simultaneously [33]. The direct transesterification of microalgae bio-
mass can also utilise ethanol as a biodegradable replacement for me-
thanol, where similar yields were reported for both fatty acid alkyl
esters [176]. The addition of carbon dioxide to supercritical methanol
lowers the reaction temperature [223]. Several researchers have re-
ported the use of supercritical methanol for microalgae biodiesel pro-
duction. Table 8 shows studies on supercritical transesterification of
microalgae biomass.
Since methanol is toxic and non-renewable, longer chain alcohols
such as ethanol, isopropanol and butanol are introduced as potential
alcohol replacements. The use of longer chain alcohols also improves
the cold flow properties and oxidation stability of the produced bio-
diesel [232]. Reddy [225] also reported that fatty acid ethyl esters have
better cetane number, oxidation stability and cold flow properties than
fatty acid methyl esters.
When supercritical methanol was compared with use of microwave
assistance for Nannochloropsis (CCMP1776) biodiesel transesterifica-
tion, the supercritical method (25 min) had longer reaction time
Table 6
Direct biodiesel synthesis using heterogeneous catalyst.
Algae species Transesterification conditions Biodiesel yield (%) Reference
Acutodesmus obliquus 1 g biomass, biomass to methanol (w/vol) ratio 1:12, 1.7% (w/w) calcium oxide catalyst from waste egg shell
mechanically stirred at 140.6 rpm for 3.6 h, 75 °C
86.41 [193]
Botryococcus sp. 0.1 g biomass, 0.5 ml of Candida antarctica lipase B (Novozyme CAL-B) immobilised on Celite with dimethyl
carbonate ultrasonicated at 40 °C for 6 h
88 [194]
Chlorella sp. 0.3 g biomass, 12 mlg−1
methanol to biomass ratio, lithium hydroxide (LiOH) pumice catalyst (20 wt%
concentration at 12 ml g−1
) mechanically stirred at 500 rpm for 3 h, 80 °C
47 (FAME) [192]
Chlorella pyrenoidosa 1 g biomass, 4 ml methanol, 4 ml chloroform, 5 wt% sulfonated graphene oxide, microwave irradiated at 90 °C for
40 min.
84.6 [169]
Nannchloropsis gaditana 3 g biomass, 13.8 cm3
, 0.32 ratio of catalyst to oil mass, lipase catalyst (Novozyme 435 (N435) from Candida
antarctica) with 21.3 cm3
t-butanol stirred and incubated at 40 °C for 56 h
99.5(FAME) [195]
Nannochloropsis sp. 1 g biomass, 45 ml mixed solvent (methanol/methylene dichloride = 3:1), 10% Mg-Zr solid catalyst heated at 65 °C
for 4 h
28 [196]
Nannochloropsis sp. (dry) 1 g biomass, methanol to chloroform (1:2 v/v), 0.3 g strontium oxide catalyst, microwave and sonication assistance
at 60 °C for 5 min
37.1 (microwave) [197]
20.9 (sonication)
Scenedesmus obliquus 0.1 g biomass, 1 ml hexane, 20:1 methanol to oil molar ratio, 15% chromium-aluminum catalyst mechanically stirred
(200 rpm) at 80 °C for 4 h
98.28 [198]
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67

10. compared to microwave method (5 min) [229]. However, the super-
critical method produces a higher quality biodiesel, free from harmful
pollutants since no catalyst was used.
In situ transesterification may reduce the number of steps needed
for biodiesel production, but it also wastes valuable nutrient in the
remnants. Zhou [233] utilised supercritical carbon dioxide to recover
high value components from the residue of Chrysophyta sp. and Chlor-
ella sp. after transesterification. Jazzar [227] also reported that super-
critical methanol was used in the single step isolation, cultivation and
transesterification of Chlorella sp. and Nannochloris sp.. For most su-
percritical cases, the optimised reaction parameters are often similar for
different species of microalgae. Therefore, the biodiesel yields are often
affected by lipid content of the microalgae species itself.
5. Challenges and future prospect of third generation biodiesel
The third generation biodiesel or microalgae derived biodiesel
avoids the use of agricultural land as microalgae can be grown in arable
land due to its robust environmental adaptability [234]. The use of
microalgae as a biodiesel feedstock is more sustainable in terms of food
security and environmental impact. However, the transition to third
generation biodiesel usage is still vague and requires more research to
become sustainable. Fig. 3 shows various obstacles to be overcome
before microalgae biodiesel can be commercialised.
An efficient and profitable microalgae biodiesel production should
utilise a high lipid productivity microalgae strain, which can be culti-
vated in a sustainable environment and with the ability to produce
other valuable by products. Therefore, it is important to reorient re-
search and development to focus on these properties. In addition, the
socioeconomic problems and opportunities of microalgae biodiesel
should be evaluated prior to commercialisation.
5.1. Technical challenges
Several factors such as environmental tolerance, high growth rate,
high lipid content and easy harvesting and extraction are important
when choosing an "all-rounder” species of microalgae [235]. The se-
lection of a suitable strain marked the start of an in-depth study into
microalgae biodiesel production. Table 9 shows the summarised se-
lection matrix for an “all-rounder” microalgae species.
A high percentage of biodiesel production cost is due to the feed-
stock. Current biodiesel production cost is calculated to be 4.4 times the
price of petroleum derived biodiesel [238]. Microalgae-based biodiesel
commercialisation faces various obstacles in terms of harsh cultivation
conditions and the complicated and often costly harvesting and oil
extraction method [239]. Lipid productivity, cultivation and down-
stream processing are the deciding factors in microalgae biodiesel
economics. Genetically modified microalgae for enhanced lipid pro-
ductivity have potential but are still in research stage. The concept of
co-cultivation between microalgae species may also help improve lipid
yields while not affecting biomass growth [240]. Microalgae can be
cultivated in wastewater or seawater, eliminating the need for fertili-
zers and at the same time can be used for wastewater remediation
[241]. Microalgae with high CO2 tolerance can also be cultivated using
industrial flue gas (high levels of carbon dioxide, sulphur and nitrogen
oxides) as a strategy for CO2 mitigation [35]. Direct biodiesel synthesis
from biomass is found to be the most cost effective process in-
tensification method because it combines oil extraction and transes-
terification into a single step [237]. Given the enormous potential of
microalgae biodiesel, it is necessary that proper policy is implemented
to support efficient commercialisation.
China, the world's second highest oil consumer, produces only 15%
of its biodiesel need due to lack of policies and regulations that en-
courage the use of biodiesel [242]. As of now, the biodiesel policies
implemented around the world are for first and second generation
biodiesels. The implications of biodiesel policies are huge, affecting not
only the oil prices, food prices and consumer welfare, but they also alter
the income distribution [243]. Given to that, any policies pertaining to
third generation biodiesel must be thoroughly studied and discussed to
prevent undesirable consequences that will impact the society [244].
Consistent monitoring on the cultivation and maintenance of micro-
algae biodiesel production plants will also prevent policy failures such
Table 7
Difference between ultrasonic and microwave assistance.
Ultrasonic assistance Microwave assistance
High temperature and pressure conditions create free radicals which cause reaction to
occur instantly [208]
Microwaves create electromagnetic fields which align polar molecules and create
heat due to friction from slower orientation of molecules and time rate change of the
fields [209].
High temperature, high pressure, acoustic microstreaming, turbulence and high shear
forces generate finer emulsions between immiscible fluids which enhance mass
transfer and transesterification reaction rates [210].
Microwave technology allows rapid, safe and cost effective microalgae biodiesel
production without the need of drying [207].
Ultrasonic can also help in the extraction of valuable components such as pigments and
carotenoids [189].
Microwave heating reduces analysis time, simplifies manipulation and creates higher
purity products [120].
Approximately 5000 K and 100 MPa produced during collapse of ultrasonic bubbles [211]. Short chain alcohols (Methanol or ethanol) have strong polarity and are active
microwave absorption media [212].
Microwave technology has been upraised to work on a continuous flow pattern in an
energy efficient manner [213].
Table 8
Supercritical transesterification of microalgae biomass.
Microalgae species Transesterification conditions Biodiesel yield (%) Reference
Nannochloropsis gaditana 255–265 °C, 50 min, supercritical methanol to algae ratio (10:1) 45.8 (FAME) [216]
Nannochloropsis gaditana (CCMP – 1775) methanol to wet biomass (vol./wt.) ratio 6:1, temperature 225 °C, and reaction time of 90 min 59.28 [224]
Nannochloropsis oculata 265 °C, 20 min, ethanol to algae ratio (9:1) at supercritical conditions 67 (FAEE) [225]
Nannochloropsis salina 280 °C, 25 min, ethanol to algae ratio (9:1) at supercritical conditions 65 (FAEE) [226]
Nannochloropsis sp. 265 °C, 50 min, methanol to algae ratio (10:1) at supercritical conditions 21.79 wt% [227]
Nannochlorpsis sp. 50 °C, 200 bar and 24 h reaction in supercritical CO2 (SC–CO2) medium 62 [228]
Nannochloropsis (CCMP1776) 1200 psi, methanol to biomass (12:1), 30 min 85.75 [229]
Chlorella sp. 265 °C, 50 min, methanol to algae ratio (10:1) at supercritical conditions 45.62 wt% [227]
Chlorella protothecoides 320 °C, 152 bar, 31 min, methanol to oil ratio (19:1) 90.8 [230]
Chlorella vulgaris CCAP (211/19) Hexane/biomass ratio (6:1) 7.06 ± 1.03 [231]
B.H.H. Goh, et al. Renewable and Sustainable Energy Reviews 107 (2019) 59–74
68

11. as India's “National Biodiesel Mission 2003”, where farmers failed to
adopt scientific cultivation and maintenance techniques for their Ja-
tropha oilseed plantations [245].
The long-term stability of microalgae products should also be stu-
died because organic compounds can easily degrade due to high tem-
perature, light and presence of other inhibitors such as oxidation ac-
celerators. Oxidative stress can cause the accumulation reactive
oxidative species in high amount that will cause damage to the mi-
croalgae cells and reduce lipid production [246]. Therefore, it is im-
portant to conduct life cycle analysis on not only the biodiesel, but on
other valuable microalgae products as well to determine the sustain-
ability of the process. Biodiesel are susceptible to oxidation during long
term storage [247] and all generations of biodiesel degrade naturally
due to oxidation, causing increase in acidity and viscosity. Recent re-
search have investigated the use of additives such as antioxidants,
phytohormone and oxygen vector to reduce oxidative damage [246].
5.2. Future prospects
The potential of modified microalgae as feedstock face numerous
challenges such as variation of culture growth conditions, low pro-
ductivity and overwhelming capital investment and operation [248].
The most common species of microalgae used for genetic engineering is
Chlamydomonas reinhardtii. This species has been studied extensively
and its genome sequenced [249]. Shin [250] increased the lipid pro-
ductivity of Chlamydomonas reinhardtii by 64.25%, achieved through
targeted knockout of phospholipase A2. The current genetic engineering
studies on microalgae lipid production have been focused on single
gene overexpression or deletion, but it is not known whether this will
be the most effective way of enhancing lipid accumulation while reg-
ulating cell metabolism. Sun et al. [251] reviewed various approaches
of microalgae lipid enhancement via metabolic engineering and found
that multi-gene manipulation is more advantageous than single gene
editing. However, multi-gen manipulation is still in the infancy stage of
research.
Microalgae utilizing its simple cell structure and rapid growth rate,
has a CO2 bio-fixation efficiency of 10–15 times higher than terrestrial
plants [252]. Wastewater loaded with nitrogen and phosphorus is sui-
table nutrient sources for microalgae growth. It is estimated that
roughly 16.67 times more carbon can be stored in diluted suspensions
of microalgae (10 kg of biomass per m3
) than in gas form CO2 [253].
However, the several challenges of wastewater based microalgae cul-
tivation include variation of wastewater composition, toxic con-
tamination and presence of suspended particles that affect light trans-
mission efficiency [34].
Mehrabadi [254] suggested that a method to reduce microalgae
biodiesel production costs is the utilisation of biomass from wastewater
treatment in high rate algae ponds. Lee [255] predicted that algal
biofuel could supply 7.1% of developed world fuel demand by 2040 and
0.5% in developing countries with strong government support. Kova-
cevic [256] estimated that algae biofuel could cost 51.60 euros/GJ by
2020 if technological advancements remained linear and crude oil re-
mained at $100/barrel.
However, lipid production from microalgae cultivated in treated
wastewater shows poor net energy recovery when both the lipid energy
content and light requirement are considered [257]. Several companies
such as Sapphire energy, Heliae and Solazyme shifted from the com-
mercialisation of microalgae biofuels to other fields (food, nutrition,
water treatment) to stay afloat [258]. This suggests that microalgae
cultivation solely for biodiesel production is impossible.
The development of microalgae biodiesel industries cannot be sus-
tained without government support. Currently, the production biodiesel
is economically viable due to various government policies that support
the commercialisation via tax credits, subsidies, import tariffs and
targets appointed [244]. Similar efforts should be plan prior to the
commercialisation of third generation biodiesel. Reduction in equip-
ment taxes, provisional training and learning sessions for farmers and
other subsidies are important for the successful industrialisation of
microalgae biodiesel [258]. Under beneficial energy policies and in-
vestments, the development of biodiesel will be encouraged, while
production and consumption will also significantly increase [242].
Brazil's “National Program for Biodiesel Production and Uses” (PNPB)
policy has recently introduced tax reductions for biodiesel production
for various feedstock, which may increase people's interest in micro-
algae cultivation due to its high lipid productivity in non-arable land
[259]. Other efforts include the United States’ introduction of title IX of
the EISA (Energy Independence and Security Act) 2017 which provides
grants and loans for the commercial production of microalgae biodiesel.
Microalgae contains high value bio products such as pigments, vi-
tamins and antioxidants that can be extracted and exploited [248].
Selection of suitable
microalgae strain with
enhanced growth
characteristics and high
lipid content
Cultivation strategy
with high biomass
productivity that
utilises wastewater for
nutrients
Harvesting strategy and lipid
extraction methods depending
on microalgae strains, and the
co-production of valuable
bioproduct
Fig. 3. Steps for efficient microalgae biodiesel production process.
Table 9
Biodiesel selection matrix for “all-rounder” microalgae species.
Factors Preferred characteristics
Biomass Growth Rate Fast growth rate for rapid accumulation of biomass which will increase the yield per harvest and reduce risks of
contamination [234].
Lipid Content High lipid content corresponds to a higher amount of biodiesel that can produced from the microalgae strain [234].
Free Fatty Acid (FFA) Content FFA content less than 0.5 wt% to avoid soap formation, reduce catalyst usage and avoid complicated phase separation [17].
Environmental Tolerance Easy acclimatization to unconducive open environments and resistant to contamination [95].
Growth Medium The use of bio-fertilizers are preferred over chemical fertilizers, and can also accelerate the microalgae metabolism [236].
Harvesting Methods Ideal harvesting methods should be suitable for most microalgae strains and achieve high biomass recovery with minimal
cost and energy use [95].
Potential for Direct Biodiesel Synthesis from
Biomass
Direct conversion of microalgae biomass into biodiesel reduces the economic and environmental impact due to reduced
processing steps and solvents used [237].
B.H.H. Goh, et al. Renewable and Sustainable Energy Reviews 107 (2019) 59–74
69

12. Biorefinery is an industrial scale facility where biomass and its inter-
mediates are converted to valuable products such as chemicals, mate-
rials and energy products [260]. For industrial scale application, mi-
croalgae strains should contain high value products and strong
environmental adaptability properties, and cost-effective biorefineries
should utilise low-cost techniques such as nutrient limitations [246].
While biofuel production is the main objective of microalgae compa-
nies, the production of other high value products is crucial for com-
panies to remain profitable in the short term [258]. The introduction of
microalgae biodiesel market may also affect conventional algae product
markets. There is no concrete evidence whether this new market will
boost or shrink conventional applications such as cosmetics and che-
micals. However, Zhu [260] stated that microalgae-based fuel will
cause competition with conventional microalgae uses, which will cause
recession in the original industrial applications. A hybrid refinery may
be the most profitable venture, rather than an exclusively product-
based or energy-based refinery [261].
6. Conclusion
The review discusses the factors that influence biodiesel production
from microalgae, the challenges it face and future prospect. All key
factors affecting microalgae cultivation, lipid accumulation, harvesting,
cell disruption, extraction and biodiesel synthesis were critically ana-
lysed. Biodiesel from microalgae also provides a more sustainable and
environmentally friendly alternative to fossil fuel. It is essential to in-
corporate these technical findings with economic analysis to ensure
feasibility of biodiesel production. For microalgae biodiesel production
to remain sustainable, the biomass should be cultivated in lipid indu-
cing culture, followed by efficient harvesting and finally, the biodiesel
synthesis that combines both lipid extraction and transesterification.
Overall, the use of microalgae as biodiesel feedstock is technically
feasible, but not economically viable. The production of microalgae
biodiesel in the form of a hybrid refinery along with production of
conventional microalgae products can improve the marketability of
microalgae. However, more research should be done on long term
stability and technical aspects of a hybrid refinery.
Acknowledgements
The authors would like to acknowledge the funding supports ob-
tained from the University of Malaya and Ministry of Education,
Malaysia under the MRSA-FRGS RU grant (grant no: MO014-2016) and
SATU joint research scheme Joint Research Scheme (grant no: ST010-
2018).
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