Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198

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Renewable and Sustainable Energy Reviews

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

A sustainable integrated in situ transesterification of microalgae for

biodiesel production and associated co˗products ˗ A review
Kamoru A. Salam a,n, Sharon B. Velasquez-Orta b, Adam P. Harvey b
a
Department of Chemical Engineering, Faculty of Engineering, University of Abuja, P.M.B. 117, Airport Road, Main campus, FCT-Abuja, Nigeria
b
School of Chemical Engineering and Advanced Materials (CEAM), Newcastle University, NE1 7RU, United Kingdom

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

Article history: Microalgae has large scale cultivation history particularly in aquaculture, pigments and nutraceutical
Received 7 February 2016 production. Despite the advantages of microalgal oil as feedstock for biodiesel production, algal biodiesel
Received in revised form is still at laboratory scale due to technical challenges required to be overcome to make it economical and
25 June 2016
sustainable. Indeed, complete drying of microalgae is energy intensive and significantly increases the
Accepted 21 July 2016
cost of algae pre-treatment. In situ transesterification is more water tolerant due to excess methanol to oil
molar ratio required by such production route. However, the need to remove unreacted methanol (494%
Keywords: of it) from the product streams certainly requires distillation heat load which increases the operating
Microalgae cost. This article reviews the key process variables affecting efficiency of in situ transesterification. These
Biodiesel
include alcohol to oil molar ratio, moisture, stirring rate, reaction time, temperature, microalgal cell wall
In situ transesterification
and catalyst type. Potential solutions of improving the efficiency/economy are discussed. Overall, an
Dimethyl ether (DME)
Biogas integrated approach of in situ dimethyl ether (DME) production along with the desired biodiesel
Bio-digestate synthesis during in situ transesterification would substantially reduce the volume of unreacted methanol
thereby reduces operating cost. Use of resulting microalgal residue for biogas (methane) production can
provide energy for biomass production/separation from the dilute algae˗water mixture. Use of bio˗di-
gestate as nutrients for supporting microalgal growth is among the probable solutions suggested for
reducing the production cost of in situ transesterification.
& 2016 Published by Elsevier Ltd.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1180
2. Alternative fuels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1180
2.1. Energy content of gasoline and alternative fuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1180
2.2. Comparative cost of gasoline and alternative fuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1182
3. Microalgae compositions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1182
3.1. Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1182
3.1.1. Effect of lipid composition on fuel quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1182
3.1.2. Effect of algae cell wall on bioprocesses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1182
3.1.3. Microalgae cell disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1183
3.1.4. Energy requirement of algae cell disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1183
4. The transesterification reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1184
4.1. Kinetics of reactive extraction (“in situ transesterification”) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185
4.2. A comparison of two step transesterification and reactive extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185
4.3. Overview of In situ transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1186
4.4. Key process variables in in situ transesterification of microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1186
4.4.1. Solvent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1187
4.4.2. Temperature and reaction time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1187
4.4.3. Agitation rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1188

n
Corresponding author.
E-mail address: kamorusalam@gmail.com (K.A. Salam).

http://dx.doi.org/10.1016/j.rser.2016.07.068
1364-0321/& 2016 Published by Elsevier Ltd.
1180 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198

4.4.4. Catalyst. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1188

4.4.5. Acid-catalysed reactive extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1188
4.4.6. Alkali-catalysed reactive extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1190
4.4.7. Heterogeneous-catalysed reactive extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1191
4.4.8. Non-catalysed reactive extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1191
4.4.9. Moisture content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1191
5. Microalgae species. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1191
5.1. Protein and carbohydrate content of algae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192
6. Probable ways of making a sustainable in situ transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192
6.1. Anaerobic digestion of algal residue after reactive extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192
6.1.1. Causes of inhibition during biogas production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192
6.2. marine species for biofuel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192
6.3. In situ co-production of diethyl-ether during in situ transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192
6.3.1. Fuel properties of DME or DME-Biodiesel blends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1194
7. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1195
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1196

1. Introduction translated to commercialisation of algal biodiesel. The problem is

Biodiesel is commonly produced from refined vegetable oil
made from soybean, rapeseed, sunflower and palm oil. In Europe
and America, it is largely produced from rapeseed and soybean,
respectively, due to their availability and the cold flow properties
of the resulting biodiesel [1]. Edible oil crops account for
95% of
worldwide biodiesel feedstocks [1]. However, using these crops for
fuel would require significant amounts of freshwater [2] and ara-
ble lands [3]. This would create undue competition between using
these resources for energy crop and agricultural or domestic pur-
poses. In Europe and America, the increased cost of food oil crops
has been strongly influenced by their use as energy crops [4]. High
prices may be beneficial to farmers, but this might lead to food
shortages in many developing countries, particularly where almost
50% of their earnings are used for food [4]. It could also lead to
global food insecurity. Competition for land between food and fuel
can also have a negative environmental implication. Recently, vast
areas of rainforests particularly in Malaysia, Indonesia and Thai-
land have been exploited for cultivation of palm oil as it is in high
demand for food and fuel [5]. This has caused deforestation and
negatively affects the forest ecosystems. In order to sustainably
provide raw materials for large-scale production of biodiesel,
non˗edible feedstocks that require marginal land and insignificant
freshwater are preferable. Such feedstocks include waste oils and
microalgae particularly the marine species [2]. Non-food oil crops
and waste oils can only supply limited quantities of biofuels, so
cannot meet world transport fuels requirements. Biodiesel can be
produced from microalgae through reactive extraction (“in situ
transesterification”) or a two˗step transesterification. In a two˗step
transesterification, pre-extracted oil from microalgae can be con-
verted into fatty acid methyl ester (FAME) (“biodiesel”) with al-
kalised or acidified methanol. Production of algal biodiesel via a
two-step transesterification usually includes dewatering, conven-
tional drying, solvent extraction, oil degumming, trans/esterifica-
tion, neutralisation and product purification. Alternatively, algal
biodiesel can be produced via in situ transesterification by con-
tacting the algal biomass directly with an alcohol containing a
catalyst [6,7]. It is potentially a cost-effective alternative way of
producing algal biodiesel due to its elimination of the solvent
extraction step and its higher water tolerance [6,7] as the solvent
extraction and drying steps account for
90% of the process en-
ergy in a two-step transesterification of algal oil to biodiesel [8].
Though microalgae has short growing time, high lipid productivity
and can be use for capturing concentrated CO2, they can be cul-
tivated on non-arable land using wastewater; and are adaptable to
harsh environments but these advantages are still yet to be
that there are still a numbers of constrains required to be over-
come to achieve commercial reality of algal biodiesel.
For instance, microalgal cell consists of phospholipid bilayer
membranes (about 7–8 nm thick) embedded with glycolipids,
glycoproteins and polysaccharides [9]. This configuration provides
structural rigidity for the cells to adapt to their environments but
adversely affects the efficiency of algal bioprocesses including
fermentation, anaerobic digestion, oil extraction and biodiesel
production. Indeed, it leads to significant high solvent require-
ments and energy load during the extraction processes [10]. A
direct biodiesel production requires a significant methanol to oil
molar ratio, usually in the range of 100:1–1000:1 methanol to oil
molar ratio but can be as high as 1570:1 [11]. Dhar and Kirtania
[12] reported that, for a transesterification process operating at 6:1
and 40:1 methanol to oil molar ratios, 662 and 6450 kW respec-
tively were required for a 95% methanol recovery. By extrapola-
tion, 16,658–171,898 kW will be required for the same methanol
recovery by a reactive extraction process operating at 100:1–
1000:1 methanol to oil molar ratio. This means the downstream
distillation heat load required to recycle unreacted methanol
(494% of it) would significantly increase the operating cost and
has been one main reason why the process is not economically
viable. In addition, FAME yield was reported to increase with rising
acid concentration during in situ transesterification [6,7,13] but the
need to neutralise the unreacted acid in the product streams will
increase the operating cost to some extent. Additionally, limited
supply of concentrated CO2, nonfully utilisation of nitrogen or
phosphorous nutrients, adverse effect of using small quantity of
fresh water even if marine algae is used, and inefficient utilisation
of algal residue after oil extraction hinder commercialisation of
algal biofuels [2]. This review provides ways of reducing the excess
methanol to oil molar ratio and efficient utilisation of residual
algae produced during in situ transesterification. Factors affecting
in situ transesterification were also critically reviewed, and a new
integrated process to improve the economy of the in situ trans-
esterification is discussed.
2. Alternative fuels
2.1. Energy content of gasoline and alternative fuels
Irreversible depletion of fossil fuel and the climatic change due
to its combustion result into a significant global interest in re-
newable and sustainable transport energy sources such as biofuels
(biodiesel and bioethanol). Similarly, use of hydrogen as energy
 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198
carrier in fuel cell vehicles (FCVs) has attracted public
attention because water and hot air are the end products of elec-
tricity generation using this approach [14]. So its emissions are not
negatively affecting human health. However, there are still some
challenges blocking wide applications of hydrogen as energy car-
rier. These include safety, high cost of production and transpor-
tation, storage problems and high cost of fuel cells. Considering
the lingering effect of hydrogen explosion during Hindenburg
tragedy, good safety techniques of hydrogen transportation and
storage are required to allay public fear and to encourage sig-
nificant sponsoring of hydrogen production as transport fuel. Most
of the well-established methods of storing hydrogen such as
compression and cryogenic or liquefaction are energy intensive,
costly and require extra safety measures. For instance liquid hy-
drogen requires cryogenic storage around  252 °C [15]. Extra cost
would be required to insulate the hydrogen tank to prevent energy
loss at this temperature.
The energy density and specific energy of gasoline and alter-
native fuels are shown in Table 1.
As can be seen in the Table, liquid hydrogen contains energy
density of 0.01 MJ L  1 which is significantly lower than energy
density of gasoline (34 MJ L  1), biodiesel (34 MJ L  1) and that of
ethanol (24 MJ L  1). This means a substantial bigger tank is re-
quired to store liquid hydrogen having the same energy contents
as the alternative fuels. On mass basis, the specific energy content
of hydrogen is 142 MJ kg  1 which is significantly greater than that
of gasoline (46 MJ kg  1), biodiesel (42 MJ kg  1) and ethanol
(30 MJ kg  1). However, storing hydrogen as gas typically requires
compressing and maintaining it at about 350–700 bar [18].
Besides extra cost of compression, the public needs to be con-
vinced on the safety in carrying such high pressure tank on board.
More research efforts are now being focused on efficient and safe
techniques of storing or producing hydrogen on board. These in-
clude metal hydrides, particularly the magnesium based one, as
they have potential of producing hydrogen capacity up to 7.6 wt%
[19,20]; glass microspheres [21] and activated carbons [22,23].
Fossil fuel is still the major source of industrial hydrogen. Ap-
proximately 96% of commercial scale hydrogen is being produced
from fossil fuel by steam reforming while about 4% is being pro-
duced through water electrolysis [24,25].
Steam reforming of hydrocarbons produces significant large
amount of CO2 rendering zero emission criteria of hydrogen ques-
tionable. Use of water electrolysis for hydrogen production requires
significant electricity consumption, causing high production cost. For
instance use of electrolysis for hydrogen generation in the United
States requires building 1800 new power plants that emit CO2 and
about $1 trillion would be needed to execute the project [26]. Pre-
sently, 168,000 gasoline fueling stations are in the United States and
a cost of $2.8 million has been projected by National Renewable
Energy Laboratory (NREL) for each hydrogen fueling station capable
of handling 100 cars per day [26]. Therefore, a total capital invest-
ment required to replicate the number of gasoline stations with
hydrogen fuel stations would be $470 billion which is quite high.
A more appealing procedure would be hydrogen production
that require no fossil fuel and which does not require significant
Table 1
Energy density and specific energy of different fuels.
Source: Gaur and Reed [16]; ETB [17].
Fuel Energy density, MJ L  1 Specific energy, MJ kg  1
Gasoline (Petrol) 34 46
Hydrogen liquid at 20 K 10 142
Hydrogen gas 0.01 143
Biodiesel 34 42
Ethanol 24 30
1181
80000
Oil Equivalent (Thousand Tonnes)
70000
60000
50000
40000
30000
20000
10000
0
Production year
Fig. 1. Global biofuels production from 2005 to 2015.
Source: British Petroleum (2016).
consumption of electricity. These include hydrogen generation
from solar energy [27]; photolysis, involving exploiting of chlor-
ophyll and enzyme in microalgae to chemically splitting sea water
into hydrogen and oxygen [28] or production of hydrogen from
starch and water [29]. While all these processes are technically
feasible, they are still at laboratory scale meaning that use of hy-
drogen as energy carrier for transport fuel may not meet the
world's energy need in the near term.
Consideration of biofuels such as biodiesel and bioethanol as
transport fuels has attracted a global interest. Between 2005 and
2015, there has been a continued increase in global production of
biodiesel and bioethanol, as shown in Fig. 1.
Global biofuels production in 2015 shows that 42.9% was ac-
counted for by North America, 27.9% by South and Central America,
18.3% by Europe and Eurasia, 10.8% by Asia while Africa contributed
0.1% to the global biofuels production [30].
Based on the highest biofuels country producer in each con-
tinent, the United State of America has the highest share (41.4%) in
the North America, Brazil produced the highest (23.6%) in the
South and Central America, Germany produced the highest (4.2%)
in the Europe and Eurasia while China was the highest producer
(3.2%) in the Asia pacific [30]. However, bioethanol is produced
mainly from food crops in these countries. For instance, bioethanol
has been produced on a commercial scale in Brazil, United State of
America and European countries solely from food crops such as
cane sugar, corn and wheat [31]. This approach is unsustainable as
every hectare of land used for cultivating these energy crops leads
to clearing of the same amount of land for food production.
Clearing rainforests, peatlands, savannas or grass lands to produce
food crop-based biofuels increases annual greenhouse gas emis-
sions [32,33] and world food prices [4,34].
Alternatively, bioethanol can be produced from agricultural by-
products such as wheat straw, sugarcane bagasse and corn stover;
forest residues including sawdust, thinning forests; energy crops
such as Salix and switch grass [31]. These feedstocks have high
content of cellulose and hemicellulose but they are difficult to
hydrolysis in a high yield into monomer sugars than obtaining
sugar from carbohydrate- or sugar-containing substances such
corn or cane sugar for bioethanol production [31].
In Europe biodiesel is largely produced from oilseed, but in-
sufficient supply from this feedstock has increased demand for
palm oil from Malaysia and Indonesia causing significant clearing
of rainforest for palm plantation [34] which can negatively affect
the ecosystem. Biodiesel from microalgae could be alternative
1182 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198
source of transport fuel. Although it has not been be commercia-
lised for some technical reasons which will be discussed later in-
side the text. Nevertheless, this can be overcome. Previous studies
by Chisti [35] has shown that a 2.8 energy ratio (i.e. the renewable
energy produced per unit of fossil fuel input) was obtained from
raceway production of microalgae biomass containing 20% (w/w),
biomass productivity of 0.025 kg m  2 d  1[36–38] and concentra-
tion of 1 kg m  3 in the ponds. An energy output of 1444 GJ ha  1
year  1 based on 90% operational factor for the production facility
has been estimated [35]. In comparison, a total energy output of
163.9 GJ ha  1 year  1 was estimated for cane sugar having total
energy yield (bioethanol and bagasse) of 218.5  10  2 GJ per tonne
of cane sugar [39] assuming a cane productivity of
75 t ha  1
[35]. In addition microalgae can be cultivated on marginal land or
wastewater. It is also non-food oil substrate, so cannot cause food
shortage.
2.2. Comparative cost of gasoline and alternative fuels
The latest national average price of transport fuels obtained
from alternative fuels data center of United State Department of
Energy [40] and the price of hydrogen reported by Dears [26] are
listed Table 2. All the prices were converted to $/gasoline gallon
equivalent (GGE) to allow comparison with the price of the
equivalent energy content of gasoline. As can be seen in the Table,
hydrogen fuel has the highest price, followed by biodiesel and
then ethanol while gasoline has the least price. While gasoline has
the lowest price, its combustion contributes significantly to cli-
mate change.
Presently there is no industrial scale production of biodiesel
from microalgae. Therefore, algal biodiesel production cost is ob-
tained from techno-economic analysis reported in the literatures
[41–44]. An updated cost estimated for in situ transesterification of
wet microalgae was carried out by Nagarajan et al. [41]. They
obtained cost of production ranged from $1.69–3.90/GGE which is
comparable to that of gasoline. As can be seen in the Table 3, they
obtained lower biodiesel production cost than other investigators.
This can be attributed to the fact that their process is wet-in situ
transesterification. Lardon et al. [8] reported that solvent extrac-
tion and drying of algae are energy intensive and contributes
significantly to the process energy. Similarly, as shown in the Table
the cost of biodiesel produced from microalgae cultivated in open
pond is generally greater than that produced from photo-bior-
eactor. This is because capital and operating cost of photo-bior-
eactor is significantly greater than open pond [44].
3. Microalgae compositions
3.1. Lipids
Lipids are broadly classified as neutral or polar. A neutral lipid
has no overall polarity. It is located inside the cells in the form of
triglycerides, mono- and diglycerides or free fatty acids. They are
more soluble in non-polar solvents such as hexane and chloro-
form. Neutral lipids are energy storage products. They are the
Table 2
Price of transport fuels.
Fuels Price ($/GGE) Reference
Gasoline 2.06 US DoE (2016)
Ethanol (E 85) 2.56 US DoE (2016)
Biodiesel (B99-B100) 3.00 US DoE (2016)
Hydrogen 14.3 Dears (2015)
common algal substrate known for biodiesel synthesis. In contrast,
polar lipids contain polar groups, such as choline, ethanolamine,
serine, water, glycerol and phosphatidyglycerol in phospholipids.
Glycolipids are other forms of polar lipid but are less polar than
phospholipids. They are simple sugar-containing lipids. They in-
clude monogalactosyl diglyceride, digalactosyl diglyceride and
sulpholipids. They are located in the cell walls. They are, as might
be expected, more soluble in polar solvents, such as methanol and
water. They give cell walls their structural rigidity. Structures of
non-polar and polar lipids are shown in Table 4.
Where R1, R2 and R3 in Table 4 denote fatty acids moieties
which could be involved in transesterification process as com-
monly observed in triglycerides. The symbol “X” in phospholipids
can be any of the substituent group listed in Table 5.
3.1.1. Effect of lipid composition on fuel quality
Microalgae are extremely diverse, with substantially different
fatty acid profiles, which means that the resulting biodiesel will
also vary substantially [19]. It has been shown by Ben-Amotz et al.
[47] that a wide range of microalgae species can synthesize C14:0,
C16:0, C18:1, C18:2 and C18:3 free fatty acids, while other fatty
acids including C16:2, C16:3, C16:4, C18:4, C20:5, C22:6 were
strain-specific. In addition, deficiency in Nitrogen caused sig-
nificant increase in the lipid content in Ankistrodesmus sp. Thomas,
Isochrysis sp., Nannochloris sp. Thomas, Nitzchia sp., Nitzchia sp.
Chapman, Botryococcus braunii Kutz except Dunaliella species that
accumulated higher carbohydrate content. Other investigations
have shown that culture, environmental conditions, habitat and
growth phase all have a significant effect on the fatty acid profile
of microalgae [48,49]. Table 6 shows the effect of growth media,
environmental stress and catalyst type on fatty acid profile of
different microalgae.
As can be seen in the Table, growth media [50,52] and varying
catalyst [51] have significant effect on fatty acid profile of algal
lipids and cause difference in FAME compositions. Variation in
fatty acid compositions due to catalyst type was attributed to
differences exhibited by alkali or acid catalyst to free fatty acids
(FFA), cell wall lipids and triglyceride during in situ transester-
ification [51]. It is well known that microalgae accumulate more
neutral lipids (triglycerides) rather that structural lipids when
cultivated on nitrogen deplete media. Since both lipids have dif-
ferent fatty acid composition, the resulting total lipids is sig-
nificantly affected by growth media [50].
Regardless of the factors, the lipids contain large amount of
polyunsaturated FAME. Significant high amount of polyunsaturated
(PUFA) reduces oxidative stability and cetane number of the resulting
algal biodiesel. Although, the oxidative stability of the lipid/FAME is
strongly dependent on the structure of the PUFA as bisallylic sites in
the PUFA are more prone to oxidation than the allylic site [53] The
bis˗allylic sites are a methylene groups (CH2) adjacent to two double
bond, while allylic sites are the one adjacent to a double bond [53].
EN 14,214 recommends that the maximum contents of linolenic
acid methyl ester should not be more than 12% (m/m) while
polyunsaturated methyl esters (with four or more double bonds)
should not be more than 1% (m/m) [54]. Probable solution to re-
duce PUFAs include addition of fuel additive containing 0.03% tert-
butyl hydroquinone (TBHQ) [55] or harvesting of microalgae using
ozone floatation [56] .
3.1.2. Effect of algae cell wall on bioprocesses
Microalgal cell wall accounts for
10% of dry algal biomass [57] It
consists of phospholipid bilayers membrane embedded with glyco-
lipids, glycoproteins and polysaccharides [9]. Eukaryotic cell's wall
typically contains fibril and mucilage such as polysaccharides, lipid
and protein [9,57,58]. Analysis of the cell wall compositions of few
microalgae conducted by Abo-shady et al. [58] are shown in Table 7.
 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198 1183

Table 3
Cost of biodiesel production from microalgae by techno-economic analysis.

Cultivation system Cost of biodiesel ($/GGE) Remarks Reference

Open pond (400 ha) 1.69–3.90 Wet in situ transesterification [41]

Ultra-sonication assisted process
Open pond (333 ha) 6.80 Monte Carlo Sampling method [42]
PBR 11.30 Monte Carlo Sampling method
Open pond 15.80 Monte Carlo Financial feasibility model [43]
PBR 40.00 Monte Carlo Financial feasibility model [43]
Open pond (1951 ha) 11.00 [44]
PBR 23 [44]

Table 4 Table 5
Structures of non-polar and polar lipids. Substituent group in phospholipids.
Source: Wood [45] Source: Wood [45]

Lipid Structure Name Structure Abbreviation for lipid

þ
Triglyceride Choline .O.CH2.CH2.N (CH3)3 P.C
Ethanolamine .OCH2.CH2.NH2 P.E
Serine P.S

Water .H P.A
Sulpholipid Glycerol .CH2(OH)CHCH2OH PG
Phosphatidyglycerol .CH2CH(OH)CH2. D.D

P.A: Phosphatidic acid; P.G: phosphatidyl glycerol; D.D: diphosphatidyl glycerol; P.

C: phosphatidyl choline; P.E: phosphatidyl ethanolamine; P.S: phosphatidyl serine.

Monogalactosyl diglyceride
wall [65]. Botryococcus braunnii is not an attractive option for
biodiesel production because it is difficult to cultivate [65]. Be-
cause microalgal neutral lipids are contained within cells they are
not readily available for extraction, so require disruption prior to
Digalactosyl diglyceride transesterification [66].

3.1.3. Microalgae cell disruption

Phospholipids
As can be seen in the Table, different microalgae contain
varying amount of carbohydrate, protein and lipids in the cell wall.
Depending on the microalgae species, a hard surface such as silica,
calcium carbonate, algaenan or sporopollelin can be present. Other
products found in the cells are listed in the Table 8.
As shown in the Table, cellulose is commonly found in the cell
wall of Chlorella vulgaris. Nannochloropsis occulata cell wall contained
polysaccharide with sugar unit as glucose (68.8%), fucose (4.4%), ga-
lactose (3.8%), mannose (6.1%), rhamnose (8.3%), ribose (4.6%), and
xylose (4.4%) [64]. In addition, algaenan (a chemical-resistant, bio-
macromolecule) are found in the cell wall of Nannochloropsis occulata
[63]. Some Chlorella species contain algaenan, albeit with a different
chemical composition to those in Nannochloropsis occulata [61,62].
Sporopollenin (a chemical-resistant bio-macromolecule) was found
in Chlorella sp.[60].
Microalgal cell wall as described above provides structural rigidity
for the cells to adapt to their environments but the cell wall resistance
adversely affects efficiency of algal bioprocessing. These includes ge-
netic transformation, fermentation, anaerobic digestion, oil extraction
and biodiesel production. Indeed, it causes significant large solvent
requirement and energy load during extraction processes [10].
There are, however, exceptions, such as Botryococcus braunnii,
that excretes oil as less oxygenated isoprenoids outside the cell
Disruption techniques are broadly classified into mechanical,
physical, chemical and enzymatic [66,67] as shown in Fig. 2.
Cell disruption enhances lipid extraction from microalgae. Lee
et al. [68] while using different cell disruption for enhancing lipids
extraction from Botryococcus sp., Chlorella vulgaris and Scenedesmus
sp. obtained for autoclaving (5.4–11.9% lipids), bead-beating (7.9–
8.1% lipids), microwave irradiation (10–28.6% lipids), sonication
(6.1–8.1% lipids) and 10% NaCl osmotic shock (6.8–10.9% lipids).
Microwave irradiation produced the highest lipid enhancement.
Surendhiran and Vijay [69] compared the effect of different cell
wall disruption (enzymatic, ultra-sonication, osmotic shock and
acid lysis) on lipid extraction from Nitrogen replete and deplete
Nannochloropsis occulata. Acid lysis at a pH of 2 for 2 h was the
most effective, producing 54.3% lipid in Nitrogen depletion but
33.2% in replete samples. This is contrary to microwave irradiation
reported to be most efficient in disrupting Chlorella vulgaris,
Botrycoccus sp. and Scenedemus sp. reported by Lee et al. [68].
Microalgae cell wall chemistries vary, consequently cell disruption
techniques should be strain and species dependent.
3.1.4. Energy requirement of algae cell disruption
Cell disruption enhances lipid extraction, but can be very energy
intensive. In addition, algal cells are unsuitable for mechanical
presses as they are too small, and can pass through unchanged [66].
An overview of energy consumptions of microalgal cell dis-
ruptions that are considered suitable for commercial scale pro-
duction [66] is shown in Table 9.
A 33 MJ kg  1 (dry cells) was the lowest energy consumed,
which is significantly greater than 27 MJ kg  1 (the estimated
combustion energy from a typical algae biomass). Furthermore,
1184 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198

Table 6
Effect of media and catalyst type on fatty acid profile of microalgal lipids.

a b c d e f
wt% C. vulgaris C. vulgaris N. occulata Z. zofingiensis D. tertiolecta I. galbana

C12:0 – – 2 (1) – – –
C14:0 2 () 2 (0.1) 6 (6) 2 () 2 (1) 17 (22)
C14:1 – 1 (0.3) 0.1 (0.3) – – –
C15:0 – – 0.2 (2) – – –
C16:0 20 (17) 6 (5) 26 (30) 18 (15) 18 (25) 9 (14)
C16:1 1 (1) 16 (11) 22 (21) 1 (1) 1 (1) 4 (2)
C16:2 11 (3) – 1 (3) 7 (4) 2 (1) –
C16:3 14 (6) – – 9 (2) 4 (2) 2 (˗)
C16:4 – – – 2 (˗) 20 (12) –
C18:0 1 (2) 10 (11) 4 (2) 2 (3) 1 (2) 1 (1)
C18:1 4 (47) 27 (25) 12 (7) 18 (47) 5 (14) 5 (27)
C18:2 19 (10) 0 (9) 5 (3) 20 (17) 11 (9) 13 (3)
C18:3 28 (14) 21(22) 0.2 (1) 18 (8) 35 (31) 9 (4)
C18:4 – – 3 (0.4) 2 () 1 (1) 10 (10)
C20:0 – –  (0.2) – – 1 (2)
C20:1 – – 4 (5) – – –
C20:4 – – – – – –
C20:5 – – 13 (18) – – –
C22:6 – –  (0.2) – – 15 (12)
∑saturated 23 (19) 18(16) 38(41) 22(18) 21(28) 28(37)
∑monounsaturated 5 (48) 44(36) 38(33) 19(48) 6 (15) 34(29)
∑polyunsaturated 67 (33) 21(31) 21(25) 51(31) 73 (56) 34(29)

ND: The conditions inside the bracket refers to the FFA inside the bracket listed in the Table.
a,d-f
Breuer et al. [50]: Nitrogen replete medium (Nitrogen deplete medium).
b
Velasquez˗orta et al. [51]: acid catalysed reactive extraction (alkali-catalysed reactive extraction).
c
Renaud et al. [52]: NO3˗containing medium (NH4˗containing medium).

Table 7 4. The transesterification reaction

Composition of the microalgae cell wall (% of cell wall dry matter).
Biodiesel is usually made by transesterification of triglycer-
Microalgae Carbohydrate (%) Protein (%) Lipids (%) ND (%)
ides or esterification of free fatty acids derived from plants or
Chlorella vulgaris (F) 30.00 2.46 15.00 52.54 animals with low molecular mass alcohols containing catalyst.
Chlorella vulgaris (S) 35.00 1.73 10.00 53.27 Transesterification involves reacting a triglyceride with an al-
Kirchneriella lunaris 75.00 3.96 12.50 8.54 cohol in the presence of alkali or acid catalyst to form alkyl
ND: Non-detected substances.
esters of the corresponding alcohol and glycerol. Methanol is
commonly used because is the cheapest alcohol [46]. If the
Table 8
reaction goes to completion, three molecules of alkyl ester
Typical cell wall composition and storage products in few algae. (biodiesel) of the corresponding alcohol are formed. For in-
stance, if methanol is used, three molecules of fatty acid methyl
Substance Chlorophyta Chor- Ochrophyta Nanno- Reference ester and one molecule of glycerol are formed as illustrated in
ella vulgaris chloropsis occulata
Fig. 3.
Phycobilins Absent Absent [9] The stoichiometric ratio of alcohol to oil needed for this reac-
Storage Starch Chrysolaminarin [9,59] tion for an alkali catalyst is 3:1, but in practice, since it is an
carbohydrate (α˗1,4˗glucan) (β˗1,3˗glucan) equilibrium reaction, an excess of alcohol of 6:1 is typically re-
Cell wall cellulose polysaccharides [9,59] quired, to increase the rate and conversion [76]. A typical alkali
a c a
Additional cell wall Sporopollelin Algaenan [60],c[63]
components b
Algaenan b
[61,62]
transesterification takes at least 3 min for two step transester-
ification [77] but 10 min for in situ transesterification of Chlorella
vulgaris[78] to reach completion. In contrast, an acid-catalysed
reaction can be
4000 times slower [79].
the energy consumption is greater than the estimated minimum The alkali reaction is faster because it involves a strong nu-
theoretical energy consumption by a factor of 105. cleophile alkoxide species. Once it is formed, it directly attacks the
There would be economic justification for this high energy carbonyl moieties in the triglyceride to form the corresponding
consumption for algal cell disruption if the lipids or pigments alkyl esters as illustrated in the Fig. 4.
The steps involved in homogeneous alkali catalysis are as
were extracted for high value commodities, such as pharma-
shown in Fig. 4 [75]. They are:
ceutical or nutraceutical products. However, it becomes difficult
to sustain if the lipids are extracted for bulk fuels such as bio-
a) formation of active alkoxide catalyst species, RO  ;
diesel [66]. In order to make algal oil economically competitive
b) nucleophilic attack of RO  to the carbonyl group on trigly-
biodiesel feedstock, a less energetic disruption technique is re- ceride (TG) producing a tetrahedral intermediate;
quired. For instance chemical pre-treatment such as surfactant/ c) breaking down of the tetrahedral intermediate;
surfactant catalyst, alkali or acid catalyst for disrupting algae d) regeneration of the alkoxide (RO  ) species. The sequences are
cells are less explored. In addition, research effort should be repeated for both diglyceride and monoglyceride formation.
directed towards the effect of these chemical pre-treatment
in depolymerising the polysaccharides and algaena on the algal It should be noted that a detailed alkali catalysed two step
cell wall. transesterification schemes contains side reactions including free
 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198
Fig. 2. Classification of cell disruption techniques. Modified from [66].
fatty acid neutralisation. saponification of triglycerides and bio-
diesel (FAME) [77].
In contrast, as shown in Fig. 5, acid catalysis involves formation
of an electrophilic species, which reacts with the alcohol to form a
tetrahedral intermediate.
As shown in Fig. 5, the homogeneous acid-catalysed reaction
scheme for triglyceride transesterification [75] is:
a) Protonation of the carbonyl group by the acid catalyst to create
an electrophilic species;
b) Nucleophilic attack by the alcohol to generate tetrahedral
intermediate;
c) Proton migration and breaking down of the intermediate. The
sequences are repeated for both diglycerides and monoglycerides.
Acid catalysis takes considerably longer than alkali catalysis.
However, acid catalysts can catalyse esterification of free fatty
acids to biodiesel, as shown in Fig. 6. This is why it is applicable for
high free fatty acid (FFA) substrates.
4.1. Kinetics of reactive extraction (“in situ transesterification”)
Reactive extraction (“In situ transesterification”) is a direct
production of fatty acid methyl ester (FAME) from oil-bearing
biomass, achieved by contacting the material directly with an al-
cohol containing a catalyst. Zakariah and Harvey [80] studied the
kinetics of reactive extraction of rapeseed to FAME with metha-
nolic NaOH. Their simulated and experimental data suggested that
the process could be either mass transfer or kinetically controlled
depending on the concentration of the catalyst. At higher catalyst
concentrations (40.1 mol/kg-solvent) the process was controlled
by diffusion rate, but when the concentration was lower, it was
kinetically controlled. However, they did not consider in their
model the competing reactions which occur alongside the desired
FAME production. In addition, the model did not include the effect
of moisture on the FAME conversion. Complete drying of micro-
algae prior to biodiesel (FAME) production is energy intensive and
has been a critical factor in blocking commercial production of
algal FAME production. Salam et al. [78] reported the kinetics of
in situ transesterification of microalgae that included side reac-
tions. Both their experiments and model showed that a maximum
96% biodiesel yield can be obtained in 10 min using excess me-
thanol to oil molar ratio of 925: 1;excess catalyst concentration of
96% NaOH w/w oil before saponification FAME losses become
significant. The process shows higher level of water tolerance than
1185
a two-step transesterification. At 600:1 methanol to oil molar ra-
tio, the process was not adversely affected by up to 20% moisture
in the microalgae. A derived numerical model fitted with experi-
mental data from their study showed that other side reactions
including FAME and triglyceride saponification; free fatty acid
neutralisation occur alongside the desired biodiesel synthesis in a
NaOH˗catalysed reactive extraction. Their findings is summarised
in the Fig. 7.
4.2. A comparison of two step transesterification and reactive
extraction
Two step transesterification requires refined oil from either ve-
getable or other oil seeds such as canola, rapeseed or soymeal. As
much as 88% of total production cost of a two˗step biodiesel pro-
duction is ascribed to the refined oil feedstock [81] . It is also im-
portant during two˗step transesterification to control the water
content in the feedstock, catalyst or methanol particularly for alkali-
catalysed process. Typically, the maximum tolerable water content
in oil is 0.3 wt% [76]. Beyond this value, there could be saponification
of the oil to soap, which reduces the biodiesel yield [82] and causes
difficulty in product separation [82,83]. In addition two step bio-
diesel production involves hexane extraction steps that are energy-
intensive and time-consuming. Up to 90% of the process energy can
be accounted for in the hexane extraction and drying steps [8]. Al-
ternatively, a reactive extraction (“in situ transesterification”) could
be used. In this process, the biomass is fed directly into the reaction
system. This eliminates the extraction steps, biomass pre-treatment
and degumming steps, and tolerates some level of water [6,7,84]
due to excess methanol used in the process. The basic differences
between the two processes are shown in the Fig. 8.
Previous studies have demonstrated the feasibility of obtaining
greater FAME conversion from such in situ transesterification than
from a two-step approach [85–87]. It is equally effective in making
biodiesel from pure algal strains [87,88] and mixed cultures of
microalgae [6,84]. However, microalgae are aquatic species. Their
dewatering usually results into 5–35% solids [89,90]. Approxi-
mately 20–30% of algal biomass production cost is accounted for
by algal recovery [91]. Therefore the cost associated with drying of
wet microalgae, if required, is prohibitively high [8].
Besides, another drawback of this method is the requirement
for large amounts of methanol to oil molar ratio which usually in
the range of 100:1–1000:1. This is necessary since methanol plays
a dual role: it acts as an oil extractor and as a reactant. Triglycides
are non-polar lipids, so not readily soluble in methanol. In
1186 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198

addition, microalgal cell wall's resistance causes large solvent re-

References
quirement during bioprocessing [10]. The main problem of such
high excesses of methanol is the capital and running (energy)

[68]

[68]
[70]
[70]

Laboratory, industrial [72]

Laboratory, industrial [73]
[71]

Laboratory, pilot scale [74]

(F)
costs associated with its recovery from the product streams, which

Laboratory, industrial
Laboratory, industrial
Laboratory, industrial

Laboratory, industrial

Laboratory, industrial
would almost certainly involve a substantial distillation column.
Scale of use

4.3. Overview of In situ transesterification

The feasibility of in situ transesterification of sunflower seed oil

(E)

was first reported in 1985 by Harrington and D’Arcy˗Evans [85].

They identified the following advantages:
Energy consumption MJ (kg dry

1. Esterification of the oil embedded in the hull, which could im-

prove the overall yield of the alkyl ester.
2. Reduction of the oil losses from the hull/kernel separation.
9.6 (Microwave only)

3. Esterification of the lipid, which may not be extracted by the

hexane due to its different solubility from triglyceride.
140 (modelled)

4. Improvement of carbohydrate digestibility of the residue by acid

or alkali catalyst interaction.
mass)  1

529

504

420
132

In addition, they observed the approach produced higher

(D)

68

33

conversion of alkyl ester than the two step transesterification. Both

processes produced the same quality of alkyl ester. The technical
Calculated energy consumption GJ m  3 cell

feasibility of making biodiesel from microalgae via in situ trans-

esterification has also been demonstrated by various investigators
[6,51,78,84].

4.4. Key process variables in in situ transesterification of microalgae

1.2 (Microwave only)

Some of the process variables that determine the FAME yield/

rate during in situ transesterification of microalgae for FAME are
1.4 (modelled)

shown in Table 10. The results of some in situ studies on plants are
suspension

also included for comparison.

Generally, the following observations can be made from the
4.5
0.7

2.5

0.3
(C)

2.1
1.1

Table:
Botryococcus, Chlorella, Scenedesmus (100 mL, 5 kg m  3, 700 W,
Botryococcus, Chlorella, Scenedesmus (100 mL, 5 kg m  3, 840 W,

1. FAME can be produced via in situ transesterification from both

Chlorococcum sp. (200 mL, 8.5 kg m  3, 2.5 kW, 6 min, High)

Hydrodynamic cavitation Saccharomyces cerevisiae (50 L, 10 kg m  3, 5.5 kW, 50 min,

Chlorococcum sp. (200 mL, 8.5 kg m  3, 750 W, 5 min, low)

Saccharomyces cerevisiae (0.8 L, 10 kg m  3, 600 W, 15 min,

freshwater and marine microalgae and other oil-bearing feedstocks.

2. The process requires either homogeneous alkali, acid, or het-
Scenedesmus (100 mL, 75 kg m  3, 1.2 kW, 1 min, high)
Overview of experimental cell disruption techniques and their energy consumptions.

erogeneous catalyst to proceed at a reasonable rate.

3. The process requires high mixing rate (150–500 rpm) to achieve
Mathematical modelling on an industrial scale

a reasonable FAME conversion.

4. When a heterogeneous catalyst is used a larger amount of
HPH: High pressure homogenization; HSH: High speed homogenization.

methanol is needed than for either homogeneous alkali or acid

Substrate & experimental conditions

catalyst, which is probably due to phase transfer limitations.

However, inclusion of co solvents such as hexane or methylene
dichloride helps to reduce the amount of methanol.
5. The reaction can proceed without catalyst with supercritical
methanol.
Column D¼ Column C/(Concentration in column B).

6. Microalgae in situ transesterification can occur at room tem-

perature particularly with alkali catalyst. Whalen et al. [6]
5 min, high)

5 min, high)

observed significant increase in FAME conversion rate with

medium)

medium)

increase in temperature from 60 to 80 °C but no significant

change in the rate was observed by increasing the temperature
(B)

from 80 to 110 °C strongly due to evaporation of methanol.

Similarly, Ehimen et al. [93] recorded significant in FAME
Microwave þ solvent

conversion rate when the temperature was increased from 30

to 60 °C, but observed no significant change in FAME conversion
Disruption technique

rate between 60 and 90 °C.

Freeze drying
Microwave
Bead mills
1. Sonication

7. FAME yield during in situ transesterification depends on a number

of variables, which include microalgae species, temperature, cat-
2. HPH
3. HSH

alyst/oil molar ratio, methanol/oil molar ratio, agitation rate,

Table 9

moisture content of the reactants or the feedstock, reaction time,

(A)

4.

5.

6.

8.
7.

phase and type of the catalyst and co solvent.

 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198 1187

Fig. 3. Overall transesterification reaction.

Fig. 4. A simplified homogeneous alkali transesterification schemes.

4.4.1. Solvent
The solvent plays a dual role during in situ transesterification. It
functions as an extractant and a reactant. Methanol is the most
commonly employed solvent because it is cheaper than all other
aliphatic alcohols. It is also less expensive to recover than ethanol
because it does not form an azeotrope with water [46]. Ordinarily,
methanol is the poorest extractant of triglycerides among aliphatic
alcohols [98]. This is because the dissolution of triglyceride in-
creases with increase chain length of the alcohol [98]. Ester yield
during acid-catalysed in situ transesterification of soy bean oil
increased with decrease in the polarity of the alcohol [98]. In
contrast, the ester yields during acid˗catalysed in situ transester-
ification of C. gracilis with methanol, ethanol, 1˗butanol, 2˗me-
thyl˗1˗propanol, and 3˗methyl˗1˗butanol did not vary significantly
[6]. This could be because soybean oil and C. gracilis are not similar
in terms of cell wall chemistry and transesterifiable lipid. Thus the
activity of the alcohol during the reactive extraction of the bio-
diesel should not be expected to be the same. In situ transester-
ification is always characterized by a large amount of methanol to
oil ratio, between 100:1–1000:1 methanol to oil molar ratio
[11,92,93].
Co-solvents can be used to reduce the methanol molar ex-
cesses. For instance, Li et al. [88] obtained 95% FAME yield during
acid-catalysed reactive extraction of Chlorella pyrenoidosa at 90 °C,
methanol to oil molar ratio of 165:1 (4 mL methanol to 1 g
microalgae) with 6 mL hexane as co-solvent. Zeng et al. [96] ob-
tained 98% FAME yield during alkali catalysed reactive extraction
of sunflower at 20 °C , 101:1 methanol to oil molar ratio with 58 ˗
1 diethoxyl methane (DEM) to methanol molar ratio as co˗solvent.
The two investigations did not include the FAME yield that would
be produced in the absence the co-solvents (i.e. hexane and DEM).
However, from the overview of parameters influencing the effi-
ciency of reactive extraction shown in Table 10, the effect of the
co˗solvent can be clearly seen, in that they have the lowest me-
thanol to oil ratio. However, the co˗solvent should be carefully
screened for health and environmental hazards. Many of these
co˗solvents have significant environmental impacts, and would
significantly adversely affect the processes’ life cycle carbon
emissions. Co˗solvents that are difficult to separate from other
species in the reaction mixture can reduce the purity of biodiesel
[84]. Furthermore, there would be an added process cost for the
separation of the co-solvent.
4.4.2. Temperature and reaction time
Temperature affects the yield and/or rate of alkyl ester depending
on the type of catalyst. It has been reported that there was no sig-
nificant difference during alkali in situ transesterification of Jatropha
curcas and soybean oil between 30 and 65 °C [94,97].
However, acid-catalysed in situ esterifications increase in rate
with temperature. Wahlen et al. [6] observed an increase in FAME
1188 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198
Fig. 5. Homogeneous acid catalysed transesterification scheme.
Fig. 6. Acid˗catalysed esterification of free fatty acid to alkyl ester.
yield/rate during acid˗catalysed in situ transesterification of C-
gracilis with increase in temperature from 20 to 150 °C. They found
that most significant changes occurred between 60 and 80 °C. Si-
milarly, Ehimen et al. [93] reported an increase in biodiesel rate
during acid-catalysed in situ transesterification of Chlorella oil from
23 to 90 °C. They also found that no significant changes occurred
in the range 60–90 °C. The reaction time during in situ transes-
terification depends also on the nature of catalyst. Just like two
step transesterification in which alkali catalyst is
4000 times
faster than acid catalysed transesterification [79], alkali in situ
transesterification is faster than its acid-catalysed counterpart. For
instance, Velasquez˗orta et al. [51] reported a FAME yield (97%) at
60 °C during acid-catalysed reactive extraction of Chlorella vulgaris
at 20 h. They obtained 78% FAME yield in 1.25 h using alkali cat-
alyst at the same conditions. Salam et al. [78] used 925:1 methanol
to oil molar ratio, 96% w/w oil alkali concentration at 60 °C to
achieve 96% FAME yield in 10 min during in situ transesterification
of Chlorella vulgaris. In practice, it is economical to operate at re-
action temperatures close to the boiling point of the alcohol [93].
4.4.3. Agitation rate
A high mixing rate should be used during in situ transester-
ification to obtain a high FAME yield. As can be seen in Table 5,
mixing rate of 150–500 rpm [51,84,93–95] are considered to be
enough to achieve a high FAME yield but some researchers always
ensure that a high mixing rate which facilitate a complete sus-
pension of particles in the reaction vessel is sufficient to achieve a
high yield [93,97] . Ehimen et al. [93] studied the effect of
continuous stirring, stirring for only 1 h, stirring for 1 h on and 1 h
off and not stirring at all on the FAME conversion during in situ
transesterification. They observed that mixing is positively affect-
ing FAME conversion and not stirring at all caused significantly
low FAME conversion. Stirring for only 1 h produced in 8 h, a
maximum 91% FAME conversion of the amount obtained in con-
tinuous stirring for 1 h. They obtained no significant difference in
FAME conversion using 1 h on and 1 h off stirring treatment and
continuous stirring. This mixing treatment is therefore suggested
for energy savings during in situ transesterification of microalgae.
Kasim and Harvey [94] studied the effect of mixing intensity (100–
400 rpm) on alkali-catalysed reactive extraction of FAME from
Jatropha curcas at 60 °C and catalyst concentration (0.1 N). They
observed that the FAME yield increased with increase in mixing
rate. The process was independent of mixing speed at 300 rpm as

90% FAME yield was obtained at that condition which was not
significantly different with that of 400 rpm. At 100 rpm, the seed
settled down at the bottle of the reaction vessel while the FAME
yield significantly reduced to 37.2%.
4.4.4. Catalyst
In situ transesterification of lipid-bearing feedstocks requires a
catalyst for it to proceed at “mild” conditions [94,95]. Various
catalysts have been used for in situ transesterification, including
homogeneous alkali acid catalysts, and heterogeneous catalysts.
4.4.5. Acid-catalysed reactive extraction
Harrington and D’Arcy˗Evan [85] in their pioneering research
demonstrated the feasibility of acid-catalysed in situ transester-
ification of sunflower oil. They obtained a 40% yield with H2SO4
concentration of 1.2% v/v of methanol in
4 h reaction. In contrast
a 30% yield was obtained with hexane-extracted oil from sun-
flower with the same process conditions. Kildiran et al. [98] con-
ducted an extraction and acid-catalysed in situ transesterification
of soybean oil using methanol, ethanol, n-propanol and n˗butanol.
They found that in situ transesterification sequentially proceeds
 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198 1189

Fig. 7. Proposed reaction scheme for NaOH  catalysed reactive extraction of microalgae for FAME production. FAME: fatty acid methyl ester (Biodiesel); TAG: triacylgly-
cerides; FFA: free fatty acid; OH  : hydroxide species; CH3O  : alkoxide species (the actual catalyst), MeOH: methanol.
Source: Salam et al. [78].

Fig. 8. Comparison between in situ and two˗step transesterification.

through oil dissolution and transesterification of triglyceride and
the triglyceride dissolution increased with increasing alkyl chain
length of the alcohol.
In microalgae research, acid˗catalysed in situ transesterification of
microalgae oil at high yields have been demonstrated by many au-
thors. Li et al. [88] reported a 95% FAME conversion in 2 h during
acid-catalysed in situ transesterification of Chlorella pyrenoidosa at
90 °C, 154:1 methanol: oil molar ratio, 0.234: 1 H2SO4: oil molar with
hexane as co-solvent. They reported hexane to be an effective co-
solvent for reducing methanol-oil molar ratio. Velasquez-orta et al.
[51] obtained a 97% FAME conversion in 20 h during reactive ex-
traction of Chlorella vulgaris at 60 °C, 600:1 methanol:oil molar ratio,
0.35:1 acid to oil molar ratio. A maximum FAME conversion of 96%
was reported by Haas and Wagner [84] who performed acid catalysed
in situ transesterifications with algae biomass containing different
moisture content at 65 °C; 308:1 alcohol to oil molar ratio. Zhao and
Liu [92] conducted acid-catalysed in situ transesterification on a di-
verse species of oil-bearing feed stocks (Lipomyces starkey, Mortierella
isabella, Rhodosporidium toruloides) and obtained a maximum FAME
conversion of 98% at 70 °C, 868:1 alcohol:oil molar ratio at 20 h.
Similarly, Wahlen et al.[6] reactively extracted FAME from dif-
ferent cultures of microalgae biomass with acid catalysts and ob-
tained a maximum FAME conversion of 77% at 0.33 h, 80 °C, and
1831:1 M ratio of methanol to oil. They found that the water tol-
erance of the process increased to as high as 400% dry weight of
Chaetoceros gracilis. However, the molar ratio of methanol to oil, at
3460:1 was very high. Downstream methanol removal would be a
significant running cost at such molar ratios. This would have to be
weighed against the running cost savings due to the reduced
feedstock drying duty.
1190 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198

Table 10
Process conditions: in situ transesterifications of algal biomass.

Feedstock Temp. (°C) Solvent catalyst (oil Mixing Molar ratio Water con- Reaction Conversion (oil Remarks References
basis) (mol/ rate (rpm) (solvent: oil) tent % (w/w) time (hr) basis) (%)
mol) dry algae

Nannochloropsis Spe- 65 Methanol Mg˗Zr – 1569:1 0 4 60 Methylene [11]

cies (biomass) oxide dichloride
1.65:1 co˗solvent
Nannochloropsis Spe- 65 Methanol Mg˗Zr – 592:1 0 4 47 Methylene [11]
cies (Oil) oxide dichloride co-
1.65:1 solvent
Chlorella pyrenoidosa 90 Methanol H2SO4 – 154:1 0 2 95 Hexane [88]
0.234:1 co˗solvent
Chlorella vulgaris 60 Methanol H2SO4 380 600:1 0 20 97 [51]
0.35:1
Chlorella vulgaris 60 Methanol NaOH 0.15:1 380 600:1 0 1.25 78 [51]
Algae biomass 65 H2SO4 150 308:1 8 2 80 [84]
0.678:1
Algae biomass 65 Methanol H2SO4 150 308:1 1 2 86 [84]
0.678:1
Algae biomass 65 Methanol H2SO4 150 308:1 0.2 2 96 [84]
0.797:1
L.starkeyi 70 Methanol H2SO4 – 868:1 0 20 97 – [92]
0.093:1
HCl
0.186:1
M. isabellina 70 Methanol H2SO4 – 868:1 0 20 91 – [92]
0.093:1
R. toruloides 70 Methanol H2SO4 – 868:1 0 20 98 – [92]
0.093:1
HCl
0.186:1
Chaetoceros gracilis 80 Methanol H2SO4 – 988:1 0 0.33 82 [6]
0.158:1
Chaetoceros gracilis 80 Methanol H2SO4 – 1,977:1 100 0.33 67 [6]
0.158:1
Chaetoceros gracilis 80 Methanol H2SO4 – 3,460:1 400 0.33 57 [6]
0.158:1
Chlorella sorokiniana 80 Methanol H2SO4 – 1,831:1 dry 0.33 77 – [6]
0.158:1
Synechococcus 80 Methanol H2SO4 – 2,354:1 dry 0.33 40 – [6]
elongatus 0.158:1
80 Methanol H2SO4 – 3,013:1 dry 0.33 74 – [6]
0.158:1
60 Methanol H2SO4 500 314:1 dry 8 92 [93]
8.49:1
Jatropha curcas 30 Methanol NaOH 300 400: 1 0.5 88 o0.71 mm [94]
2.4:1 Particle size
Rapeseed 30–60 Methanol NaOH 200 600 :1 o 6.7 wt% 1 85 [95]
2.1:1
Sunflower 20 Methanol NaOH – 101:1 4.6% 0.2 98 DEM co˗solvent [96]
0.5:1
Soybean NaOH – [97]
23 Methanol 2:1 543:1 8
60 Methanol 1.6:1 226:1 8 84

DEM: Diethoxymethane.

It is interesting to note that the times to reach a maximum
FAME conversion vary between the microalgae species, as shown
in Table 10. The times range from 0.33 to 20 h. This is expected
since microalgae are very diverse (more diverse than plant and
animal kingdoms), and in particular have a wide range of cell wall
compositions. The difference in their cell wall compositions could
have a significant effect on the time to reach optimum FAME
conversion. Another major advantage of using acid catalysts for
in situ transesterification is that they are more tolerant to high free
fatty acid concentrations.
4.4.6. Alkali-catalysed reactive extraction
A number of researchers have obtained high FAME conversions
in alkali˗catalysed in situ transesterification of oil-bearing feed-
stocks. Haas et al. [97] reported catalysed reactive extraction of
soybean oil, where he obtained an 84% FAME conversion with no
significant difference when 23 °C or 60 °C was used. However, they
found that operating at 23 °C required larger methanol oil molar
ratios (543:1) than (226:1), which was needed for 60 °C. Other
researchers also observed no significant change in FAME conver-
sion between 23 °C and 60 °C in alkali-catalysed reactive extrac-
tion of FAME from Jatropha curcas [94] and rapeseed [95]. An
87.8% FAME conversion was obtained by Kasim and Harvey [94] for
Jatropha curcas L. Similarly, Zakaria and Harvey [72] who worked
on rapeseed/NaOH/methanol system obtained an 85% FAME con-
version at optimum conditions. Zeng et al. [96] observed a 97.7%
FAME conversion with sunflower/NaOH/methanol/DEM. However,
this entailed the inclusion of an extra solvent, which will lead to
increased complexity in the separations train. While several pub-
lications have been conducted on the alkali reactive extraction of
oil seeds as indicated above, few studies have been conducted
using alkali catalyst for reactive extraction of microalgae, which
 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198
has created a gap in generalising the schemes of reaction exhibit
by alkali reactive extraction. Velasquez-orta et al. [51] reported a
78% FAME at 60 °C during alkali-catalysed reactive extraction of
Chlorella vulgaris in 1.25 h. Salam et al. [78] used 925:1 methanol
to oil molar ratio, 96% w/(w oil) alkali concentration at 60 °C to
achieve 96% FAME yield in 10 min during in situ transesterification
of Chlorella vulgaris. They achieved this high FAME yield despite
high levels of free fatty acid (6% lipid) in Chlorella vulgaris. This
shows high catalyst concentration and excess methanol to oil
molar ratio but short reaction time can be used to avoid saponi-
fication FAME losses and to produce enhanced rate during alkali-
catalysed in situ transesterification of microalgae.
4.4.7. Heterogeneous-catalysed reactive extraction
A heterogeneous catalyst has also been used to promote in situ
transesterification [11]. They compared the FAME yield obtained
during in situ transesterification of Nannochloropsis and two step
transesterification of Nannochloropsis oil with a heterogeneous
(Mg2Zr5O12) catalyst using methylene dichloride as a co-solvent. A
maximum FAME yield of 60% was observed at 65 °C, 4 h, 10 wt%
catalyst and 45 mL of mixed solvent (3:1 v/v methanol/methylene
chloride). A lower FAME yield of 47% was obtained with two step
transesterification using the same process conditions as the in situ
transesterification. The authors found that the FAME yield increased
with increased catalyst concentration and volume of mixed solvent
but further increases in these two parameters lead to reduced yields.
Recently, micro-algal biodiesel production via a two-step in situ
transesterification was reported by Dong et al. [99]. This process in-
volved a two-step in situ transesterification, where the algae free fatty
acid was reduced with Amberlyst-15 before alkali in situ transester-
ification. They obtained a maximum FAME conversion of 94.9%.
4.4.8. Non-catalysed reactive extraction
It is possible for two step and in situ transesterifications to
proceed without catalyst in supercritical water or alcohol. Saka
and Kusdiana [100] reported transesterification of rapeseed oil in
supercritical methanol. They obtained a
95% FAME yield in 4 h at
350 °C, 45 Mpa and 1:42 oil to methanol molar ratio. Similarly, Lim
et al. [101] conducted in situ transesterification of Jatropha curcas L
seeds with supercritical methanol with the aid of hexane as co-
solvent. A maximum FAME yield of 103% (w/w) was obtained at
300 °C, 240 MPa, 10 mL/g methanol to solid ratio and 2.5 mL/g
hexane to seed ratio. This FAME yield was greater than theoretical
FAME yield obtained from n-hexane Soxhlet extraction. This in-
dicates that FAME was extracted from components of the biomass
besides triacylglycerides possibly polar lipids such phospholipids.
The advantages of this method over catalysed transesterification,
as reported by Saka and Kusdiana [100], include:
1. Shorter time to completion.
2. Simpler process, requiring fewer purification steps.
3. The ester yield is greater than catalysed process.
The major drawbacks of the process are that it operates at high
temperature and pressure, leading to significant increases in capital
cost, and increased costs associated with safety and monitoring.
4.4.9. Moisture content
One major challenge in biodiesel production is the need for dry
feedstocks. The moisture limit for common biofuel feedstocks is
0.5 wt% [83], and in practice lower moisture limits are preferred.
Water, in the feedstock or the methanol causes a significant re-
duction in the yield of biodiesel [83]. It also results in soap for-
mation in alkali-catalysed transesterification, leading to increased
complexity in the product separation train [83]. However, in situ
transesterification has been shown to be more water-tolerant than
1191
two step transesterification. Velasquez˗orta et al. [7] found that a
reactively extracted wet Nannochloropsis cell at 1.5% moisture
content has equal FAME yield or higher than a dried cell with both
acid and methoxide catalyst. However, they observed a decrease in
the FAME yield at 10% moisture. They found similarly moisture
tolerances with Chlorella cells for a moisture content which was
not greater than 1.5%. Salam et al. [78] observed during alkali-
catalysed in situ transesterification of Chlorella vulgaris that at
600:1 methanol to oil molar ratio, the process was not adversely
affected by up to 20% moisture in the microalgae. Similarly, during
acid˗catalysed in situ transesterification of C. gracilis, Wahlen et al.
[6] achieved a 57% FAME yield with 3460:1 methanol: oil mole
ratio at 400 wt% moisture content compared to a 82% FAME yield
with 988:1 methanol: oil mole for fully dried cells. The difference
in the moisture tolerance obtained by these authors is possibly
due to techniques used to quantify the moisture content. While
Velasquez-orta et al. [7] used bound water in the algae, Salam et al.
[78] and Wahlen et al. [6] used free water. Zakariah and Harvey
[95] also observed some level of moisture tolerance during alkali-
catalysed reactive extraction of rapeseed oil for FAME. However,
they observed a drastic reduction in the FAME yield when the
moisture content was greater than 6.7 wt%. Water tolerance of
in situ transesterification is caused by excess methanol to oil molar
ratio but removal of the unreacted methanol (
94% of it) usually
by distillation would increase operating costs. It has been shown
that reducing the water content reduces the excess methanol re-
quired: Haas and Scott [102] reported that a 60% reduction in
methanol and a 56% reduction in NaOH were achieved when fully
dried soybean was used than when the bean contained 2.6 wt%
moisture content. No researchers have investigated the effect of
including SDS in H2SO4 on the water tolerance of the in situ
transesterification of microalgae. This is important, as the sig-
nificant amounts of energy required to completely dry microalgal
biomass or microalgal oil to the levels required in two steps bio-
diesel production render the process uneconomic. This is currently
one of the major technical challenges to microalgal biodiesel
production. Complete drying of algae is energy intensive, which
significantly increases the cost of algae pre-treatment.
5. Microalgae species
In a practical biodiesel production, selection of a suitable algal
species is an important factor, since there are differences between
lipid content and biomass productivity among different species and
even within the same algal species. It can be seen from Table 11, that
the Nannochloropsis species is a competitive biodiesel candidate
among marine microalgae, as it is relatively high-yielding and
productive. Nannochloropsis occulata have been reported to accu-
mulate as much as 60% lipid (w/w dry algae) but this high lipid yield
was achieved only in a nitrogen-depleted medium [103]. However,
lipid content of 8–35% lipid (w/w dry algae) is common in this
species if the cells are not environmentally stressed [103] Amongst
freshwater microalgae, Chlorella species are suitable due to their
substantial lipid accumulation. Chlorella species have been reported
to accumulate 19–66% lipid content (w/w dry algae) but this high
yield is achieved under heterotrophic conditions using urea [104] or
rice straw hydrolysate [103] as carbon source. Marine microalgae
have additional advantages over their freshwater counterparts as
they do not require fresh water, so could not compete with food
crops for this resource. Some types of microalgae, such as Nanno-
chloropsis occulata, Dunaliella tertiolecta [105], Pavlova lutheri, Ske-
letonema sp, Chaetoceros calcitrans, Tetraselmis sp. and Isochrysis sp.
[103] as can be seen in Table 11, can be cultivated on brackish or sea
water, thereby posing no threat to freshwater for human con-
sumption or for agricultural use.
1192 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198
5.1. Protein and carbohydrate content of algae
In addition to lipids, microalgae contain substantial levels of
proteins and carbohydrates [57]. Results of the study conducted by
Becker [57] on the biochemical compositions of some microalgae
are illustrated in the Table 12. It shows that significant portions of
algal biomass were carbohydrate and protein, which means if
utilise after the reactive extraction as added value products could
improve the process's economy.
6. Probable ways of making a sustainable in situ
transesterification
6.1. Anaerobic digestion of algal residue after reactive extraction
Algal residue after reactive extraction may contain carbohy-
drate, protein, and unconverted lipids. Therefore, it can be used as
feed for either bioethanol or biogas production. If the residue
contains no toxic substances, it could serve as supplement for
animal feeds. Biogas production through anaerobic digestion is
considered as better option due to its efficient conversion of all the
residual carbohydrate, proteins and unconverted lipids to final
products [108] as shown in the Fig. 9.
Anaerobic digestion is a biological breakdown (bio˗depoly-
merisation) of complex organic compounds by consortium of
bacteria in the absence of oxygen to produce biogas. It occurs
naturally in a landfill or artificially in a digester under a control
conditions. The resulting biogas usually contains methane
(
60%), CO2 (
40%) and other trace gases [109]. Due to occur-
rence of a naturally induced anaerobic digestion in a landfill,
spent microalgae or degradable organic compounds should be
disposed of in an environmentally sustainable way.as methane's
greenhouse gas effect was
20 times greater than CO2 [110].
Whole algae and spent algal residue of lipid extraction can be
digested anaerobically to produce biogas [111,112] but little or no
information are available in the literature on the use of algal
residue resulting from in situ transesterification of microalgae for
biogas production. Apparently algal residues obtained from lipid-
extracted microalgae and reactive extracted (in situ transester-
ification) microalgae would be different due to varying process
conditions. One major difference is that in situ transesterification
involved methanol containing catalyst (acid or alkali) which
might cause disruption of the algae cell wall resulting in en-
hancement of biogas production. More research effort should be
directed towards utilisation of such residue for biogas produc-
tion. It could contribute to the improvement of the economy of
in situ transesterification. Besides, the biodigestate can be re-
covered as manure to support algal cultivation [113] and the
biogas (methane) produced can be burnt to supply the electrical
energy required for separating or concentrating the dilute algal
biomass-water mixture [114,115]. Burning of methane can also
supply the energy required for drying the biomass particular in a
region where sun˗drying is not a feasible option.
6.1.1. Causes of inhibition during biogas production
The yield of methane produced during anaerobic digestion of
microalgae strongly depends on a number of inhibitory agents and
conditions. These inhibitors include carbon/nitrogen (C/N) ratio [108],
long chain free fatty acids (LCFAs), [116–120], extraction solvents
[111], salinity [121,122] particularly for marine species. Becker [57]
showed that most microalgae contained protein more than carbo-
hydrate. Considering the fact that spent residue after reactive ex-
traction (in situ transesterification) would certainly have low lipid
content due to high percentage of it being converted to biodiesel, C/N
ratio is therefore a major concern when such residue is used as feed
in anaerobic digestion. Approximately 20–30C/N ratio is considered
as optimum for anaerobic digestion process [123]. A significant high
C/N ratio leads to rapid consumption of nitrogen by methanogens for
its metabolic protein requirement resulting into low biogas produc-
tion while a low C/N causes accumulation of ammonium ion (NH4)
[108]. Excess NH4 raises the pH of the bio˗digestate above the opti-
mum (6.7–7.5 pH) causes inhibition of methane production [108].
Co˗digestion of low C/N substrate with a high C/N substrate is one
probable solution to reduce its inhibitory effect. Yen and Brune [124]
achieved a significant increase in methane production from 573775
to 1170775 mL CH4/day when algal sludge was co-digested with
waste paper.
When whole algae is used for anaerobic digestion, effect of long
chain free fatty acids (LCFAs) should be considered as they inhibit
biodegradability of hydrolytic bacteria, acidogens, acetogens, and
methanogens community resulting into low biogas production
[116–120]. Their inhibitory effects increase with increase in the
chain length [125], presence of double bond [126,127], and in-
crease in the number of double bonds [125,128]. Use of adequate
amount of inoculum to substrate (I/S) ratio has been effective for
reducing LCFAs inhibitory effects [111,129,130]. A I/S ratio of 1–2 is
considered to be optimum [111]. Similarly, addition of calcium ion
[111,130] has been shown to partially reduce the LCFAs inhibition
particularly when the I/S ratio met the threshold level [130].
However, inclusion of calcium ion has no significant effect on
LCFAs when residual algae was used [111] obviously due to low
level of LCFAs in the algal residue. Therefore when spent algal
residue after reactive extraction serve as feed in anaerobic diges-
tion, LCFAs inhibition should not be a major concern as both the
neutral lipids (TAG) and part of the membrane lipids would have
been converted into biodiesel causing significant low level of
LCFAs in the residue.
6.2. marine species for biofuel production
Using fresh water microalgae are still not economically attrac-
tive and cannot support a large scale biodiesel production [2] due
to a huge fresh water footprint required [131]. A water footprint of

3700 kg kg  1 of biodiesel is estimated for algae cultivated in
open pond in fresh water without water recycling but recycling
reduced the water requirement to 600 kg.kg–1 of biodiesel. In
contrast, the water footprint plus the compensated fresh water for
evaporation for algae cultivated in open pond in seawater was
370 kg kg–1[131]. Since there was a significant decrease in the
freshwater footprint when marine species was used, this species is
a good alternative to freshwater species to achieve commercial
reality of algal fuel. Apparently, residue from marine species will
contain high salt concentration resulting in salinity inhibition
during anaerobic digestion [121,122]. This causes bacteria cells
death and inhibition of reaction pathways in anaerobic digestion
resulting to low biogas production [132]. Probable techniques of
reducing salinity inhibition include using municipal bio-solids
digestate as inoculum [133], use of halophilic microorganisms or
halo-tolerant microorganism obtained from a long acclimation as
inoculum [132] or washing of the biomass with fresh water to
reduce its salinity [2]. Although utilisation of residue for biogas
production will improve the economy of the process, significant
cost during in situ transesterification resulting from algae drying
and recycling of the excess unreacted methanol.
6.3. In situ co-production of diethyl-ether during in situ
transesterification
In situ transesterification of microalgae for biodiesel production
requires excess methanol due to mass transfer limitation resulting
from solid (algae) and liquid (catalyst plus methanol) involved.
Table 11

K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198
Different microalgae species, habitat, lipid content, biomass productivity and applications.

Algae species Habitat Lipid content % w/ Biomass productivity mg/L/ Applications Remarks References
w day

Nannochloropsis species Marine, fresh and brackish 60 300 Biofuel and feed supplement Nitrogen deprived medium promoted lipid content [103]
water
Nannochloropsis oculata Marine, fresh and brackish 7.90–15.86 – Biofuel Nitrogen deprived medium and temperature in- [103]
water fluence lipid content
Nannochloropsis species Marine , fresh and brackish 29.6–35.5 170–210 Biofuel CO2 enriched air; continuous illumination for [103]
water cultivation
Dunaliella tertiolecta Marine 15.20 28 CO2 capturing; Biofuel/ wastewater treatment – [105]
Pavlova lutheri Marine 35.50 140 Biofuel CO2 enriched air; continuous illumination for cul- [103]
tivation 2
Skeletonema species Marine 31.80 90 Biofuel CO2 enriched air; continuous illumination [103]
Chaetoceros Calcitrans Marine 39.80 40 Biofuel CO2 enriched air; continuous illumination for [103]
cultivation
Tetraselmis sp. species Marine 12.90–14.70 280–300 Biofuel CO2 enriched air; continuous illumination for [103]
cultivation
Isochrysis species Marine 22.90–27.40 140–170 Biofuel CO2 enriched air; continuous illumination for [103]
cultivation
Scenedesmus species Freshwater 12.80–21.10 126.54–260 Waste treatment/Biofuel;CO2 mitigation – [103,106]
Chlorella protothecoides Freshwater 50.30–55.20 2020–7300 Biofuel Heterotrophic condition enhanced lipid [107]
accumulation
Chlorella species Freshwater 19.30–66.10 230 Biofuel, food supplement, sorption of toxic chemical Urea was used as a source of low cost Nitrogen [103,104]
Chlorococcum species Freshwater 19.30 280 CO2 mitigation CO2 enriched air; continuous illumination for [103]
cultivation
Chlorella pyrenoidosa Freshwater 56.30 1100 Biofuel Rice straw hydrolysate served as carbon source [103]

1193
1194 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198

After completion of the reaction the unreacted methanol (
94% of pressure (
5 MPa) there could be additional capital cost and in-
it) needs to be removed from the product streams and this re- creased cost associated to safety and monitoring. However, this
quires substantial distillation heat load. Removal of this excess cost will be substantially less than producing biodiesel from pre-
methanol has been a major drawback of in situ transesterification. extracted oil at supercritical condition of methanol such as 350 °C
It significantly contributes to the high operating cost of this pro- and 45 MPa reported by Saka and Kusdiana [100] and in situ
cess rendering it uneconomical. transesterification of the algae at supercritical methanol condi-
A more appealing process will be to co-produce dimethyl ether tions such as 300 °C and 240 MPa reported by Lim et al. [101].
(DME) in situ along with biodiesel during in situ transesterification
of microalgae. This would require conducting the in situ transes- 6.3.1. Fuel properties of DME or DME-Biodiesel blends
terification at conditions favourable to methanol etherification. Apart from potential improve in the economy of in situ trans-
DME can be produced in good yield at 260 °C and 5 MPa [134]. esterification, blending DME with biodiesel would also improve
Conducting the reaction at this conditions can also serve as pre- the fuel quality. For instance, blending DME (5–15% by volume)
treatment for the residue when used in biogas production. Com- with biodiesel obtained from Jatropha oil has been shown to in-
bination of acid/alkali with thermal pre-treatment [135] as well as crease the brakes thermal efficiency (BTE), decreased the brakes
thermal pre-treatment alone has been shown to cause enhance- specific fuel consumption (BSFC) and decrease the emission levels
ment in biogas production [135,136]. This enhancement occur due of all blends as shown in Table 13 by Loganathan et al. [137].
to significant increase in carbohydrate dissolution (
5 fold) with DME is a green fuel. It is non corrosive to any metals, contains
thermal pre-treatment alone and (
7 fold) with combination of neither sulphur non nitrogen, non-harmful to human body and
thermal and acid. Pre-treatment. Although the use of thermal pre- has physical and combustion characteristics comparable to other
treatment for enhancing biogas production alone is energy in- fuels. As can be seen in Table 14, it has high cetane number
tensive and resulted in negative energy balance [135], considering (55  60) comparable to diesel cetane number (40 55) so it can be
the fact that dimethyl ether would be produced along with bio- used as perfect alternative for diesel or as blends [134].
diesel might improve the energy gain as there will not be the need Handling of DME does not pose serious environmental concern.
to recycle significant amount of unreacted methanol. Since the Studies have shown that DME has low toxicity potential, no gen-
process would operate at high temperature (
260 °C) and high otoxicity potential in vitro and sub˗chronic and no significant
toxicity effect as shown in cancerogenicity inhalation studies by
Table 12 European Food Safety Authority [138].
Major chemical composition of different algae. Fig. 10. shows a schematic diagram of the proposed new in-
tegrated process of co˗producing DME in situ along with biodiesel
Algae Protein (%) Carbohydrate (%) Lipids (%) Nucleic
acid (%) production via in situ transesterification as described in Section 6.3.
Fig. 10: shows a proposed new way of efficient utilising algae
Anabaena cylindrica 43–56 25–30 4–7 – residue for biogas generation and bio˗digestate for supporting al-
Aphanizomenon flos- 62 23 3 –
gae cultivation. As shown in the Fig. DME would be produced
aquae
Chlamydomonas 48 17 21 –
rheinhardii Table 13
Chlorella pyrenoidosa 57 26 2 – Engine performance and emission level of DME- Biodiesel blends.
Chlorella vulgaris 51–58 12–17 14–22 4–5
Dunaliella salina 57 32 6 – Blends BTE in- BSFC in- HC reduc- CO reduc- NOx reduc-
Euglena gracilis 39–61 14–18 14–20 – crease (%) crease (%) tion (%) tion (%) tion (%)
Porphyridium 28–39 40–57 9–14 –
cruentum BDE 5 6 8 42 67 17
Scenedesmus obliquus 50–56 10–17 12–14 3–6 BDE 10 10 12 36 51 28
Spirogyra sp. 6–20 33–64 11–21 – BDE 15 3 17 26 35 35
Arthrospira maxima 60–71 13–16 6–7 3–4.5
Spirulina platensis 46–63 8–14 4–9 2–5 BDE 5 (DME, 5% and biodiesel, 95%); BDE 10 (DME, 10% and biodiesel, 90%); BDE 15
Synechococcus sp. 63 15 11 5 (DME, 15% and biodiesel, 85%); BTE: Brake thermal efficiency; BSFC: Brake specific
fuel consumption.

Fig. 9. Methane and alcoholic route of conversion of algal residue. Modified from Chandra et al. [108].
 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198 1195

Table 14
Physical properties of DME and other fuels.
Source: Ogawa et al. [134].

Properties Chemical Boiling Liquid Specific Heat of vapor- Vapour Ignition Explosion Cetane nob Net calorific va- Net calorific
formula Point (K) Density Gravity ization (kJ kg  1) Pressure Temp. (K) limit lue value
(g cm  3)a (VS. air) (atm.)a 106 J (Nm)  2 106 J kg  1

DME CH3OCH3 247.9 0.67 1.59 467 6.1 623 3.4–17 55–60 599.44 28.90
Propane C3H8 231.0 0.49 1.52 426 9.3 777 2.1–9.4 (5)b 91.25 46.46
Methane CH4 111.5 – 0.55 510 – 905 5–15 0 36.0 50.23
Methanol CH3OH 337.6 0.79 – 1097 – 743 5.5–36 5 – 21.10
Diesel – 180.4 0.84 – – – – 0.6–6.5 40–55 – 41.86

a
(293 K).
b
(estimated value).

Fig. 10. Proposed in situ co-production of DME during in situ transesterification.

in situ along with biodiesel during in situ transesterification of
microalgae using conditions favourable to biodiesel and DME
synthesis. Therefore, there may be less or no methanol remain in
the product streams resulting in significant reduction in distilla-
tion heat that might be require to remove the unreacted methanol.
Apparently acid catalyst would be used as it promotes etherifica-
tion. The high temperature required would help in disrupting the
microalgal cells, and it will also shorten the reaction time. Me-
thane produced can be burnt to provide energy for algal biomass
processing such as concentration and separation from dilute algae-
water mixture [2,114]. The bio˗digestate can serve as manure to
support microalgae cultivation [113]. This approach could produce
a sustainable integrated in situ transesterification with a potential
improve in the process economy.
7. Concluding remarks
 In situ transesterification of microalgae to produce biodiesel has
been demonstrated to be technically feasible for a range of
marine and freshwater species. However, one major drawback is
the relatively high molar ratio of methanol to oil (range: 100:1
to 1000:1) required for desired yield. In addition complete
drying of microalgae to a level required by two step transes-
terification is energy intensive and significantly hinders the
commercial production of large scale algae biofuels.
 The possible cost savings due to the increased water tolerance
of reactive extraction of microalgae oil for FAME are consider-
able [8], but they must be weighed against the costs of
regenerating the alcohol (almost certainly by distillation).
 Co-solvent use can reduce the molar excess to as low as 101:1
(dimethoxymethane). However, this introduces extra costs for
the co-solvent itself and extra downstream separation duties.
Furthermore, dimethoxymethane, which has been evaluated for
this application is not a “green” solvent. It should also be borne
in mind that co-solvents can alter the range of products.
 Microalgae lipids are bound by a cell wall that inhibits FAME
extraction. Cell disruptions have been shown to be effective in
enhancing lipids extraction but are prohibitively energy in-
tensive. The resistance provided by the cell wall causes addi-
tional excess requirement of solvent which translates to extra
production cost. A cost effective pre-treatment technique will
substantially improve the process economy.
 Sulphuric acid is the most common homogeneous catalyst for
reactive extraction of microalgae. Heterogeneous catalysts have
also been shown to be effective, but require co-solvents or even
greater excesses of methanol. When H2SO4 is used in reactive
extraction, a high concentration of the catalyst is always required
to achieve high yield [6,7]. However, the need to neutralise the
unreacted acid in the product streams will increase operating
costs. Inclusion of SDS in H2SO4 catalyst for FAME enhancement
has not been explored in reactive extraction of microalgae.
Considering the fact that surfactants can disrupt cell walls, their
use could lead to enhancement of FAME yield and/or rate.
 Unlike acid catalysts, there are handful publications on use of
alkali catalyst for reactive extraction of microalgae. Salam et al.
[78] has shown that a 96% FAME yield can be obtained during
alkali-catalysed in situ transesterification in 10 min using excess
methanol and alkali concentration but short reaction time. This
significant reduction in reaction time can save operating cost.
 Overall, an integrated approach of producing dimethyl ether
(DME) in situ along with the desired biodiesel and utilisation of
algae residue after reactive extraction for methane production
and the biodigestate for supporting algal growth may further
reduce the cost of in situ transesterification. Becker (1994)
1196 K.A. Salam et al. / Renewable and Sustainable Energy Reviews 65 (2016) 1179–1198
reported that microalgae contained substantial carbohydrate
and protein besides the lipids used as the transesterification
substrate. This makes microalgae residue after reactive extrac-
tion an attractive candidate for biogas production. Reactive ex-
traction/in situ transesterification of microalgae to biodiesel
may well be a good basis for a microalgae-based bio˗refinery.
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