Applied Energy 179 (2016) 609–625

Contents lists available at ScienceDirect

Applied Energy
journal homepage: www.elsevier.com/locate/apenergy

Cultivation in wastewaters for energy: A microalgae platform

Wai Yan Cheah a, Tau Chuan Ling a, Pau Loke Show b,c, Joon Ching Juan d,e, Jo-Shu Chang f,g,⇑,
Duu-Jong Lee h,⇑⇑
a
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
b
Department of Chemical and Environmental Engineering, Faculty of Engineering, University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih,
Selangor Darul Ehsan, Malaysia
c
Manufacturing and Industrial Processes Division, Faculty of Engineering, Centre for Food and Bioproduct Processing, University of Nottingham Malaysia Campus, Jalan Broga,
43500 Semenyih, Selangor Darul Ehsan, Malaysia
d
Laboratory of Advanced Catalysis and Environmental Technology, Monash University Sunway Campus, Malaysia
e
Nanotechnology & Catalysis Research Centre (NANOCAT), University of Malaya, 50603 Kuala Lumpur, Malaysia
f
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan
g
Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan 701, Taiwan
h
Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan

h i g h l i g h t s

 Utilization of microalgae grown on wastewater for biofuels production is reviewed.

 Microalgae performed high pollutants removal efficiencies in wastewaters.
 Multiple forms of biofuels can be produced by using microalgal feedstock.
 Challenges of biofuels production with microalgae from wastewater are discussed.

a r t i c l e i n f o a b s t r a c t

Article history: The use of microalgae for wastewater treatment has recently attracted increased interest due to the
Received 24 December 2015 effective nutrient removal abilities of such microorganism, and additional benefits of biofuel production.
Received in revised form 5 July 2016 Microalgae also have the advantages of high growth rates, high cellular lipid productivities, capability to
Accepted 6 July 2016
bio-sequester carbon dioxide, capability to remove pollutants from wastewater and their abilities to
produce biofuels and bio-based chemicals. However, large scale, cost-effective and sustainable
microalgae biofuel production remains still uncertain. In this review, the pathway of integrating
Keywords:
wastewaters (including palm oil mill effluent (POME), sewage and landfill leachate) as the medium for
Biofuel
Microalgae
microalgal cultivation is comprehensively discussed. The technology/potential of using microalgae to
Pollutants removal remove pollutants from wastewaters as well as converting the resulting microalgal biomass for biofuel
Wastewater production, are critically reviewed.
Ó 2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
2. Wastewater characteristics and energy content. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
2.1. POME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
2.2. Sewage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
2.3. Landfill leachate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
3. Microalgal growth with wastewater and pollutant removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612
3.1. POME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612
3.2. Sewage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612
3.3. Landfill leachate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613

⇑ Corresponding author at: Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan.
⇑⇑ Corresponding author.
E-mail addresses: changjs@mail.ncku.edu.tw (J.-S. Chang), djlee@ntu.edu.tw (D.-J. Lee).

http://dx.doi.org/10.1016/j.apenergy.2016.07.015
0306-2619/Ó 2016 Elsevier Ltd. All rights reserved.
610 W.Y. Cheah et al. / Applied Energy 179 (2016) 609–625

4. Converting microalgal biomass to energy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615

4.1. Biodiesel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615
4.1.1. Harvesting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615
4.1.2. Lipid extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
4.1.3. Transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
4.2. Fermentative biofuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617
4.2.1. Saccharification of microalgae-based carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619
4.2.2. Fermentative production of liquid biofuels using microalgal feedstock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619
4.2.3. Gaseous biofuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620
5. Potential of using microalgae as feedstock for biofuel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621
6. Challenges and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622
7. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623

1. Introduction
The United States Energy Information Administration (EIA)
claimed that global petroleum consumption increased by 6.3%
from 2009 to 2013, rising risen more rapidly than its production.
Additionally, world-wide energy consumption is estimated to rise
by 49% from 2007 to 2035, due to the demand from an expanding
world population, economic growth and social pressures [1]. The
adverse impacts caused by intensive utilization of fossil fuels are
also increasingly alarming, as it contributes to greenhouse gas
emissions and environmental damage. Global concerns for energy
security and environmental sustainability have thus raised interest
in alternative energy resources, with the aim of substituting the
utilization of finite fossil fuels. The production of biofuels is
expected to play a key role in this process.
The main contemporary biofuels are bioethanol and biodiesel
produced from sugary, starchy and lipid-rich feedstock, respec-
tively, due to the simple and relatively easy processing steps [2].
Microalgae have been identified as a promising alternative for
biofuel production, primarily due to their high photosynthetic
efficiency with regard to bio-sequestering carbon dioxide, high
biomass productivity, high lipids/carbohydrates accumulation,
better tolerance towards diverse environments, resistance to con-
tamination and their valuable components, which show potentially
for the production of non-fuel bio-products [1,3–8]. Although large
commercial scale cultivation, harvesting, post-processing and
purification system are undeniably needed to produce microalgal-
based biofuel in the amounts needed to meet a significant portion
of global energy demand, there are many challenges that hinder
the scaling up and commercialization of microalgal biofuel manu-
facturing processes. For instance, culturing of algae on a large scale
requires a large amount of freshwater, growth nutrients, and elite
microalgae strains with the highest lipid content and biomass pro-
ductivities. Downstream processing is very costly and requires
intensive energy consumption for harvesting and oil conversion.
These challenges are mainly related to the economic viability of this
technology [6,9]. Cultivating microalgae with wastewater is
recognized as one way to substantially reduce the nutrient expenses
and water resources required for commercial scale microalgal
cultivation, and could thus contribute to successful large scale
microalgal-based biofuel production.
The annual worldwide freshwater consumption was estimated
at 3908.3 billion m3 in 2009 [10]. The large amounts of freshwater
that are consumed then becomes wastewater which requires treat-
ment. The integration of wastewater as a medium for microalgal
cultivation thus offers wastewater treatment together with a
cost-effectively form of sustainable biofuel production [3,6].
Fig. 1 shows the relationship between microalgal-based wastewa-
ter treatment and biofuel production. Wastewater generally con-
tains an abundance of organics and nutrients, and if these are
discharged directly to the rivers then the resulting organics and
nutrients loads may cause oxygen depletion and eutrophication,
thus threatening the ecosystem [3]. Microalgae are able to survive
in wastewater due to their capability of assimilating the organics
and nutrients needed for growth [11,12]. It has been observed that
microalgal-based wastewater treatment can remove pollutants
more efficiently from settled domestic sewage compared to the
conventional activated sewage process [6,13]. Additionally, the
energy required to remove nutrients in an algal pond could also
be offset when compared to using chemical fertilizers, which are
needed for the biological nutrients removal processes in typical
treatment plants [14].
Cultivating microalgae in municipal wastewaters for biofuel
production and pollutant removal has been discussed in many

Microalgal -biofuel productions

• Biodiesel
Wastewater • Fermentative liquid biofuels
• POME Microalgal • Gaseous biofuel
• Sewage Cultivation
• Landfill leachate Bioremediation
• Pollutants
removal

Fig. 1. The relationship between microalgal-based wastewater treatment and biofuel production.
 W.Y. Cheah et al. / Applied Energy 179 (2016) 609–625 611

Table 1 lowed by thermal treatment for sludge management and biogas

Wastewater characteristics of POME, sewage and landfill leachate. production. The energy content of POME is estimated to range from
Parametersa POME Sewage Landfill leachate 210 to 1400 kJ, assuming that the COD biomass is equivalent to
pH 3.4–5.2 7.11–7.59 7.86–8.31 total COD [23]. The exact energy content is depends on the sludge
Temperature (°C) 80–90 28–29 – and moisture contents, and the loads of POME. The use of microal-
BOD 5 days at 20 °C 10,250–43,750b 30–50 97–184 gae for POME treatment is an alternative wastewater treatment,
COD 15,000–100,000 95.33–126.33 680–950 although with the aim of efficient biofuel production rather than
Total solids 11,500–79,000 – –
Suspended solids 5000–54,000 80–130 151–278
digesting POME for biogas. The organics and nutrients available
Volatile solids 9000–72,000 – – in POME are readily assimilated by microalgae for their growth,
Oil and grease 130–18,000 – – thus leading to lipid and carbohydrate accumulation for biofuel
Total nitrogen 180–1400 – – production.
Ammoniacal nitrogen 4–80 8.7–40.0 410–1185
Sulphate – – 22.47–175.07
Copper – – 0.002–0.15 2.2. Sewage
Iron 170.105 – 0.96–3.65
Manganese 14.447 – 0–0.21 The sewage system is designed to carry wastewater and foul
Nickel – – 0.09–0.23 sewage for treatment prior to disposal in the receiving water bod-
Zinc 2.785 – 0.14–0.34
ies. Sewage systems range from unconnected simple toilets in rural
Energy content (kJ)c 210–1400 1.33–1.76 9.52–13.3
Reference [14,21] [24] [28] areas, to modern and connected public sewage treatment plants.
a
Table 1 illustrates the typical raw sewage characteristics for a sam-
Units in mg L 1 except for pH, temperature and energy content.
b ple collected from sewage a oxidation pond in Universiti Sains
The sample for BOD analysis is incubated at 30 °C for 3 days.
c
Unit in kJ, estimated data from Kampschreur et al. [23], energy content in Malaysia (USM) engineering campus, with a total of 4193 users
organic carbon in wastewater is 14 kJ per g COD and assuming total COD is [24]. As shown in Table 1, the sewage generated is high in organics
equivalent to biomass COD. and ammonia nitrogen, which requires further treatment. The
ammonia nitrogen, organics and suspended solids are primarily
derived from the waste, whereas 50% or more of phosphorus came
studies [6,15–17]. This review article thus attempts to summarize from synthetic detergents flushing together to the sewage treat-
and discuss the feasibility of microalgal growth in palm oil mill ment plant [25]. The energy generated from sewage sludge by solid
effluent (POME), sewage and landfill leachate that is produced in recovery fuel production had a calorific value of 14.25 MJ kg 1,
large quantities around the world. Research into the best medium with all wastewater evaporated and discharged [26]. Although
for microalgal cultivation and the subsequent downstream energy the energy content of sewage wastewater is low, its characteristics
production are also discussed. are always uniform and stable, and thus it can serve as a source of
nutrients for microalgal cultivation for biofuel productions.
2. Wastewater characteristics and energy content Microalgal-based sewage treatment has been commonly prac-
ticed due to the low maintenance cost and sufficient land availabil-
Microalgae can grow in various types of water resources, such ity. Previous research also showed that the microalgal pond
as fresh and marine waters, agricultural wastewater, industrial process could remove pollutants, especially N and P, more effi-
wastewater and municipal wastewater; and these are all vary in ciently compared to conventional activated sewage process [6].
their chemical profiles and characteristics [9,10,18]. Microalgae This encourages microalgal cultivation for biofuel production in
have high tolerance level against adverse environmental stresses, sewage treatment stabilisation ponds, due to the large open ponds
resulting their survival in wastewater. With regard to biofuel which are readily available, with suitable nutrients available in the
productions, the microalgae used for this process have to be mass wastewater. This will thus offset the operational costs of biomass
produced using wastewater. The following section provides infor- lipid or carbohydrate content and nutrients recovery, for biofuel,
mation on POME, sewage and landfill leachate characteristics for biofertilisers and biochemicals, such as proteins [27].
microalgal cultivation.
2.3. Landfill leachate
2.1. POME
Urbanisation, industrialisation, growth in the human popula-
POME is a polluting agro-industrial effluent that generates high tion and the consumption patterns of modern citizens have
pollution loads in rivers. It is mainly generated in the fresh fruit resulted in an increase rise in the generation of solid waste.
bunches sterilisation and clarification process [19]. Approximately Landfilling is the most commonly used approach for solid waste
0.9, 1.5 and 0.1 m3 of POME are generated from steriliser conden- disposal, but this results in the generation of huge amount of land-
sate, sludge separator and hydrocyclone waste for every tonne of fill leachate daily. Leachate generally contains lots of pollutants
crude palm oil processed [14]. For each tonne of crude palm oil which are highly toxic, including recalcitrant organic substances,
produced, 5–7.5 tonnes of water are required, and 50% of water ammonical nitrogen and heavy metals [28]. The composition of
ends up in POME [20]. This large proportion of water has resulted landfill leachate may vary depending on the composition of solid
in massive amounts of POME being generated per day. waste and the landfill conditions. Table 1 shows the typical charac-
The typical POME characteristics are listed in Table 1 [14,21]. As teristics of raw leachate at the Kuala Sepetang landfill site (KSLS),
shown in Table 1, POME is typically high in nitrogen, organics and Malaysia, in 2012 [28].
solids, which are contributed by fertilizer application and the fruit The concentrations of ammonia nitrogen and organics are very
bunches themselves. POME typically contains 2–4% suspended high in landfill leachate. Previous research showed that ammonia
solids, 95–96% of water and 1% of oil [21]. Additionally, it contains removal by microalgal assimilation during the growth and deposi-
carbohydrates, proteins, lipids and nitrogenous compounds that tion of organic nitrogen in a stabilisation pond was able to remove
make up the high pollutants profile of POME and the energy con- 65–85% of nitrogen from leachate [29]. A microalgae-bacteria con-
tent. POME is commonly treated in anaerobic facultative ponds sortium has removed 90% of total nitrogen in 10% landfill leachate
and digestive tank systems [19,22]. Anaerobic facultative ponds [30]. Although microalgal growth is affected by presence of toxic
use microorganisms to biodegrade the pollutants in POME, and fol- heavy metals in landfill leachate, trace metals such as Fe, Cu, Mn
612 W.Y. Cheah et al. / Applied Energy 179 (2016) 609–625
and Zn are essential elements for the photosynthesis of microalgae
[5]. Heavy metals, though present in leachate, may be variable, but
usually in fairly low concentrations [31]. The forms of energy gen-
erated in sanitary landfills are at present mainly solar and biogas.
However, the leachate could also be a medium for microalgal cul-
tivation and thus biofuel productions, provided that the heavy
metal concentrations are not in the range that would inhibit
growth.
3. Microalgal growth with wastewater and pollutant removal
Microalgae cultivation in wastewater has been widely discussed
by researchers worldwide, as this is the most cost-effective way of
biofuel production. Microalgae species are generally sensitive to
different types of wastewaters with imbalanced nutrient profiles,
the presence of inhibiting pollutants in wastewater, and deficien-
cies in certain essential trace elements. Typically, Chlorella sp. and
Scenedesmus sp. are more tolerant towards different types of
wastewaters [4]. The efficiency of microalgal growth by assimilat-
ing these nutrients is indicated by the microalgal biomass, lipid
content, carbohydrate content, dried cell weight and biomass
productivities. The performance of microalgae cells is strongly
dependent on the microalgal species itself and the growth
conditions. The following sections focus on the growth of microal-
gae cultivated in wastewaters, as well as their pollutant removal
efficiencies.
3.1. POME
Successful microalgae cultivation needs a growth medium
which provides all the necessary nutrients for growth. Numerous
researchers have reported that POME serves as the effective
wastewater source that promotes microalgae growth [32–35].
For example, Esa et al. [24] stated that the dried cell weight and
lipid content of Chlorella sorokiniana, harvested from 75% of POME
cultivation medium were the highest when compared to 50% and
25% of POME. 75% of filtered sterilised POME was able to produce
the highest microalgal biomass of 1070 ± 30 mg L 1, with a lipid
content of 156 mg per g of cell. Conversely, Nur et al. [33] evalu-
ated the variation of POME for Chlorella sp. growth and the addition
of synthetic nutrients in the medium. They found out that a high
concentration of POME took a longer time to reach stationary
phase, thus inhibiting microalgae growth. Chlorella sp. cultivated
in 20% of POME with 40% of synthetic nutrients, which are
45 mg L 1 urea and 4 mg L 1 of triple super phosphate, gave the
highest lipid productivity of 34 mg L 1 day 1. The marine
microalgae Isochrysis sp., cultivated in 5% digested POME and a
supplement of 0.075% of NPK inorganic fertilisers, yields
119.17 mg g 1 and 104.50 mg g 1 fatty acids per gram of algal cells
in photobioreactor and outdoor culture, respectively [34]. These
studies indicate that POME is effective for use in algal cultivation
and causes high lipid accumulation; however, it is the POME con-
centration and the addition of supplements that determine the
success of microalgae cultivation in this context. Moreover, the
POME and supplements needed vary among microalgae species,
due to their tolerance to surviving when assimilating these
nutrients.
Carbon, nitrogen and phosphorus are the three essential
nutrients for biomass growth, and the empirical formula for
microalgae can be expressed as C106H263O110N16P [36]. The N/P
ratio can be 250 for freshwater while 4–5 for most wastewater. A
lower nitrogen concentration may lower the microalgal growth
rate and biomass [34,35]. Under stress conditions with low
nitrogen in POME, microalgae require more energy to carry out
photosynthetic reaction, thus lowering the growth rate. However,
a lower growth rate does not reflect a low cellular lipid accumula-
tion. For instance, while heterotrophic microalgae Chlorella
protothecoides have a higher lipid content per cell, this does not
translate into high lipid productivity [37]. Selmani et al. [35] also
stated that Botryoccocus sudeticus cultivated in POME has a lower
dried biomass of 1.03 g L 1, but higher lipid content of 26.6%, as
compared to Chlorella vulgaris cultivated in POME, with 1.47 g L 1
dried cells and 15.06% lipid content. A study was carried out to
investigate effects of the growth and starvation phases of microal-
gae with regard to lipid productivity when using domestic
wastewater. It was observed that the growth phase with N and P
at their maximum concentrations produced higher biomass, while
higher lipid productivity was observed in the starvation phase with
the maximum concentration of organics [25]. This occurs when the
nitrogen in the medium is consumed and reaches the exhaustion
stage, while the carbon metabolism continues, making a diversion
of carbon to lipid as well as carbohydrate accumulation [38]. Nasci-
mento et al. [4] also reported that the production of cellular lipids
happens during the stationary phase, and this is when nitrogen
moves towards exhaustion and the microalgal cells have sufficient
biosynthetic capacity for the production of lipids. These studies
reflect that nutrient-rich POME is suitable for use as a microalgal
cultivation medium, but the cultivation strategies used have to
be suitable for the changes in C, N and P concentrations that occur
throughout the growth cycle. For example, a hybrid-system of PBR-
open pond cultivation was used whereby the cell yield was
enhanced in enclosed PBR with maximum N. This was followed
by transferring to the open pond system for second stage cultiva-
tion, to provide the stress conditions needed for maximum lipid
accumulation [5].
Moreover, the effects of various sterilisation methods for POME
have also been investigated, with the results showing that filter
sterilisation with 0.22 lm membrane filtration would be the best
option to cultivate microalgae on the lab scale, compared to auto-
claving, 50 lg mL 1 chloramphenicol addition and without any
sterilisation [32]. Sterilisation is carried out to prevent bacterial
contamination of microalgal culture on the lab scale, and this
may not be applicable for open pond systems with a large volume
of POME. Although some researchers stated that the microalgal-
bacteria symbiotic relationship is important for effective treatment
of municipal wastewater, the appropriate methods to prevent
bacterial contamination for microalgal cultivation have not yet
been determined.
POME has received considerable attention in recent, especially
from palm oil exporters, due to the fact that it creates pollution
loads in rivers. A mixed culture of macroalgae and microalgae iso-
lated from an open pond system subjected to POME treatment was
shown to be efficient in removing COD in POME. The COD removal
from digested POME reached 97.8% using algae grown in a faculta-
tive pond of an anaerobic ponding system [39]. C. sorokiniana cul-
tivated in filtered sterilised POME is capable of removing 63 ± 3% of
COD [32]. Moreover, Nur et al. [33] studied phytoremediation of
POME using aquatic plants, followed by the use of Spirulina sp.
for COD and nutrient removal. The COD, N and P removal rates
achieved with aquatic plants are 50%, 88% and 64% respectively,
while microalgae Spirulina sp. removed 50.79%, 96.5% and
85.92%. This clearly indicates that the latter is more effective in
removing organics and nutrients from POME.
3.2. Sewage
The utilization of microalgal-based wastewater treatment is
more effective in removing pollutants from domestic settled sew-
age when compared to the activated sludge process [4,6]. This
reflects the great potential of achieving both microalgae treatment
together with microalgae cultivation in sewage. Sydney et al. [40]
 W.Y. Cheah et al. / Applied Energy 179 (2016) 609–625
has screened thirteen microalgae strains which are capable of
removing pollutants while accumulating lipids from effluent
generated in treated or secondary domestic sewage. Botryococcus
braunii produced the highest lipid content, at 36.14%, with biomass
production of 1.88 g L 1. However, the lipid accumulation of B.
braunii is was only 17.85% in urban wastewater, indicating this
microalga is more suitable to be cultivated in sewage.
According to Mahapatra et al. [27], Chlorella minutissima had a
380 mg L 1 growth rate when grown in raw sewage under hetero-
trophic conditions. Heterotrophic microalgal cultivation means
dark cultivation, which requires an organic carbon source to be
added to the microalgal nutrient medium [17,41]. It was found that
microalgae cultivated under heterotrophic conditions exhibit
higher lipid content than that cultivated under autotrophic condi-
tions [42]. Moreover, mixotrophic cultivation means a combination
of heterotrophic and autotrophic conditions, whereby microalgae
grow by photosynthesis and use both inorganic and organic carbon
sources, like glucose and glycerol [16,43–45]. A mixotrophic
system was found to enhance the growth of Chlorella sp. and Botry-
ococcus sp. [16]. Euglene sp. has a high tolerance for raw sewage
and is able to grow under mixotrophic conditions, with 24.6% lipid
content and 1.24 g L 1 biomass density [27]. Ramachandra et al.
[46] also reported that Euglene sp. isolated from a sewage treat-
ment plant gave highest lipid content of 24.6% compared to Spir-
ogyra sp. and Phormidium sp. isolated from sewage fed urban
lakes at 18.4% and 8.8%, respectively. This could be due to the
higher nutrient and organic concentrations in a sewage treatment
plant when comparing to sewage fed urban lakes, which are prob-
ably polluted by other toxic substances.
In addition to raw sewage, according to Gao et al. [47] treated
sewage, which is clearer in colour, usually contains a suitable
amount of nutrients for microalgal cultivation. This research team
grew microalgae C. vulgaris in treated sewage using membrane
photobioreactor, with a productivity of 39.93 mg L 1 day 1. de
Souza Silva et al. [48] harvested mixed culture microalgae, in the
classes of Chlorophyceae (Micractinium sp., Eudorina sp., Chlorella
sp., Pandorina sp. and Carteria sp.), Cyanophyceae (Aphanocapsa
sp., Microcystis sp., Oscillatoria sp. and Planktothrix sp.), Eugleno-
phyceae (Hyalophacus sp. and Phacus sp.) and Bacillariophyceae
(Cyclotella sp.), from a sewage stabilisation pond. They reported
that the lipid content of isolates could reach up to 33.7%. These
studies obviously revealed that using raw and treated sewage for
single and mixed microalgae cultivation can produce promising
results.
Microalgae, such as Chlorella sp., Chlamydomonas sp., Spirulina
sp., Scendesmus sp., Nostoc sp. and Oscillatoria sp., have been used
for sewage treatment over the last few decades [49]. For instance,
microalgae can often be found living on the surfaces of the waste
stabilisation ponds that are commonly used for sewage treatment.
These microalgae work closely with bacteria, using a symbiotic
relationship to treat sewage [50]. The bacteria aerobically bio-
degrade organic and inorganic pollutants in the sewage, producing
carbon dioxide and simpler pollutants for microalgae to assimilate.
Microalgae grow by taking up these nutrients, using the available
sunlight and carbon dioxide for photosynthetic activity. This
ultimately provides oxygen back to the bacteria in the pond for
oxidation of organic matter [12,51]. This completes the
microalgal-bacteria cycle for stabilisation of the pond system. This
microalgal-bacteria relationship reveals the potential of substitut-
ing an activated sludge process to secondary or tertiary sewage
treatment, due to the benefits of nutrient reduction and biomass
production [13].
C. vulgaris cultivated in treated domestic sewage achieved the
maximal nitrogen and phosphorus reductions of 53.8% and 49.6%,
respectively [40]. Scenedesmus quadricauda shows excellent perfor-
mance in phosphorus removal [52]. Blue green algae, Oscillatoria
613
sp. is efficient in reducing BOD by up to 98% in 50% of sewage,
while Nostoc sp. reduces 96% of BOD in 40% of sewage. The BOD
reduction of distillery effluent can reach up to 53% with Nostoc
sp. [49]. This proves that Nostoc sp. is more capable of growing
and treating pollutants efficiently in sewage, rather than in
distillery effluent. Gao et al. [47] evaluated the nutrient removal
rate of C. vulgaris in treated sewage by using a membrane
photobioreactor and conventional photobioreactor. They found
out that the membrane photobioreactor had a removal rate of
4.13 mg N L 1 d 1 and 0.43 mg P L 1 d 1, which was higher the fig-
ures found for a conventional photobioreactor, which were
0.59 mg N L 1 d 1 and 0.08 mg P L 1 d 1. Overall, microalgae have
been shown to be competent in treating sewage, in terms of
organic and inorganic pollutant removal. However, the selection
of microalgae species, sewage concentration and culturing meth-
ods have to be considered if successful microalgal-based sewage
treatment is to be achieved.
3.3. Landfill leachate
Landfills are natural bioreactors that are used to treat municipal
solid wastes. Landfill leachate is produced in huge amounts, espe-
cially in urban areas, and thus there is interest in using it for
microalgae cultivation. High rate algal ponds are commonly
applied for secondary treatment of the leachate generated in land-
fills. A consortium of mixed algae species, Ankistrodesmus convolu-
tus, Euglene gracilis, C. vulgaris, S. quadricauda and Chlorococcum
oviforme were used in a high rate algal pond, and it was reported
that this produced 2.00–5.54 g dried weight L 1 [53]. Edmundson
et al. [54] reported that the pH of the raw leachate has to be
adjusted with hydrochloric acid, to pH 7.0 ± 0.1, before microalgal
cultivation begins. This aims to reduce the toxicity of the leachate,
thus preventing growth inhibition. A maximum productivity of
0.55 g L 1 day 1 and cell yield of 91% can be achieved by Scenedes-
mus sp. grown in HCl adjusted leachate. Conversely, leachate which
is in extreme alkaline conditions if no HCl adjustment is used
might only be suitable for culturing specific microalgae, such as
the alkaline Spirulina sp., due to the advantage of reducing contam-
ination risk for outdoor cultivation. Moreover, microalgae have tol-
erance against adverse environment stresses, for example Chlorella
sp. and Scenedesmus sp. are known as acid-tolerant and heavy
metal tolerant species, respectively [55]. Richards and Mullins
[56] stated that leachate inhibited the growth of Scenedesmus sp.
and Dunaliella sp., but noted that a metals bioaccumulator, Chlor-
ella sp., can grow and produce lipid yields in toxic leachate. Heavy
metals, though present in leachate, may be variable, although usu-
ally in low concentrations [31]. These studies concluded that using
leachate as a nutrient source and medium for microalgal cultiva-
tion is a workable approach.
Conversely, Lin et al. [55] examined the idea of using leachate to
cultivate microalgae. The growth of Chlorella pyrenoidosa and
Chlamydomonas snowiae were found to be inhibited by a high
ammonical nitrogen concentration of above 670 mg L 1. C. pyrenoi-
dosa can only survive in 10% of leachate with ammonical nitrogen
of 134 ppm. Additionally, wastewater containing 39–143 ppm of
ammonical nitrogen showed a decline in the specific growth rate
of C. vulgaris [57]. Leachate was found to inhibit growth of Chlorella
sp., Scenedesmus sp. and Dunaliella sp. [56]. A leachate spike ratio of
10% (v/v) has produced a microalgae-bacteria consortium with a
maximum biomass of 1.58 g L 1 and 24.1 mg L 1 day 1 lipid pro-
ductivity [30]. These studies suggest that leachate dilution and
extensive treatment are needed for microalgae cultivation. Landfill
leachate can be purified to reduce turbidity, so as to enhance light
penetration, for autotrophic microalgae cultivation [55]. This
causes the cultivation in pre-treated leachate to be more tedious
614 W.Y. Cheah et al. / Applied Energy 179 (2016) 609–625

Table 2
Microalgal biomass, lipid content, and calorific value of microalgae species grown on wastewater resources.

Wastewater Species Dried cell weight (g L 1)a Biomass Lipid content (%) Gross calorific Reference
productivity (mg L 1 day 1)b Growth rate g L 1c
valued (kJ g 1)
POME Botryoccocus sudeticus 1.03a 26.6% 12.24 [35]
Chlorella sorokiana 1.07a 15.6% 7.18 [32]
Chlorella sp. 0.034b – – [33]
Chlorella vulgaris 1.47a 15.1% 6.95 [35]
Isochrysis sp. – 10.5–11.9% 4.83–5.74 [34]
Sewage Botryococcus Braunii 1.88a 36.14% 16.62 [40]
Chlorella minutissima 0.38c – – [27]
Chlorella vulgaris 0.04a – – [47]
Euglene sp. 1.24a 24.6% 11.32 [27]
Euglene sp. – 24.6% 11.32 [46]
Spirogyra sp. – 18.4% 8.46 [46]
Phormidium sp. – 8.8% 4.05 [46]
Mixed culture (Refer 3.2) – 33.7% 15.5 [48]
Leachate Mixed culture (Refer 3.3) 2.00–5.54a – – [53]
Scenedesmus sp. 0.55 b – – [54]
a
Data in dried cell weight.
b
Data in biomass productivity.
c
Data in growth rate.
d 1
Estimated from Illman et al. [59], 1% of lipid producing 0.46 kJ g .

than that in sewage and POME, due to the complex constituents
present in leachate.
Landfill leachate is a complex mixture of organic and inorganic
pollutants. It is more toxic and variable in pollutant profiles when
compared to POME and sewage. Waste stabilisation ponds which
are applied for leachate treatment, consist of conventional,
aeration and recirculation stages. Claudia et al. [29] reported that
the ammonia removal rate achieved with microalgae can reach
75–99% with 64–79% of the nitrogen was removed by dead and
inert algae settling, 1–6% by assimilation of microalgae biomass
and 10% by vaporisation of ammonia due to the aeration process.
The isolated algae were dominated by the Chlamydonomas genera.
Moreover, Mustafa et al. [53] studied the efficiency of using a high-
rate algal pond to treat leachate. One high-rate algal pond was
grown with mixed microalgae species of S. quadricauda, E. gracilis,
C. vulgaris, Ankistrodesmus convolutus and Chlorococcum oviforme.
Another pond was filled with natural lake water which contained
mixed indigenous algae. Their results showed that while the first
pond gave higher biomass the pollutant removal rates of the two
ponds were similar, with COD and ammonical nitrogen reductions
of 91% and 99% respectively. Although both ponds performed pol-
lutant removal, only the microalgae cultured in the first pond are
considered for biofuel production, unless the indigenous microal-
gae in the second pond are screened for oleaginous microalgae
strains.
Table 2 summarises the microalgal biomass and lipid content of
microalgae species grown in wastewater resources. As shown in
Table 2, Chlorella sp. and Botryococcus sp. were grown in POME
and sewage, producing high biomass production and lipid content.
Chlorella sp. achieved a higher dried cell weight when grown in
POME rather than sewage. This indicates that the nutrients in
POME are more favourable to be assimilated than those in sewage.
In contrast, Botryococcus sp. has shown higher biomass production
and lipid content in sewage compared to POME. This indicates that
biomass production and lipid accumulation vary significantly,
depending on the microalgae species. Moreover, the mixed species
showed a higher biomass production than individual strains when
grown in leachate medium. This could be due to the increase in the
heavy metal resistance of mixed species in the leachate medium.
Nevertheless, Chen et al. [58] reported that a high diversity of
microalgae may not be translated into high cellular lipid or
carbohydrate contents, when compared to the those seen with a
monoculture.
The calorific values of lipid-containing microalgae were deter-
mined by combusting microalgae in a bomb calorimeter. Illman
et al. [59] claimed that there is strong correlation between calorific
value with lipid content, with Chlorella emersonii cultivated in a
low nitrogen medium having a maximum calorific value of
29 kJ g 1 with a lipid content of 63%. The gross calorific values
are estimated in Table 2. As shown in Table 2, B. Braunii, which
achieved the highest lipid content in sewage, was estimated to
have a gross calorific value of 16.62 kJ per g of microalgae. This
calorific value is somewhat lower than that seen with biodiesel
and rapeseed oil, as 43 kJ g 1 and 39.5 kJ g 1, respectively, making
biofuel production impractical [59]. However, a recent study
claimed that Chlorella sp. culturing under axenic conditions (with
the presence of bacteria) in aquaculture wastewater achieved
50.4% lipid content with 23.6 MJ g 1 calorific content [60]. This
indicates that the cultivation medium and growth conditions sig-
nificantly affect the microalgal biomass production, cellular lipid
content and the subsequent energy productions. Overall, the sum-
mary of these results presented here serves as a survey of the liter-
ature, rather than a strict comparison pf the performances of
microalgae species grown in these media. Factors such as nutrient
profile, supplement addition, light supply, CO2 supply and cultiva-
tion system still remain to be investigated.
The pollutant removal efficiencies in POME, sewage and lea-
chate by microalgae are summarised in Table 3. Overall, the pollu-
tant removal efficiencies were higher in POME and sewage. This
indicates that nutrients in POME and sewage are favourable for
microalgae assimilation. Microalgae grown in leachate can obtain
high pollutant removal provided that the microalgae are mixed
with an indigenous culture. Additionally, the pollutant removal
with a mixed culture also achieved a higher pollutant removal rate
compared to that seen with a single species in POME. Mixed cul-
tures can remove organics in POME and leachate, achieving COD
removal of up to 97.8% and 82%, respectively. The percentage of
BOD removal reached more than 95% when using microalgae in
sewage, indicating the efficiency of Oscillatoria sp. and Nostoc sp.
to assimilate organics. Spirulina sp. has also shown to have a high
capability of removing pollutants in POME. However, further
research is needed to identify the potential indigenous microalgae
 W.Y. Cheah et al. / Applied Energy 179 (2016) 609–625 615

Table 3
Pollutant removal efficiencies of microalgae species.

Wastewater Microalgae species Nitrogen or ammonia-nitrogen (%) Phosphate (%) COD (%) BOD (%) Reference
POME Mixed species – – 97.8 – [39]
Chlorella sorokiniana – – 60–66 – [32]
Spirulina sp. 96.5 85.92 50.79 – [33]
Sewage Chlorella vulgaris 53.8 49.6 – – [40]
Oscillatoria sp. – – – 98 [49]
Nostoc sp. – – – 96 [49]
Chlorella vulgaris 4.13a 0.43a – – [47]
Scenedesmus quadricauda SDEC-13 70 100 – – [13]
Leachate Mixed isolates (Chlamydonomas as dominant) 75–99 – 35–82 – [29]
Mixed culture (Refer 3.3) & Mixed isolates 99 – 91 – [53]
a 1 1
Data in removal rate, mg L d .

Lipid extraction Transesterification Biodiesel

Residual biomass Bioethanol &

Biobutanol
Harvested Algae Biochemical
Biomass conversion Biohydrogen

Residual biomass Residual biomass

Biochemical Biomethane
conversion

Fig. 2. The microalgal biomass processing systems for bioenergy production.

isolates with appropriate nutrient profiles, so as to boost lipid pro-
duction and pollutant removal simultaneously.
4. Converting microalgal biomass to energy
Microalgal biomass with high lipid and carbohydrate contents
can be used for biofuel feedstock. Multiple biofuels are produced
from one biomass source. The various microalgal biomass process-
ing systems for energy production are outlined in Fig. 2. Mass cul-
tivation of microalgal cells has to be supported with efficient
microalgal cell harvesting and energy conversion processes, to
ensure successful biofuel production. In particular, to cultivate
microalgal biomass from wastewater is regarded an emergent plat-
form for converting energy in wastewaters to biomass energy. The
following sections summarise the available and latest techniques
for biofuel downstream processes for the production of biodiesel,
fermentative liquid biofuels and gaseous biofuels.
4.1. Biodiesel
The lipid content in microalgal biomass can be processed to
become biodiesel. The following subsections describe the conver-
sion of microalgal biomass for biodiesel production.
4.1.1. Harvesting
Microalgal harvesting is one of the main challenges towards
biofuel production, due to the high energy input and recovery cost
for microscopic microalgae and from diluted microalgal suspen-
sion [9]. Biomass bulk harvesting and thickening processes are
used to separate biomass cells from the suspension and concen-
trate them by centrifugation, filtration and flocculation, and so
on prior to biofuel conversion [17]. The choice of harvesting
technique is greatly dependent on the microalgae species, desired
quality of the products, harvesting efficiency and economic factors
[17]. If the harvested biomass cells have a high moisture content
then this may cause spoilage within hours, and add extra costs
and energy requirements to the subsequent lipid extraction pro-
cess [61]. Indeed, the harvesting cost can reach up to 20–30% of
total production cost [62,63]. The optimal harvesting techniques
used should thus be: (i) species-independent; (ii) require low con-
sumption of energy and chemicals; (iii) rapid; (iv) cost-effective;
(v) maximize yields; and (vi) minimize lipid loss.
Sedimentation is one of the simplest biomass harvesting tech-
niques used, as it is based on gravitational force, with biomass cells
left to settle out of the cultivation medium. This is the most com-
monly used biomass separation method with large volumes of
wastewater or mass cultivation. Sedimentation is a low cost and
low energy method, and has been proven effective for harvesting
large microalgae cells like Spirulina sp. [9]. However, the settling
rate varies depending on species, cell size, density, light and nutri-
ent availability. The average settling velocity for green microalgae
and diatoms are 0.1 m day 1 and 0.2 m day 1, respectively. How-
ever, poor light availability and nutrient deficiencies in the med-
ium result in low settling rates [61].
Flocculation is an enhanced process of sedimentation, with the
addition of flocculants to improve the biomass settling rate.
Microalgal cells are originally negatively charged on their surfaces
to prevent self-aggregation [63]. The positive-charged flocculants
can disrupt these negative charges, flocculating these cells and
then enhancing sedimentation due to higher floc density. The ideal
flocculants should be non-toxic, low cost, effective in low concen-
trations and have no effect on medium recycling, like, for example,
aluminium sulphate, ferric chloride and polyelectrolytes [9,63]. In
addition to chemical flocculation, microalgal harvesting involves
bio-flocculation, whereby microalgae spontaneously flocculate
with bacteria and settle in the ponds. Microalgal bacterial floc from
sewage secondary treatment can remove 97.5% of biomass from
616 W.Y. Cheah et al. / Applied Energy 179 (2016) 609–625
the culture medium in 30 min [61]. In contrast, Ho et al. [64] stated
that flocculation is ineffective for harvesting in large scale pond
systems and oceanic cultivation.
Flotation is another method of harvesting biomass cells. While
sedimentation is effective for harvesting large-sized and heavier
cells, dissolved air flotation (DAF) can be applied for smaller-
sized and lighter cells [17]. This is also a simple and cost-
effective method, whereby air in small bubbles is injected into
the culture medium, enabling cells to float due to the small bubbles
rising to the surface of the culture medium. Biomass cells are then
be removed, and thus harvested. Wastewater treatment plants in
the USA usually apply coagulation followed by DAF, with, for
example, DAF found to be effective in harvesting microalgae grown
in pig slurry with alum [61]. Additionally, DAF is usually applied in
treating wastewater with high concentrations of oil and grease, as
these can easily float on the surface of wastewater. The drawbacks
of this approach include the high cost and energy intensity, due to
the high pressure required to generate small air bubbles. Neverthe-
less, fluidic oscillation can be used to generate small bubbles using
less energy.
Filtration has been shown to be effective in recovering biomass
cells, by separating these from the suspension through retention
on filters. Approaches used included those applying macrofiltra-
tion, microfiltration, ultra filtration and reverse osmosis, with pore
sizes >10 lm, 0.1–10 lm, 0.02–0.2 lm and <0.001 lm, respec-
tively. Microalgal cells are thus easily harvested via microfiltration
and macrofiltration, in the same way as harvesting flocculated
cells. For example, Chlorella sp. with the size of 5–6 lm can be har-
vested using microfiltration [61]. The drawbacks of filtration
include membrane fouling or clogging, energy consumption with
pump operation, limited to a relatively small volume of culture
medium, and the shear stress imposed on the biomass cells. The
solutions to these drawbacks are fouling removal using NaClO
and suitable flowing velocities [65].
Another alternative for microalgal harvesting is the use of
microalgal cells immobilisation in suspended media. According to
Zhou et al. [6], pellet-forming filamentous fungi can be cultured
together with microalgae, forming larger microalgae-fungi pellets.
These pellets are subsequently removed by filtration, as these pel-
lets are now larger in size for retainability. Additionally, microalgal
cells can also be immobilised in natural or synthetic polymers
embedded and continuously grow within the matrix. After
reaching stationary phase, immobilised microalgal cell beads are
separated out through filtration [66]. Moreover, microalgae
immobilisation can also be carried out by attaching the cells to a
support and then scraping them off for harvesting [67]. This can
prevent microalgal cells from moving freely within the suspension,
thus contributing to a high recovery rate, preventing microalgal
cell washout, and making recycling of the cultivation medium
easier due to cell-free effluent production [36]. However, this
immobilisation system requires special technology to be
incorporated in the design of cultivation system or bioreactor, to
allow cell adsorption, confinement in liquid suspension, entrap-
ment and so on.
4.1.2. Lipid extraction
As discussed in Section 3, microalgae are capable of accumulat-
ing a maximum of more than 50% of lipid per dried cell weight. The
release of these cellular lipids from microalgal cells after cultiva-
tion and harvesting is required for biofuel production. The com-
monly used methods for lipid extraction include physical
extraction, chemical (solvent) extraction and supercritical fluid
extraction. Effective lipid extraction should be fast, lipid-specific,
and prevent lipid loss and cellular material denaturation, which
would affect the subsequent lipid conversion process, as well as
the quantity and quality of biofuel.
The simplest method used for lipid extraction is mechanical
crushing of microalgal cells by using glass beads, a screw press,
extruder and pulverisation. Additional machinery is thus used in
lipid extraction, allowing both small scale and large scale lipid
extraction for biofuel production. The extracted oil can reach up
to 70–75% of dried biomass cells using mechanical pressing [9].
Effective cell disruption may offset the subsequent high tempera-
ture and pressure required for solvent and biomolecular contact
[68]. Though this physical extraction is a simple, cheap and
chemical-free method, it may not be suitable for microalgal cells
with rigid cell walls. Moreover, some cells easily wash off with
moisture, and thus the mechanical rupturing step can be skipped
[9]. Low heat pretreatment might be necessary for drying microal-
gal cells, as extraction from dried biomass is more efficient than
wet biomass, although this imposes higher cost and energy
consumption [50,69]. Apart from mechanical extraction, non-
mechanical approaches, like autoclaving, sonication, freezing,
microwaving and osmotic shock, can also be employed, but these
are less practical for commercial scale biofuel production.
The Bligh and Dryer method is the most commonly-used lipid
extraction approach, using solvents like n-hexane, chloroform,
methanol, acetone and benzene. The solvents used should be
cost-effective, have high selectivity to penetrate lipid-containing
cells, solvate lipids effectively, and be less toxic. A combination
of non-polar (chloroform) and polar (methanol) solvents is usually
applied. The hydrophobic interactions between non-polar and neu-
tral lipids are disrupted by the use of a non-polar solvent; and the
hydrogen bonds in polar lipids are disrupted by a polar solvent
[62]. Makareviciene et al. [50] reported that using a chloroform
methanol mixture with the Bligh and Dryer method could extract
lipid two times more efficiently than when using n-hexane with
the Soxhlet extraction method. Large scale microalgal lipid extrac-
tion is usually accomplished by mechanical cell disruption, fol-
lowed by solvent extraction. Mechanical cell disruption is carried
out to ensure the solvent diffuses into the inner cell for lipid
extraction.
Lipid extraction using supercritical CO2 is known to be a greener
approach due to the elimination of a large amount of toxic sol-
vents. The inert and low cost CO2 is heated and compressed beyond
the critical temperature and pressure, until it reaches a liquid-gas
state, and then it is added to the biomass cells [66]. This method
provides favourable mass transfer rates due to better diffusion in
liquid-gas states and low toxicity compared to organic solvents.
Additionally, it works better in wet biomass than in dry biomass,
thus eliminating the drying or dewatering step of harvesting bio-
mass cells. A period of 80 min of supercritical CO2 extraction is
1.8 times more efficient than 330 min Soxhlet extraction using
hexane [17,50]. However, the drawbacks of this approach include
the high cost due to separating and re-compressing CO2 after each
time of lipid extraction. Therefore, future studies should carry out
an energy and cost analysis of large scale biofuel production using
this method.
4.1.3. Transesterification
Fatty acid methyl esters (FAME) are obtained via a simple and
commonly used chemical transesterification process. High viscos-
ity crude microalgal oil (triglycerides) requires chemical conver-
sion into low molecular weight, non-toxic, biodegradable FAME
(biofuel), for direct use in engines. This production process uses
excess methanol or ethanol in the presence of a catalyst, to main-
tain the equilibrium shift towards fatty acid esters production and
accelerate the reaction rate [9]. Transesterification can also be
known as alcoholysis, and this is when triglycerides with short
chain alcohol are displaced by alcohol for fatty acid esters and glyc-
erol formation. Fig. 3 shows the triglycerides transesterification
reaction producing fatty acid esters. In general, transesterification
 W.Y. Cheah et al. / Applied Energy 179 (2016) 609–625
O O
CH 2 O C R1
O O
CH O C R2 + 3CH 3OH
O Catalyst
CH 2 O C R 3
Triglyceride Methanol
Fig. 3. The triglycerides transesterification reaction producing fatty acid esters.
can be divided into stages, whereby triglycerides are converted
into diglycerides, followed by diglycerides converting into
monoglycerides, and finally into FAME and glycerol [17,50]. The
efficiency of biofuel production is determined by the reaction
temperature, reaction duration, alcohol quantity and catalyst
quantity.
There are alkaline and acid catalysts, such as sodium hydroxide
in methanol and sodium methoxide for the former, and
hydrochloric and sulphuric acid in methanol for the latter
[69,70]. Acid catalytic activity has a low reaction rate and needs
to operate at a high temperature, and it serves as the first step prior
to transesterification. However, alkaline catalysis is widely used in
larger and commercial scale operations, as it enhances the reaction
rate. While alkaline catalysis is a faster reaction, it is limited by the
free fatty acids content. The presence of water can hydrolyse
triglycerides into diglycerides, forming free fatty acids, which will
then react with the alkaline catalyst allowing saponification.
Saponification is undesirable, as it reduces the biofuel yield and
quality, as well as causing catalyst loss. Acid pre-treatment is
essential to convert free fatty acids, and then water removal is
followed by converting triglycerides to diglycerides to produce bio-
fuel using an alkaline catalyst, so as to inhibit the saponification
reaction [63,71]. However, enzymatic (lipase) catalysis is feasible
with transesterification carried out at 35–45 °C, and this enables
catalytic activities without the formation of any foams. The
enzyme used has to be tolerant to methanol inactivation, but the
key factor here is the high enzyme production cost, which makes
it economically impractical for industrial scale operations [62].
Alternatively, industrial scale transesterification reaction can be
enhanced by the use of solid catalysts, such as zeolites, metal oxi-
des and ion-exchange resins, as these solid catalysts can prevent
water formation and saponification, and are active, selective and
stable at high temperatures. Additionally, the of ultrasonic mixing
along with transesterification has been shown to significantly
reduce the processing time from hours to minutes, thus reducing
operating costs and energy consumption [72].
In-situ transesterification or direct transesterification is a rapid
method combining extraction and transesterification into a
single-step process. It facilitates direct conversion of fatty acid
esters from biomass cells, eliminating the conventional lipid
extraction step. It involves the addition of organic solvent,
methanol and catalyst to dried microalgal biomass for direct trans-
esterification. The catalytic activities are similar to those seen in
the typical transesterification process, whereby it can be acidic,
alkaline or enzymatic catalysis. Enzyme lipase, as mentioned above
or in the immobilised form with lipase in cells, can also be
employed as a biocatalyst [69]. This single-step process can signif-
icantly reduce the production cost and time, simplifying biofuel
production while improving FAME production. A yield of more
than 98% FAME is obtained through in-situ transesterification, with
n-hexane and chloroform as solvents, and methanol and sulphuric
617
CH 3 O C R1 CH 2 OH
CH 3 O C R 2 + CH OH
O
CH 3 O CH 2 OH
C R3
Methyl Ester Glycerol
acid as reactant and acidic catalyst, respectively [50]. Saharan et al.
reported that when using acid as a catalyst a second less polar,
co-solvent, like toluene, can be mixed with methanol to modify
the polarity of the reaction medium, to enhance the reaction rate
[68]. Despite the advantages of this approaches, challenges for
large scale in-situ transesterification include the high chemical
consumption rate, high moisture level of biomass cells, the need
for an appropriate temperature and control of the stirring speed
[9].
The most commonly adopted fuel conversion process is chemi-
cal transesterification. Though it is an effective process it is also
energy intensive, while glycerol is difficult to recover, and if
wastewaters or moisture are present with the biomass cells then
this may result in variations in pH, thus affecting catalytic effi-
ciency [71]. Alternative thermochemical conversion methods,
including hydrothermal liquefaction and pyrolysis, might also pro-
duce a high biofuel yield, but they are not currently being
employed in commercial scale production [1].
4.2. Fermentative biofuels
In addition to microalgal cellular lipids, another exploitable
energy resource is fermentative biofuels, such as bioethanol and
biobutanol, which are derived from microalgal carbohydrate con-
tent. Bioethanol is commonly applied as a substitute for gasoline
in combustion engines, as it is cheaper, and its production is
expected to reach 100 billion liters in 2015 [73,74]. Biobutanol
derived from biomass feedstock has several advantages over
bioethanol, due to its higher energy content, higher blending per-
centage with gasoline, lower vapour pressure, and low volatility
[38,75]. Microalgae are rich in carbohydrates, which are the major
products formed by photosynthetic activity and CO2 fixation. Car-
bohydrates are found mainly in the cellulose-based cell wall and
starch reserves stored in plastids, allowing them to be readily used
as carbon source or substrate for the fermentation process in
bioethanol and biobutanol productions [2,38]. Microalgae have a
low percentage of lignin cross-linking molecules in the cellulose
structures, which enables them to float and grow in aquatic
environments, and they also contain a great amount of sugars for
fermentation [76]. These characteristics mean that only a simple
pre-treatment step which is needed to break down lignin if ligno-
cellulosic biomass is used as substrates.
The carbohydrate contents and compositions differ significantly
depending on the species. Chorella sp., Scenedesmus sp.,
Chlamydomonas sp., Dunaliella sp., Anabaena sp., Porphyrydium
sp., Spirogyra sp. and Spirulina sp. have been used as the microalgal
carbohydrate feedstocks for fermentative biofuel production, with
8–64% of starch per dry cell weight [38,73,77–79]. Green algae,
including Chlorococum sp. and Spirogyra sp., have shown high
polysaccharide accumulation in cell walls. Chlorococum sp. gener-
ates 60% more bioethanol from samples that are pre-extracted
618 W.Y. Cheah et al. / Applied Energy 179 (2016) 609–625
Table 4
Carbohydrate or starch content of microalgal species.
Growth medium Microalgae species
Freshwater Chlorella vulgaris
Chlorella vulgaris
Seawater Chlorella vulgaris UTEX 2247
Dunaliella tertiolecta
Tetraselmis sp. CS-362
Tetraselmis subcordiformis
Lab scale medium Botryococcus Braunii
Chlorococcum sp.
Chlorella vulgaris FSP-E
Chlorella vulgaris LEB-104
Chlamydomonas reinhardtii UTEX 90
Scenedesmus obliquus CNW-N
Scenedesmus obliquus CNW-N
Scenedesmus obliquus CNW-N
Spirulina platensis LEB-52
a
Starch content.
for lipids compared to dried unextracted cells [76]. Lipid extraction
for microalgal biomass can serve as the pretreatment step to
release carbohydrates for bioethanol or biobutanol fermentation,
and this encourages biodiesel and fermentative biofuel production
consecutively. Table 4 summarises the carbohydrate or starch con-
tent of microalgae species. This data focuses on the carbohydrate
content in some microalgae species rather than the microalgal
growth in specific wastewaters, due to the lack of details on this
in the literature.
As shown in Table 4, the overall carbohydrate content of
microalgae species is up to 60% for C. vulgaris and Tetraselmis
subcordiformis grown in freshwater and seawater, respectively.
This shows that these wastewaters, as well as cultivation condi-
tions, are favourable for the carbohydrate accumulation of these
species, rather than the use of controlled nutrients in a lab scale
medium. Ho et al. [80] reported that Scenedesmus obliquus CNW-
N could accumulate up to 51.8% of carbohydrates under 1–3 day
nitrogen starvation, and that the glucose content accounted for
73–80% out of total carbohydrates. This revealed that S obliquus
is a potential bioethanol-producing feedstock, with high glucose
content recovery [81]. C. vulgaris FSP-E has also been shown to
accumulate 51.3% of carbohydrates after 4 days of nitrogen
starvation [82]. These examples show the high correlation between
accumulated products and the starvation period. Carbohydrate
accumulation reached a maximal level under an appropriate nitro-
gen starvation period, and the optimal nitrogen starvation duration
seems to be microalgal strain dependent. It has been widely
reported that under stress conditions microalgal transformation
of cellular proteins and peptides to carbohydrates and lipids as
energy storage may also occur [38,81]. Dragone et al. [83] noted
that high starch accumulation was observed in two-stage cultiva-
tion, the first stage with nitrogen and iron supplementation, and
the second with a nitrogen- and iron-free medium. The first and
second stages aim to improve biomass cell growth and enhance
carbohydrate accumulation, respectively. After carbohydrate accu-
mulation reaches a maximum with the rising nitrogen starvation
period, extension of this may result in a slight reduction of carbo-
hydrate content with an increase in lipid content [81]. There is a
carbon flux shift from starch to lipid biosynthesis in microalgae
species, and the starchless mutant Chlamydomonas reinhardtii
was found to have enhanced lipid accumulation in a stress envi-
ronment [81]. There is indeed competition that occurs between
carbohydrate and lipids biosynthesis, due to their closely related
metabolic pathways, although this competition differs among
strains [38,77]. The duration of nitrogen depletion in relation to
Carbohydrate content (%) Reference
60 [125]
41 [83]
45 [126]
14.0 [127]
26 [128]
62.1 [84]
2.4 [127]
32.5 [38]
51.3 [82]
16.7 [127]
52a [129]
51.8 [80]
46.7 [80]
49.4 [81]
11.0 [127]
carbohydrate accumulation in microalgae species has to be further
investigated, so as to ensure maximal carbohydrate content, and
thus the highest fermentative biofuel yield.
Carbohydrate is the major microalgal component for biofuel
production via fermentation. Carbohydrate accumulation can also
be improved by various cultivation strategies, such as by altering
the pH, CO2 supply, nutrient profile, irradiance and temperature.
As shown in Table 4, T. subcordiformis grown in seawater achieved
up to 62.1% and 49.3% starch content under irradiance of 200 and
50 lmol m 2 s 1, respectively [84]. Chen et al. also claimed that
increasing the light intensity from 215 to 330 lmol m 2 s 1 caused
a 31.5% increase in starch content [38]. The carbohydrate content
was 4% higher for Chlorella sp. cultivated in a low nitrogen medium
compared to a control batch [59]. Additionally, biomass production
together with carbohydrate accumulation can be enhanced by a
greater CO2 supply. For instance, the carbohydrate contents
increased by 11.7% and 4.21% for C. pyrenoidosa and Chorella
reinhardtii with the addition of 183 lmol/L of CO2 supply [38].
Moreover, Choix et al. [85] claimed that Azospirillum basilense
bacteria co-cultured with Chlorella sp. promoted both microalgal
growth and the accumulation pf starch and carbohydrates.
S. obliquus CNW-N cultivated under semi-batch conditions with
50% medium replacement achieved the highest carbohydrate pro-
ductivity, and thus bioethanol yield [81]. While these cultivation
parameters undeniably enhanced the carbohydrate accumulation
in microalgal cells, the fermentative biofuel yield is also greatly
affected by the conversion methods used.
The conversion of microalgal carbohydrates into bioethanol
begins with biomass pretreatment. This allows cellulose to be
extracted from the microalgal biomass structure, so as to ease
the breakdown of cellulose and release fermentable sugars in the
subsequent saccharification process. Pretreatments include
mechanical grinding, alkali or acid pre-treatment, microwave
pre-treatment, ultrasound pre-treatment and steam explosion.
However, the pretreatment step has to be cost-effective, with high
efficiency requiring a high biomass surface area to allow chemicals
and enzymes access during the hydrolysis reaction. The hydrolysis
methods used with microalgae are classified under two categories,
namely enzymatic saccharification and chemical saccharification,
followed by alcoholic fermentation, which are similar to the pro-
cesses used with any other starch-based feedstock. This can be car-
ried out by sequential steps (known as separate hydrolysis and
fermentation; SHF) or a single step (known as simultaneous sac-
charification and fermentation; SFF). These processes are described
in detail in the following subsections.
 W.Y. Cheah et al. / Applied Energy 179 (2016) 609–625
4.2.1. Saccharification of microalgae-based carbohydrates
The advantage of using microalgae as the source of carbohy-
drates for fermentation is mainly the absence of lignin in
microalgae-based carbohydrates. The carbohydrates produced in
microalgae are primarily in the form of starch or glycogen stored
in the chloroplast and the cellulose located in the inner cell walls
[80,86]. Therefore, when compared with lignocellulosic feedstock,
it is much easier to convert microalgal carbohydrates into mono-
sugars, as no intensive pretreatment procedures (such as steam
explosion, acid/alkali pretreatment, and so on) are required for
the saccharification of microalgal carbohydrates. In addition, only
mild conditions are required (in contrast to those needed with lig-
nocellulose hydrolysis) for the hydrolysis of microalgae-based car-
bohydrates to yield fermentable reducing sugars using enzymatic
and chemical saccharification methods. This form of hydrolysis is
vital, as it could enhance fermentation efficiency and ethanol pro-
duction by more than 33% and 60%, respectively [74].
Enzymatic saccharification of microalgal-based carbohydrates
involves the application of enzyme amylases, celluloses and
glucoamylases to hydrolyse microalgal-based carbohydrates for
glucose units. These enzymes hydrolyse carbohydrates and as well
cleaving the glycosidic linkage between glucose subunits in
cellulose and starch molecules. Cellulase, and particularly
Endo-b-1,4-D-glucanase, hydrolyses cellulose, breaking the non-
covalent interactions within the cellulose crystalline structure
and producing smaller fragments. Exo-b-1,4-D-glucanase will fur-
ther hydrolyse these fragments into soluble cellodextrin, and this
is subsequently further degraded by b-glucosidase releasing
glucose monomers as end products [87]. These processes are also
be known as cellulolysis. Cellulase can be easily obtained from
bacteria and fungi due to their high growth rates in less severe
environments. The starch reserves which are stored in plastids
inside the microalgal cells can be hydrolysed by amylase followed
by glucoamylase, producing glucose and other monosaccharides.
The glucose units produced after enzymatic saccharification are
then used as the substrates for subsequent fermentation. Enzy-
matic saccharification of C. vulgaris FSP-E biomass, with enzyme
mixture derived from Pseudomonas sp., has been shown to produce
51% carbohydrate per dry weight and a 90.4% glucose yield [77].
Additionally, while chemical saccharification is a fast reaction it
involves the application of pressure and a high temperature, as
well as the addition of an acid and base. The generation of inhibi-
tors during the chemical hydrolysis process, like furfural, can
potentially suppress fermentation, thus making downstream waste
treatment more costly. Therefore, parameters such as moisture
content, temperature, reaction duration, reactant concentrations
and pressure have to be properly controlled, in order to improve
saccharification efficiency and achieve high glucose production
[38]. Sulphuric acid and acetic acid are most commonly used for
chemical saccharification [74]. Diluted acid hydrolysis pre-
treatment was carried out in an earlier work using 1.0 M sulphuric
acid, and 120 min at 80–90 °C as the optimal conditions, and a sig-
nificant amount of 166.1 g sugar content per kg dried microalgae
was thus obtained [88]. Alkaline treatment with 0.75% sodium
hydroxide at 120 °C and for 30 min achieved a sugar yield of
0.35 g per g of microalgal biomass [38]. The glucose content
accounted for 93% of total carbohydrate of C. vulgaris FSP-E after
primary hydrolysis (72% sulphuric acid, 30 min, 30 °C) followed
by secondary hydrolysis (4% sulphuric acid, 20 min, 121 °C) [82].
Acid hydrolysis of 2% sulphuric acid followed by 2% sodium
hydroxide was found to have the highest biobutanol yield, rather
than using water, acid, alkaline or cellulose alone [89].
Carbohydrate-rich microalgal cells can be saccharified efficiently
by using 2% sulphuric acid, giving a better glucose yield than
achieved with other forms of enzymatic and alkaline saccharifica-
tion [90].
619
Chemical saccharification offers efficient, rapid and cheap
hydrolysis reactions. More than 50% of glucose can be released
from microalgal biomass slurry after chemical hydrolysis [74,77].
In contrast, concentrated acid utilization may lead to corrosion of
the container and formation of inhibiting components that may
inhibit the fermentation process [79]. Enzymatic saccharification
is slower and requires less energy consumption, as a relatively
mild temperature is sufficient to breakdown microalgal-based car-
bohydrates. This process is thus still widely used as it provides a
high glucose yield without producing inhibitory compounds under
mild temperature operations and without the addition of chemi-
cals. Nevertheless, the drawbacks of enzymatic saccharification
include the high cost due to the use of expensive enzymes and
the requirement of energy-consuming pretreatment prior to
enzymatic hydrolysis to ensure high saccharification efficiency
[74,77,79]. Despite these factors, Suali et al. [74] claimed that pre-
treatment using dilute acid hydrolysis followed by enzymatic
hydrolysis can be applied to gain a higher glucose yield. Overall,
hydrolysis strategies, enzyme composition, composition of chemi-
cals added, biomass and reagent loadings and hydrolysis time all
need to be further studied to ensure maximal hydrolytic efficiency
and therefore the highest fermentative fuel production.
4.2.2. Fermentative production of liquid biofuels using microalgal
feedstock
At present, bioethanol is the most commonly used liquid bio-
fuel, which can be produced by microalgal-based carbohydrates
through saccharification followed by alcoholic fermentation [80].
Alcoholic fermentation is the conversion of cellulose, starch and
sugars from microalgal biomass into fermentative fuels by the
metabolic activities of bacteria, yeast or fungi under anaerobic con-
ditions [78,91,92]. Commercially, Saccharomyces cerevisiae is the
dominant strain for fermentation [74,93]. After the above-
described saccharification process, the fermentation reaction
applies S. cerevisiae as the fermenting yeast and uses water to start
up the fermentation process. The culture is then kept warm in a
large fermenter for an efficient reaction [79,92,94]. The yeast
assimilates the sugars, converting them into pyruvate at the
glycolytic pathway, followed by acetaldehyde formation from
pyruvate and the release of CO2 as by-products. Finally, the
acetaldehydes are reduced to form ethanol [74,78,95]. Both CO2
and water are generated in the fermentation process, and thus
the liquid biofuel has to be subsequently fed into a distillation unit
for high purity biofuel production. The concentrated biofuel will
then be condensed and stored in liquid form for engine usage. The-
oretically, the maximum yield is 0.51 kg of ethanol with 0.49 kg of
CO2 per kg of glucose [78,91,96]. The by-product CO2 can be
recovered for re-use in microalgal cultivation, whereas the residual
biomass can become the substrate for anaerobic digestion.
C. vulgaris and Chlamydomonas sp. are proficient microalgal spe-
cies for bioethanol production via starch fermentation [68]. It was
reported that using C. vulgaris with 37% starch content per dry cell
weight could achieve 65% ethanol conversion [92]. Approximately
0.235 g of bioethanol was produced from 1.0 g of C. reinhardtii bio-
mass using a separated hydrolysis and fermentation (SHF) method
[97]. Two types of fermentation process, namely SHF and
simultaneous hydrolysis and fermentation (SSF), are described
here. SHF is the more commonly applied process, allowing enzy-
matic and chemical hydrolysis to be performed in its optimum
conditions in each unit, and to produce a high glucose yield prior
to fermentation [89,95]. Nevertheless, the drawback of SHF is that
the increased amount of glucose and cellodextrin may have an
inhibition effect on cellulose activity [98]. This challenge can be
resolved in SSF, which provides a single-step method to convert
microalgal-based carbohydrates into fermentative biofuel. Saccha-
rification and fermentation occur in the same vessel, thus reducing
620 W.Y. Cheah et al. / Applied Energy 179 (2016) 609–625
the inhibiting effect seen with SHF, as glucose and cellodextrin are
progressively assimilated by yeast or bacteria in the fermentation
process. SSF also offers a shorter reaction time, lower equipment
cost, and less contamination due to the elimination of some steps
[89,98]. The pretreatment required for the SHF process may
increase energy consumption by up to 30%, and thus SSF also
seems to be favourable in terms of cost [74]. However, high solid
retention may occur in the fermenter during the SSF process. It
may also be difficult to find the optimal temperature for both sac-
charification and fermentation processes in an SSF operation, thus
reducing its performance [93].
In general, SHF may provide higher efficiency in fermentative
biofuel production as compared to SFF, due to the need for less glu-
cose substrate in SFF than in SHF [89]. Harun et al. [93] applied
both SHF and SFF processes to Chlorococcum sp. for bioethanol pro-
duction. They claimed that enzymatic activity was still inhibited in
SSF, although there was no separate hydrolysis stage. The bioetha-
nol yield in SHF was greater than that in SSF due to the lack of an
optimum working temperature and changes in pH in the SSF fer-
menter. In contrast, Ho et al. [77] used an enzymatic hydrolysate
of C. vulgaris FSP-E for bioethanol production via both SHF and
SSF methods, and found that the bioethanol yield was 92.3% in
SSF as compared to 79.9% in SHF. Moreover, a marine algae, Lami-
naria japonica, produced 0.4 g ethanol per g of carbohydrate after
acid hydrolysis followed by SSF when used together with a hydro-
lytic enzyme [99]. A bioethanol yield of 11.8 g L 1 was generated
by using 25.7 g L 1 of glucose from Schizochytrium sp. via the SSF
process [100]. Moreover, Chen et al. [38] suggested that for mixed
substrates of primary feedstock, like corn with microalgal biomass,
liquefaction and pre-saccharification can be carried out, followed
by SSF. There are also reports on bioethanol production from ligno-
cellulosic feedstocks and industrial sweet potatoes via SSF
[98,101]. Anaerobic-based self-fermentation of algae for bioetha-
nol production has also been suggested, as it requires a shorter fer-
mentation time and simpler process [102–104]. Genetically-
modified Chlamydomonas perigranulat was also used for bioethanol
production through self-fermentation of microalgal carbohydrates
[79].
Biobutanol also offers a high energy content for use as a liquid
biofuel, and is even expected to replace bioethanol [38]. Although
biobutanol production through ABE (acetone-butanol-ethanol) fer-
mentation has been performed on a large scale using a sugar- or
starch-based feedstock since World War II, current commercial
production of butanol is still mainly based on chemical synthesis
using petroleum [105]. In particular, investigations of biobutanol
production from microalgal-based carbohydrates are still in an
early stage, with very few reports available in the literature. Biobu-
tanol fermentation is considered as having low efficiency and low
productivity, mainly due to the inhibition effects of biobutanol
itself in relation to the producing microbes, mostly saccharolytic
species, Clostridium sp. Unlike yeast, Clostridium sp., especially
the commonly used Clostridium acetobutylicum, are capable of
digesting biomass feedstock, including cellulose and starch, yield-
ing biobutanol and other organic compounds, like acetone, ethanol,
organic acids and gases [89]. The generation of these organic com-
pounds in the medium along with the reduction in pH are the main
reason for the low efficiency of biobutanol fermentation [106].
According to Chen et al. [38], theoretically the maximal biobutanol
yield is 0.41 g per g of glucose under controlled fermentation
conditions. This yield is lower than the bioethanol yield, which is
0.5 g per g of glucose.
Biobutanol is generated through ABE fermentation with a ratio
of approximately 3:6:1 [89]. ABE fermentation can be categorized
under two distinct phases, namely acidogenesis and solventogene-
sis. The first phase of acidogenesis occurs in the microbial
exponential growth phase, along with rapid production of organic
acids like acetate, butyrate, and lactate, as well as hydrogen and
ethanol. A significant fall in pH occurs due to the generation of
these organic acids. Subsequently, the proton gradient of the mem-
brane is destroyed, allowing the occurrence of the second phase,
solventogenesis. In solventogenesis, acetone, butanol and ethanol
are generated in late growth phase to early stationary phase
[89,106]. Wang et al. [107] claimed that 111 g of acid-pretreated
C. vulgaris yielded 3.37 g L 1 of butanol by ABE fermentation, and
the use of more than 2% sulphuric acid was suggested to hydrolyse
microalgae prior to ABE fermentation. Diluted acid hydrolysis pre-
treatment with 1.0 M sulphuric acid for 120 min at 80–90 °C are
the optimal conditions for preparing the feedstock for ABE fermen-
tation, resulting in 3.74 g L 1 of biobutanol [88]. Moreover, the
main constraints with regard to low biobutanol production are
the reduction in pH reduction and change in medium composition
due to the generation of inhibitory organics. Therefore, Tsai et al.
[106] claimed that adding ammonium acetate into medium at a
concentration of 6 g L 1 could enhance biobutanol concentration,
with an increase of 1.6 g L 1. An acetate buffer of 100 mM added
into the culture caused an increase in biobutanol concentration
from 2 g L 1 to 9.8 g L 1. The best pH for biobutanol production
was 4.5, which can be maintained by calcium carbonate neutralisa-
tion. Moreover, the addition of butyrate into ABE fermentation can
improve biobutanol production, as butyrate triggers the metabolic
switch from acidogenesis to solventogenesis and induces aceto-
acetyl Co-A to be converted to butyl Co-A rather than acetoacetate
[89,108]. However, the butyrate concentration in the culture
should be kept at a non-inhibitory level to maintain the activity
of Clostridium sp. In addition, the critical problem with ABE fer-
mentation is solvent toxicity, which limits carbon substrate fer-
mentation by Clostridium sp. Continuous fermentation with in-
situ biobutanol removal by a vacuum membrane distillation unit
has been applied to reduce the solvent concentration in the cul-
ture, reducing the inhibitory effect on microbial activity and there-
fore boosting the biobutanol yield from 0.18 g butanol per g of
glucose to 0.21 g [106].
4.2.3. Gaseous biofuels
Biohydrogen and biomethane are two major alternative gaseous
biofuels produced mainly by anaerobic digestion of carbohydrate-
rich microalgal biomass grown on organic wastewater. The conver-
sion of biohydrogen and biomethane from microalgal biomass is
discussed as follows.
4.2.3.1. Biohydrogen. Biohydrogen serves as an efficient energy car-
rier with a calorific value of 242 kJ mol 1, which can be produced
by energy-intensive conventional processes like reverse water
gas shift reaction, water electrolysis, steam methane reforming
and gasification [58,109]. Hydrogen gas can also be produced via
biological pathways. For instance, hydrogen can be produced
directly through the metabolic activities of microalgae, although
the production efficiency is quite low. Alternatively, biohydrogen
can be produced via a biological route through anaerobic fermen-
tation, which includes both photofermentation and dark fermenta-
tion. Photofermentation involves using photosynthetic
microorganisms (such as Rhodobacter sphaeroides and
Rhodopseudomonas palustris) to metabolise organic substrates, like
microalgal-based glucose, producing CO2 and hydrogen [2].
Additionally, photosynthetic microalgae are able to generate
biohydrogen directly via photofermentation under anaerobic
conditions involving ferredoxin oxidation by hydrogenase [76].
Dunaliella tertiolecta and C. reinhardtii have achieved biohydrogen
yields of 61% and 52%, respectively, by photofermentation [110].
The limitations of photofermentation include the high bioreactor
cost, low light conversion efficiency, accumulation of the proton
 W.Y. Cheah et al. / Applied Energy 179 (2016) 609–625
gradient, and inhibition of enzyme activities due to the generation
of photosynthetic oxygen [2,68,92].
Dark fermentation carried out by acidogenic bacteria is more
favourable than photofermentation, due to having a much higher
production rate and fewer limitations. Acidogenic bacteria like
Enterobacter sp., Bacillus sp. and Clostridium sp. ferment substrates
without sunlight, producing biohydrogen and soluble metabolites
like alcohols and volatile fatty acids, together with CO2 [111]. In
fact, biohydrogen production occurs along with ABE fermentation,
which produces both liquid (bioethanol and biobutanol) and gas-
eous biofuels (biohydrogen) simultaneously [79]. Continuous dark
fermentation with Clostridium butyricum using carbohydrate-rich
C. vulgaris biomass exhibited a hydrogen production rate of
200 mL L 1 h 1 and a biohydrogen content accounts for 66% of
the total biogas [111]. C. acetobutylicum immobilised in poly (vinyl
alcohol) cryogel was shown to be highly efficient in biocatalyzing
anaerobic fermentation for biohydrogen production [112]. Heat
pretreatment of sludge substrate that helps in producing a high
hydrogen yield has been applied to preserve hydrogen-producing
bacteria, mainly Clostridium sp., and can also be applied to microal-
gal biomass substrate [113]. Factors affecting the efficiency of bio-
hydrogen production via dark fermentation include the substrates,
pH, temperature, nutrients and inhibitors [113]. Safety issues and
proper storage of biohydrogen, bioreactor design and the retention
time in the fermenter are identified as the main constraints to large
scale biohydrogen production, and thus more work is required in
these areas.
Large scale biohydrogen generation remains unrealistic due to
the low conversion rate. Another alternative for biohydrogen pro-
duction is through the combination of dark fermentation followed
by photofermentation, thus eliminating the constraints of any indi-
vidual process. This integrated system begins with dark fermenta-
tion to break down carbohydrates to organic acids, followed by
photofermentation to further degrade organic acids to biohydro-
gen. Furthermore, dark fermentation can also be integrated with
anaerobic digestion, which is a methanogenic process, to achieve
biomethane production [2]. For instance, Xia et al. [109] claimed
that a combination system comprising dark fermentation,
photofermentation and methanogenesis could enhance the biohy-
drogen and energy production when using Nannochloropsis ocean-
ica biomass as the feedstock. The biohydrogen and energy yield
increased significantly by 1.7- and 1.3-fold when compared to
using dark fermentation-methanogenesis and methanogenesis
process alone, respectively [109]. Therefore, these integrated sys-
tems might be key steps towards commercial biohydrogen produc-
tion, although more research is need here.
4.2.3.2. Biomethane. Biomethane is another gaseous fuel source
that can be used to generate electricity by anaerobic digestion of
microalgal biomass, while the spent biomass can be used for
biofertilisers [62]. According to Pragya et al. [94], if the lipid con-
tent of microalgae is lower than 40%, anaerobic digestion is the
best approach with respect to energetic recovery and energy bal-
ance of the biomass.
Anaerobic digestion is the process of converting organic wastes
into biogas including carbon dioxide, methane, trace gases and par-
ticulates. Anaerobic digestion occurs in three stages, namely
hydrolysis, fermentation and methanogenesis. Complex com-
pounds are broken down into simple sugars in the hydrolysis stage.
Subsequently, simple sugars will be converted by fermentative
bacteria to produce alcohols, acetic acid, volatile fatty acids and
gases containing hydrogen and carbon compounds. Finally, metha-
nogens will metabolize these compounds producing gases, mainly
methane and carbon dioxide [92]. This process takes place in a
closed digester with the temperature ranging from 25 to 50 °C.
Microalgal biomass and the residues can be digested for bio-
621
methane production to recover the remaining energy. A high bio-
methane content of more than 60% can be produced from
microalgal biomass [38], and this also serves as an approach to
treat organic wastes.
Biomethane production is directly affected by the quality of the
microalgal substrates (i.e., organic loadings), temperature, pH, and
retention time in the digester, and also requires a significant land
area and infrastructure investment [62,68]. The longer the reten-
tion time and the higher the organic loadings, the greater the bio-
gas yields. Microalgal biomass with a low C/N ratio may reduce the
biomethane production, and thus co-digestion can take place by
digesting microalgal biomass together with cellulosic materials,
like agricultural wastes, to obtain the optimal C/N ratio of
20:1–25:1 [38,92]. Moreover, biogas production has shown to be
species-dependent based on cell degradation efficiency [76]. A
study found that 50% of the tested biomass with Phaeodactylum tri-
cornutum was not converted into methane, while Scenedesmus sp.
AMDD was identified as a species with effective methane produc-
tion [114]. Nevertheless, it is important to note that anaerobic
digestion cannot be economically competitive, unless it targets
the residual biomass after biodiesel and bioethanol production.
together with microalgal biorefinery applications.
5. Potential of using microalgae as feedstock for biofuel
production
Microalgae generally accumulate up to 20–75% of cellular lipid
content per g of dried biomass cell [67]. Microalgal lipids serve as
clean-burning alternative fuels which are biodegradable, user-
friendly, non-toxic, and free from sulphur and odour. The resulting
materials can be easily used by blending with petroleum fuel to
create biofuel blends. Large scale microalgal cultivation is essential
for the process to be cost-effective and minimal inputs to be used,
so as to achieve economical mass production of feedstock for bio-
fuel production. Microalgae cultivation for biofuel production
requires a great quantity of water, with nutrients and organics pre-
sent in it. Wastewaters such as POME, sewage and leachate are
notable media to work with in this context, as they are nutrient-
rich and produced in huge supply daily. Microalgae B. braunii cul-
tivated in sewage shows the best results in biomass production
and lipid content [37,40] (Table 2), and such results have made
microalgae cultivation in wastewater for biofuel production
increasingly attractive. This is because the pollutants profile in
sewage wastewater is more uniform and at lower strength than
that seen in some other wastewaters, without causing growth inhi-
bition to microalgae.
Sewage treatment plants can be the best places for cultivation
in the most economical and practical way. The waste stabilisation
ponds originally function as sewage treatment plants, and can be
used as open ponds and for commercial scale microalgae cultiva-
tion for biofuel production [6]. In addition to the combination of
wastewaters for microalgal cultivation, flue gas cultivation has also
been recommended and received much research attention in
recent years. This approach can further reduce the cultivation cost,
and most importantly contribute to carbon dioxide biosequestra-
tion [115–119]. This method has improved biomass production
and lipid accumulation of Auxenochlorella protothecoides cultivated
in municipal wastewater [10]. However, the tolerance of microal-
gae towards SOx and NOx, as well as the high temperature of flue
gas, varies significantly depending on the microalgae species. Fur-
ther studies on flue gas utilization remain to be carried out.
Furthermore, microalgae species, either single or mixed, work
well in removing organic and inorganic pollutants in the POME,
sewage and leachate (Table 3). For instance, indigenous microalgae
isolated from landfill leachate are competent in removing up to
622 W.Y. Cheah et al. / Applied Energy 179 (2016) 609–625
90% of organic and inorganic pollutants in the leachate. Landfill
leachate is usually toxic and less stable in terms of pollutant pro-
files when compared to POME and sewage. However, indigenous
microalgae which have adapted to the growth conditions are able
to eliminate pollutants. In order to achieve biofuel production, the
introduction of oleaginous single species, such as B. braunii, into
the leachate may not be possible, due to the toxic conditions.
Therefore, strong, adapted indigenous microalgae species ought
to be screened for strains possessing high biomass and lipid or car-
bohydrate production. Microalgae cultivation could thus serve dual
roles in this case, for both nutrient reduction and biofuel
production.
As shown in Fig. 2, the lipid and carbohydrate contents of
microalgal feedstock can be converted in biodiesel and fermenta-
tive biofuels, respectively. The residual biomass after these
conversion methods can be applied for biohydrogen and
biomethane production. A single microalgal feedstock source can
produce multiple biofuels. The efficiency of the downstream
processes significantly contributes to the potential of microalgal
biomass with regard to producing biofuel. In the subsequent
downstream process, sedimentation incorporated with floccula-
tion will the most feasible harvesting method. These processes
are widely used in wastewater treatment plants, as they are simple
and cost-effective. As suggested earlier in this paper, sewage treat-
ment plant can be employed as an open pond cultivation system.
The integration of a sedimentation tank with the addition of a floc-
culant to boost settling might be the most practical approach. Fur-
thermore, immobilisation of microalgae can also contribute to
successive cultivation and harvesting. The working principle here
is similar to that seen with the attached growth cultivation system,
such as with the use of a trickling filter and rotating biological con-
tactor, with bacteria-microalgae attached to the support media
treating wastewater by bio-decomposition of organics. The process
of microalgal immobilisation might have to be further improved in
the design of a cultivation system by integrating some support
media. Additionally, in-situ transesterification is favourable to
enhancing microalgal-based biodiesel conversion.
Biobutanol produced from microalgal-based carbohydrates via
ABE fermentation can serve as an alternate fuel, as it offers several
advantages over bioethanol. It contains more energy, is less corro-
sive, less volatile, more soluble, less hygroscopic and mixes better
with gasoline [38]. This allows substitution of bioethanol by biobu-
tanol for transportation purposes. However, more efforts are
required to enhance the biobutanol fermentation efficiency and
yield. The residual microalgal biomass can be fed into an integrated
system with dark fermentation-anaerobic digestion, to produce
biohydrogen and biomethane simultaneously. The integrated sys-
tem, which is was proposed for wastewaters and organic solid
waste digestion, can also be applicable for biofuel conversion of
microalgal feedstock. With this the number of steps in microalgal
fuel processing can be reduced, resulting in low cost processing
systems [68]. Further cost and energy analyses are required to
ensure the economic and practical feasibility of biofuel production.
Moreover, economic biofuel production would be even more
meaningful if associated with the production and recovery of valu-
able bioproducts [16]. For example, microalgal biomass residue
after lipid extraction still contains a significant amount of protein
or other valuable components, which can be subjected to biorefin-
ing to generate more profits.
6. Challenges and perspectives
Microalgal cultivation in wastewaters, apart from wastewater
treatment, has contributed both resources and energy reduction
for biomass cultivation and processing. Despite the multiple
advantages of this microalgal cultivation approach, a number of
challenges still exist, as follows: (i) variations in wastewater com-
position; (ii) wastewater flow rate; (iii) fluctuation in pH and tem-
perature; (iv) nutrient limitations; (v) light intensity; (vi) CO2
concentration; (vii) bioreactor design; (viii) contamination risk;
(ix) the microalgae species used, and (x) economic aspects
[9,10,67,71,120,121].
Changes of any of the growth conditions greatly affect the bio-
mass production, and most importantly the lipid productivity.
High turbidity or coloured wastewater blocks the sunlight penetra-
tion to the open pond, thus reducing the growth of autotrophic
microalgae. pH regulation is essential for microalgal cultivation
in countries with distinct seasons [122]. Extremely high nutrient
concentrations in wastewater may inhibit the growth of certain
microalgae species, so dilution of wastewater is required [123].
Nutrient starvation, after medium dilution, may be favourable for
lipid accumulation with certain microalgal species. However, the
responses towards nutrient limitations differ in relation to those
seen with other indigenous microalgae species present in the cul-
tivation medium [10]. Moreover, open pond systems may face the
problem of water loss due to wastewater evaporation, as well as a
greater contamination risk. This may lead to higher costs and
energy consumption associated with replenishment of lost water
and selection of risk-tolerance species [1]. Finally, the land avail-
ability for commercial scale microalgal cultivation is a constraint,
which may even cause microalgae biofuel production to be unsus-
tainable [10].
The major challenges of scaling up downstream processes, such
as harvesting, extraction and fuel conversion, are still economic
ones, such as the operating cost and intensive energy requirement.
Ribeiro and da Silva [124] noted that while many good technolo-
gies have been developed for microalgal-based biofuel conversion,
scalability remains a challenges before they are economically fea-
sible. The efficiency of biofuel conversion also needs to be
improved with respect to the biofuel yield. Life cycle and economic
assessments to promote CO2 capture, solar energy utilization,
wastewater cultivation, biorefineries and bioenergy generation
are all needed to reduce the overall cost of biofuel production.
However, the literature has mainly focussed on upstream process-
ing to boost microalgal biomass and lipid productivity, rather than
making the downstream process more cost-effective [2]. Moreover,
the results of biofuel production on the lab scale may not be com-
patible with real commercial scale production.
Overall, the survival of microalgae in wastewater is dependent
on the species’ tolerance with regard to the environmental stress,
as well as the cultivation conditions. Metabolic engineering or
genetic modification can be applied to optimize biofuel producing
strains, improving the quality of biofuels and the production quan-
tities [76]. The issues of water and nutrient availability for microal-
gal cultivation can be solved with microalgae-wastewater
cultivation. However, more efforts are required to further screen
high performance microalgae species grown in wastewaters in
relation to the appropriate nutrient profile of the medium. Down-
stream processes also have to be further researched and modified,
to ensure minimum input with effective conversion of microalgal
biomass for maximal biofuel production.
7. Conclusions
Wastewater based microalgal cultivation is an ideal platform to
simultaneously achieve waste reduction and renewable energy
production when the yielded microalgal biomass is further con-
verted to biofuels via multiple-step processes. However, growing
microalgae with wastewaters is very challenging. It would still
require further investigations to improve knowledge about high
 W.Y. Cheah et al. / Applied Energy 179 (2016) 609–625 623

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