Energy Conversion and Management 173 (2018) 81–94

Contents lists available at ScienceDirect

Energy Conversion and Management

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

Review

Overview: Comparison of pretreatment technologies and fermentation T

processes of bioethanol from microalgae
⁎
Chai Kee Phwana, Hwai Chyuan Ongb, , Wei-Hsin Chenc,d, Tau Chuan Linga, Eng Poh Nge,
⁎
Pau Loke Showf,
a
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
b
Department of Mechanical Engineering, Faculty of Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia
c
Department of Aeronautics and Astronautics, National Cheng Kung University, Tainan 701, Taiwan
d
Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan 701, Taiwan
e
School of Chemical Sciences, Universiti Sains Malaysia, USM, Malaysia
f
Bioseparation Research Group, Department of Chemical and Environmental Engineering, Faculty of Engineering, University of Nottingham Malaysia Campus, Jalan Broga,
43500 Semenyih, Selangor Darul Ehsan, Malaysia

A R T I C LE I N FO A B S T R A C T

Keywords: Continuous exploitation of natural resources especially in fossil fuels to fulfil the market demand has jeopardized
Microalgae the natural resources in the Earth. Bioethanol produced by microalgae is one of the promising biofuel for energy
Bioethanol security and ecological sustainability. Yet, to date only a modest review has been reported on the bioethanol
Pretreatment production technology from microalgae by using various pretreatment methods, fermentative microorganisms,
Fermentation
fermentation processes, commercialization techniques and its environmental perspectives. This review paper
Economic and environmental vitality
aims to provide a comprehensive information by comparing the recent pretreatment technologies by using
different types of ethanologenic microorganisms in various fermentation processes to maximize the bioethanol
production. Studies of the economic viability and environmental perspectives of bioethanol from microalgae
have been carried out to investigate its potentialities to eliminate environmental issues and towards commer-
cialization. Therefore, this review is essential in providing ideas for the future researches in renewable energy
resources technology to develop a more efficient way to scale-up the bioethanol production from microalgae for
commercial and industrial application.

1. Introduction
Depletion of natural resources due to the gradual growth of the
worldwide population has resulted in the current resource depredation
with significant environmental impacts in energy shortage, worsening
climate change and increasing greenhouse gasses emission [14]. Fossil
fuels which are the primary energy source for the world are non-re-
newable and estimated to be used up by middle of the century [5,28]. In
addition, increasing global population which is projected to exceed 9
billion by 2050 will lead to overexploitation of the resources and drives
the scarcity of arable land to its limit [42]. Thus, it is a critical concern
to develop the alternative energy resources and adopt policies to
minimize the utilization of fossil reserves, maintain the environmental
sustainability and cost-effective, and reduce the releases of greenhouse
gas [8,71,132]. Recent statistical report of International Energy Agency
(IEA) revealed that the total primary energy supplied by fuel showed
the energy produced from biofuels and waste increased steadily from
2.3% in 1973 to 5.7% in 2016 with the total of 3740 and 5257 (Mtoe)
respectively [67]. Therefore, it is expected that biofuels will emerge to
one of the most strategically sustainable energy source [109]. Biofuels
are biological sources generally derived from primary fuels such as
firewood, wood pellets, wood chips, animal waste, crop residues and
landfill gas; while secondary fuels which consists of bioethanol, bu-
tanol, biodiesel, and biohydrogen [95,137].
Biofuels are categorized into first-, second-, third-, and fourth-gen-
eration biofuels based on the type of feedstock used [121,129]. Re-
search and developments in biofuels initiated from the first-generation
fuel which used the sources of food (corn, wheat, barley and sugarcane)
as feedstock has evolved to fourth-generation algae metabolic en-
gineering [42]. The production of first-generation fuel has raised con-
troversial debates on food supply, food security and land use changes
due to the large conversion of agricultural crops to biofuels

⁎
Corresponding authors.
E-mail addresses: onghc@um.edu.my (H.C. Ong), PauLoke.Show@nottingham.edu.my (P.L. Show).

https://doi.org/10.1016/j.enconman.2018.07.054
Received 8 May 2018; Received in revised form 8 July 2018; Accepted 17 July 2018
0196-8904/ © 2018 Elsevier Ltd. All rights reserved.
C.K. Phwan et al. Energy Conversion and Management 173 (2018) 81–94

[83,142,147]. First-generation bioethanol not only lead to the growing Table 1

concern of the environmental issue but also causes dilemma of unstable Carbohydrates content in different species of microalgae (all results are pre-
food prices in the market because they are food sources and feed crops sented in % dry weight).
globally [146]. Nevertheless, the growing interest in biofuels has been Biomass Carbohydrate References
switched to second and third generation which have no food-fuel con-
flict and better environmental performance in terms of reducing Anabaena cylindrica 25–30 [41]
Aphanizomenon flos-aquae 23 [102]
greenhouse gasses emission [49]. Second-generation biofuels are
Chlamydomonas reinhardtii 17 [41]
mainly produced from lignocellulosic materials from forest and agri- Chlorella sp. 19.5 [116]
culture residues and industrial organic wastes such as straw, grass, Chlorella sorokiniana 35.67 [24]
woods, sawdust and others [88,146]. However, using the agricultural Chlorella vulgaris 20.99 [154]
Chloroccum sp. 32.50 [57]
residues or industrial wastes still facing the limitations, due to difficulty
Dunaliella salina 32.00 [41]
and high costs to convert lignocellulosic biomass into biofuel in pre- Dunaliella tertiolecta 21.69 [130]
treatment process [64,88,146]. Third-generation biofuels produced Euglena gracilis 14–18 [41]
from microalgae as feedstocks such as bioethanol, biodiesel, biohy- Isochrysis zhangjiangensis 23.2–47.7 [47]
drogen and biomethane have been garnering interest worldwide Isochrysis galbana 7.7–13.6 [48]
Isochrysis sp. 5.2–16.4 [98]
[25,85,146]. Microalgae have been recognized as a more promising
Nannochloropsis oceanica 22.70 [26]
alternative feedstock that do not require arable land, not competing Nannochloropsis oculata 8 [16]
with food cultures, high growth rate, high photosynthetic efficiency, Pavlova lutheri 28.25 [118]
potentially to cultivate in offshore marine environment and they are Porphyridium cruentum 40 [16]
Prymnesium parvum 25–33 [41]
easy to be cultivated in larger quantity [18,89,159]. Algae can be
Scenedesmus dimorphus 21–52 [41]
grouped into prokaryotic microalgae (cyanobacteria Chloroxybacteria), Scenedesmus obliquus 10–17 [41]
eukaryotic microalgae (green algae Chlorophyta), red algae (Rhodo- Spirulina platensis 31.20 [70]
phyta), and diatoms (Bacillariophyta) [122]. Microalgae are microscopic Spirogyra sp. 33–64 [102]
algae or photosynthetic unicellular microorganism that generally found Spirulina sp. 20 [16]
Tetraselmis maculate 15 [98]
in ocean and fresh water environment.
Tetraselmis suecica 15–50 [17]
Microalgae are convincing alternative resources for bioethanol Tetraselmis sp. 24 [125]
production in comparison with conventional plant crops and their
carbohydrate contents are mainly in the form of starch and cellulose
which are easily break down to fermentable sugars via microbial fer- microalgae. In contrast, cyanobacteria commonly referred to as blue-
mentation [23,44]. The cell wall composition of microalgae are also green algae which is a division Cyanophyta are also considered as
different from the lignocellulosic crops due to low content or absence of photosynthetic protists which contain a group of oxygenic bacteria that
the lignin [37]. Bioethanol has been recognized as a clean and sus- obtain energy by photosynthesis process [21,91]. The smallest cell size
tainable fuel because it is non-toxic, biodegradable, and produce almost of cyanobacteria can be found on picoplankton (0.2–2 μm) and the
zero pollutant to the environment [109,133]. In addition, the global largest unicellular organisms being of about 500 μm [91]. Most studies
production of bioethanol showed a rapid growth from 17.25 billion of cyanobacteria and microalgae have been investigated to produce
liters in 2000 to over 46 billion liters in 2007 [10] in a few years and it efficient sustainable energy like hydrogen (direct synthesis in cyano-
is projected to exceed 160 billion liters by 2020 [15]. There are many bacteria), lipids for biodiesel, and carbohydrates for ethanol production
pretreatment methods to break down the carbohydrates of microalgae [113]. Table 2 shows the composition of protein and carbohydrates of
and convert them to bioethanol. To ensure microalgae completely break 13 cyanobacterial species, n = 3 [107].
down into simple sugars or monomers, pretreatment for saccharifica- Prokaryotic cells (cyanobacteria) are more similar to bacteria cell
tion is done before fermentation. Among of these methods, one of the because they lack of membrane-bound organelles such as plastids, mi-
most popular method is fermentation process. The bioethanol produc- tochondria, nuclei, Golgi bodies and flagella. Eukaryotic cells (micro-
tion of microalgae biomass is greatly influenced by the degree of dif- algae) which consist of these organelles can control the functions of the
ficulties in pretreatments and the type of fermentation process [30]. cell, allowing it to survive and reproduce [19]. In the cell of microalgae,
Currently, there is no other comprehensive review reported about the carbohydrates usually existed in the outer cell wall (pectin, agar, algi-
pretreatment of microalgae, importance of fermentative microorganism nate), inner cell wall (cellulose and hemicellulose) and inside the cell as
to bioethanol production, various fermentation processes and com-
mercialize value of bioethanol produced from microalgae. Therefore,
this review aims to discuss the recent information on the pretreatment Table 2
technologies, production of bioethanol with different fermentative mi- Composition of protein and carbohydrates of 13 cyanobacterial species, n = 3
[107].
croorganisms in various fermentation processes and the potential ap-
plications of bioethanol production from microalgae to the environ- Species Protein (% DW) Carbohydrates (% DW)
mental and economic impacts.
Calothrix crustacea (HF) 21.50 ± 0.40 7.60 ± 0.50
Calothrix contarenii (HF) 27.43 ± 0.47 8.23 ± 0.65
2. Microalgae and cyanobacteria cells Gloeocapsa crepidinum (C) 56.46 ± 0.25 7.63 ± 0.55
Lyngbya martensiana (F) 18.86 ± 0.65 5.43 ± 0.41
The advantages of algae, microalgae and cyanobacteria for the Lyngbya semiplena (F) 27.50 ± 0.45 8.93 ± 0.15
Phormidium corium (F) 49.56 ± 0.55 16.46 ± 0.45
production of third-generation biofuels are higher than agricultural
Phormidium tenue (F) 62.96 ± 0.55 15.46 ± 0.40
crops or lignocellulosic biomass in view of producing first- and second- Spirulina subsalsa (F) 70.76 ± 0.90 16.63 ± 0.56
generation biofuels [21]. Algae are primitive plants (thallophytes) Spirulina labyrinthiformis (F) 68.03 ± 0.85 14.73 ± 0.66
which have no sterile covering of cells around the reproductive cells but Synechococcus sp. (C) 63.56 ± 0.60 8.56 ± 0.56
they have chlorophyll a as their primary photosynthetic pigment [19]. Oscillatoria formosa (F) 50.85 ± 0.79 9.46 ± 0.45
Oscillatoria salina (F) 41.80 ± 0.81 11.20 ± 0.36
Microalgae have cell size between 2 to 200 μm and considered as a Oscillatoria subbrevis (F) 45.16 ± 0.41 11.53 ± 0.68
photosynthetic microorganism which capable of producing large
amounts of biomass containing lipids, proteins, or carbohydrates (HF) Heterocystous filamentous cyanobacteria; (F) Non-heterocystous fila-
[5,149]. Table 1 shows the carbohydrates content in different species of mentous cyanobacteria; (C) Unicellular cyanobacteria.

82
C.K. Phwan et al.
Fig. 1. General outline of carbon and energy storage routes in microalgae cell [98].
storage of products (such as starch found in microalgae and glycogen in
cyanobacteria) as seen in Fig. 1 [98]. Microalgae cell wall generally
quite resistant to pretreatment methods employed for disruption when
comparing to peptidoglycan-based cell wall which is more easier to
disrupt [52]. Monosaccharides glucose produce from microalgae during
fermentation will be used as an energy and carbon sources to produce
carbohydrates, lipids, and proteins. Excessive irradiance or “nitrogen
stress” happened due to insufficient inorganic nutrients supply will
cause the rate of glucose production during photosynthesis exceed the
rate of glucose consumption by the cell [98]. However, both of the
microalgae and cyanobacteria gaining the attraction as feedstock used
to produce bioethanol. Both of the microorganisms have potential to
reach 50% of their dry weight (DW) in carbohydrates which can be
fermented with high ethanol yields [21]. Common carbohydrates can
be found in cyanobacteria and microalgae used for the production of
bioethanol are starch, glycogen and cellulose [21].
3. Pretreatment and saccharification of microalgae biomass
Pretreatment is an important step to break down carbohydrates or
starch polymers into monomers such as glucose to produce bioethanol
[138]. Then, the fermentable sugars will be metabolized by micro-
organisms that carry out bioconversion into bioethanol via fermenta-
tion [103]. Fig. 2 shows a general description of different pretreatments
and fermentation processes to convert microalgae into bioethanol.
Pretreatment is one of the most crucial steps to minimize the crystal-
linity degree of the cellulose matrix [45]. Furthermore, it also increases
the surface area and improves the digestibility of the substrate or bio-
availability for bacterial enzymes to hydrolyze the biomass more re-
sourcefully [45,59]. Many methods, including chemical methods, en-
zymatic hydrolysis and mechanical methods, have been conducted to
disrupt cell wall of microalgae in order to release more carbohydrates
and process them into monosaccharides [74,127,162].
3.1. Chemical method
Chemical pretreatment is one of the most accessible methods be-
cause chemical substances are more accessible and cost effective in
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Energy Conversion and Management 173 (2018) 81–94
comparison to engineered enzymes. When chemical substances are
stored properly, they are easier to handle as they have higher durability
and less hassle than enzymes [4]. In addition, it is relatively easier to
carry out the mild condition chemical hydrolysis of microalgae-based
cellulose which is not associated with lignin as compared to lig-
nocelluloses [23].
3.1.1. Acid hydrolysis
The release of monosaccharaides molecules from long chains of
polysaccharides can be enhanced by using chemicals such as acid to
break their bonds [68]. In most studies, the common acids used to
convert feedstock to fermentable sugars are sulfuric acid (H2SO4) and
hydrochloric acid (HCl) [100,106,163]. A study was conducted to
evaluate the efficiency of acid hydrolysis on the sugar extraction from
mixed microalgae with different concentrations of HCl (0.5, 1, 2 M) and
H2SO4 (0.5, 1, 2 M), 0.5 M H2SO4 and 2.5% (w/v) MgSO4, 0.5 M H2SO4
and 2.5% (w/v) CaCI2 respectively, autoclaved at 121 °C with different
reaction times (10, 20, 30, 40 min) [127]. The result indicated a higher
acid concentration improves the sugar extraction yield [127]. Miranda
et al. [103] reported that acid pretreatment was more efficient than
physical treatment because physical disruption method did not really
create a great interruption impact on the microalgae cell wall to release
fermentable sugars. The study also highlighted the acid treatments
(H2SO4 and HCl 2 N) had achieved higher sugar extraction efficiency
which were 8.2% and 8.1% respectively, at temperature 120 °C. Dilute
acid pretreatment was applied widely because strong acid would cause
an excessive degradation of the complex substrate and consequential in
loss of fermentable sugars [114]. Severe acid pretreatment will de-
graded the fermentable sugars and formed high concentration of fur-
fural (from pentoses) and hydroxymethyl furfural (from hexoses) [82].
According to Miranda et al. [103], sugar degradation happened during
the acid concentration higher than 2 N. Therefore, it was not a surprise
that unwanted compounds such as weak acids, furaldehyde and phe-
nolic compounds were found in acid hydrolysate which could interfere
the growth and glucose conversion reaction of S. cerevisiae during fer-
mentation process [153]. The formation of inhibitors after acid pre-
treatment can have a negative effects on the fermentation process
[135]. Thus, acid hydrolysate need to be neutralized before
C.K. Phwan et al. Energy Conversion and Management 173 (2018) 81–94

Microalgae
Biomass

Pretreatment/ Saccharification

Chemical Enzymatic Combined Mechanical

Bead beating
Acid hydrolysis
High pressure
Alkaline hydrolysis homogenization
Supercritical CO2 (HPH)
Ammonia Fiber Ultra-sonication
Explosion (AFEX) Autoclaving

SHF SSF SSCF Dark Photo CBP

fermentation fermentation

Bioethanol

Fig. 2. General description of bioethanol production from microalgae with different pretreatments and fermentation processes.

fermentation process and it could directly increase the production cost. facilitates the separation of the desirable products [35]. Research on
Many experimental studies have been carried out to identify the effi- supercritical CO2 to extract carbohydrate from microalgae for bioe-
cient pretreatment methods without the use of acid hydrolysis Kim thanol production has been reported [58]. After the pretreatment which
et al. [81,123]. carried out at 60 °C with 400 mL min−1 of carbon dioxide, the cell wall
of the Chlorococum sp. was disrupted due to the high pressure and
temperature. The disruption lead to the release of carbohydrates en-
3.1.2. Alkaline hydrolysis
trapped within cell wall. This study indicated microalgae pretreated
Different concentrations of NaOH solutions 0.5%, 0.75%, 1%, 2%,
with supercritical CO2 gave 60% higher ethanol concentration for all
and 3% (w/v) with specific temperature and specific period of time
samples than the biomass without any pretreatment. It was noticed
were investigated in order to free and breakdown the Chlorococcum
from most of the current reports that related to utilization of super-
infusionum cell wall for fermentation process [59]. The highest bioe-
critical CO2 were mainly applied for lipid extraction instead of carbo-
thanol yield was 26.1% (g ethanol /g algae) with 0.75% (w/v) NaOH at
hydrates extractions. Thus, more studies could be conducted on ex-
120 °C for 30 min while the lowest bioethanol yield was 10.66% with
traction of carbohydrates from microalgae with the employment of
1% (w/v) of NaOH at 100 °C for 60 min. This study indicated it was
supercritical CO2.
feasible to produce bioethanol from microalgae through alkaline pre-
treatment. Besides, four different alkaline agents (potassium hydroxide
(KOH), sodium hydroxide (NaOH), sodium carbonate (Na2CO3) and
3.1.4. Ammonia fiber explosion (AFEX)
aqueous ammonia (NH4OH)) were used to pretreat T. suecica and
During the pretreatment, ammonia is allowed to penetrate into cell
Chlorella sp. biomass with molarity of 0.3 M [73]. The highest reducing
wall with the presence of water, creating a cleavage of diferulate lin-
sugar concentration for T. suecica and Chlorella sp. were obtained by
kages which cross link polysaccharides, lignin ferulate and lignin di-
using 0.3 M (at 1.68% w/v) of KOH and 0.3 M (1.19% w/v) NaOH,
ferulate linkages [99]. In AFEX pretreatment, biomass is treated with
respectively at 90 °C for 75 min. However, insignificant effects on re-
liquid anhydrous ammonia at a specific temperature (normally range
ducing sugar production during the alkaline pretreatment process were
between 60 °C and 100 °C) under a high pressure (about 250–300 psi)
found by using Na2CO3 and NH4OH. The amount of reducing sugar
for a limited period of time (30–60 min) [76,139]. Liquid ammonia will
produced was similar to that in the control sample (with the presence of
be vaporized to allow its recovery and recycling [141]. At the end of
alkali solution in room temperature). This study indicates the pre-
pretreatment, large pores are formed in the middle lamella and outer
treatment conditions such as selection of alkaline reagent, concentra-
cell wall are due to the expeditious pressure release. Then, it causing
tion and temperature of alkaline are actually play an important role in
the decompression of ammonia at cell wall periphery which lead to an
the process.
increase in enzyme activity on AFEX treated biomass [99]. AFEX pre-
treatment shows a significant improvement in saccharification rates
3.1.3. Supercritical carbon dioxide (CO2) especially in various herbaceous crops and grasses [158]. Furthermore,
Supercritical fluid method displays the interaction between fluid one of the benefits of AFEX pretreatment is no formation of by-products
properties (density, diffusivity, surface tension, viscosity) and opera- inhibitors such as furans [29]. In general, ammonia is a commodity
tional conditions (temperature, pressure, concentration of biomass) chemical that is widely applied, and the cost is cheaper than H2SO4, at

84
C.K. Phwan et al.
about one-fourth of the price on a molar basis. This implies that AFEX
pretreatment is more economically viable than dilute acid pretreatment
[80]. However, one concern of AFEX process is the recovery of am-
monia after pretreatment which may increase both capital and opera-
tional costs [13]. Till date, most of the AFEX pretreatment experimental
studies are focused on lignocellulosic biomass for lipid extraction in-
stead of bioethanol production from microalgae.
3.2. Enzymatic hydrolysis
Recently, the production of bioethanol from enzymatic hydrolysis
has greater potential as compared to acid hydrolysis [68]. Enzymatic
hydrolysis of cell wall utilizes less energy than chemical hydrolysis
although the amount of selective enzyme used for effective sacchar-
ification is generally high [131]. In addition, Choi et al. [31] had per-
formed enzymatic pretreatment of algal biomass Chlamydomonas re-
inhardtii UTEX 90 which consisted of two parts, liquefaction by α-
amylase from B. licheniformis at pH 6.0 from 70 °C to 90 °C followed by
saccharification amyloglucosidase from Aspergillus Niger. Findings of
the study reported 235 mg ethanol/g algae was produced under optimal
condition of 0.2% (v/w) enzyme at pH 4.5 under 55 °C in 30 min. Based
on the experimental studies, parameters such as enzyme concentration,
pH value, temperature and reaction time appeared to have great in-
fluence on the productivity of bioethanol in enzymatic hydrolysis.
Moreover, Harun & Danquah [57] had investigated the effects of
Chlorococum humicola concentration which was hydrolyzed by cellulose
from Trichoderma reesei in at different temperatures (28–60 °C) and pH
(2.5–7.5) on saccharification yield in a reaction time of 72 h. Sacchar-
ification yield of 68.2% (w/w) was obtained under the condition of
40 °C, pH 4.8 with a microalgal biomass concentration of 10 g/L. In
general, comparing to the acid pretreatment, enzymatic hydrolysis has
more advantages than acid pretreatment. It is because higher glucose
yield could be achieved by enzymatic hydrolysis at mild conditions in
addition to the absence of toxic by-products such as formic acid, le-
vulinic acid, hydroxymethyl furfural (HMF), furfural and other phenolic
compounds that may degrade the production of bioethanol during
fermentation process [23,161]. However, the selection of the pre-
treatment technology for a specific raw material depends on several
factors which directly and indirectly affects the enzymatic hydrolysis
step. Factors such as sugar-release patterns, combination of the sub-
strate, types of pretreatment and concentration and efficiency of the
enzymes used for the hydrolysis will have great impact on the biomass
digestibility [6].
3.3. Mechanical methods
Mechanical pretreatment increases the substrate accessibility to
enzymes by breaking the cell wall through a combination of chipping,
grinding, or milling [6]. Destruction of the cell wall through mechanical
forces can be divided into solid-shear forces (bead beating), liquid-shear
forces (high pressure homogenization), wave transmission (ultra-soni-
cation) and thermal heat [53]. An integration of process steps in mi-
croalgae to bioethanol via different mechanical pretreatments is illu-
strated in Fig. 3. Mechanical disruption of cells are gaining the interest
of the researchers as this physical method provides an approach to
preserve most of the functionality of the materials within the cell and
prevents further chemical contamination during the biomass algae
preparation [128]. Mechanical disruption not only improves the effi-
ciency of microalgae and increases cell area surfaces make it easier to
handle. However, this pretreatment requires higher cost and energy-
intensive in rupturing cell process as compared with chemical methods
[112,124].
3.3.1. Bead beating
Bead beating is a type of mechanical force and shear stress on
biomass by the contact with beads in movement to break down the
85
Energy Conversion and Management 173 (2018) 81–94
recalcitrant cell walls in an easy way [98]. This technique comprises of
two main parts which are shaking vessels and agitated beads [112].
Cells are disrupt through the shaking vessels which usually make up of
multiple containers or wall-plates on a vibrating platform, whereas
agitated beads rupture the microalgae cells by a rotating agitator in a
fixed vessels consisted with beads and cell culture and equipped with
cooling jacket to prevent the denaturing of protein [112]. Microalgae
Chlorococcum sp. stock cultures were mixed with two different glass
beads (1 mm diameter) with volumetric ratio of glass beads to sus-
pension cells of either 1:3 or 1:2 [55]. The mixtures were placed inside
a blender type beaker and grinded for 4 min at full impeller speed to
rupture the Chlorococcum sp. microalgae. Cell disruption was found to
be more pronounced in the presence of increasing bead loadings
probably due to higher solid surface area leading to more frequent
collision of the beads within the cell wall [55]. In order to increase the
product efficiency, bead beating is commonly combined with other
pretreatments in the production of bioethanol from microalgae. In a
previous study conducted by Kim et al. [79] reported combination of
pretreatment bead beating and enzyme saccharification was able to
destroy the cell wall of C. vulgaris to release the glucose and produced a
maximum sugar conversion yield of 69.9% at 72 h. In comparison of the
sugar conversion rate at 72 h, the beat milling samples were 25% more
efficient than the non-pretreated (control) samples.
3.3.2. High pressure homogenization (HPH)
High pressure homogenization (HPH) is a mechanical cell disrup-
tion process through high-pressure impingement of accelerated cellular
jet on the stationary valve surface with the assistance of pressure-drop-
induced shear stress on the cell suspension [55]. High pressure homo-
genization process was used to produce high shear stress on Chlorella sp.
cells under optimal condition at a pressure of 206.84 MPa with two
cycles. By-product of Chlorella sp. biomass was then added with fer-
mentative microorganism Saccharomyces cerevisiae cultures to produce
bioethanol and obtained an ethanol yield of approximately 47.3% [32].
Halim et al. [55] reported 200 mL of Chloroccum sp. suspension was
homogenized by HPH at 2.5 kW, 85 MPa for 6 min had achieved a
disruption efficiency of 80% via cell count measurement. Both experi-
ments indicated that high-pressure homogenization process is one of
the effective pretreatment methods to break the cell wall. However, the
disadvantage of this technique is it depends on the controllable para-
meters such as the pressure applies on the medium.
3.3.3. Ultrasonication
The energy of high frequency acoustic waves initiates a cavitation
process and a propagating shock wave, results the formation of jet
streams in the surrounding medium of the biomass during ultra-soni-
cation treatment leading to the cell rupturing by high shear forces [53].
An ultra-sonication pretreatment had been conducted on the micro-
algae biomass Scenedesmus obliquus suspended in 5 mL water under
condition at 200 W, 30 s followed by 10 min ice bath for 5 cycles re-
sulted conversion of complex sugars to fermentable sugars which
yielded 0.025 g glucose/g biomass [103]. Furthermore, Byong-Hun
Jeon & Kim [20] have conducted a study of ultrasonication with the
used of microalgae as feedstock. Scenedesmus obliquus YSW15 was so-
nicated for 10, 15 or 60 min at 45 °C and 60 °C, respectively at a con-
stant frequency of 40 kHz and an output power of 2.2 kW. The ultra-
sound energy was applied continuously mode obtained significant
result at 15 min sonication treatment increased the dissolved fraction of
total carbohydrates from 3% to 32% and also increased the production
of ethanol during microbial fermentation [20].
3.4. Combined pretreatment
Physical methods require higher energy consumption and complex
operations [97]. Chemicals methods are widely used but they will
produce many inhibitors after pretreatment which interrupt the
C.K. Phwan et al.
Fermentative
Microalgal biomass
microorganisms
Bead beating
Solid shear
High pressure
Mechanical Liquid shear homogenization
cell (HPH)
disruption
Wave Ultrasonication
Heat Autoclave
Fig. 3. Integration of process steps in microalgae to bioethanol via different mechanical pretreatment.
following fermentation process. Even though enzymatic hydrolysis has
its inherent advantages however, the costs and highly specificity en-
zyme applied to certain algal may hinder its commercial application
[160]. Compared with the aforementioned, there were some reports in
literatures applied the combined pretreatment to study the efficiency of
bioethanol production. A summary of several studies about the com-
bined pretreatment and the results are stated in Table 3.
Combined sonication and enzymatic hydrolysis on microalgal
Chlamydomonas mexicana to determine the efficiency of bioethanol
production via SHF and SSF was conducted by El-Dalatony et al. [44].
Finding of this study indicated that 88.2% theoretical bioethanol yield
was obtained after 48 h SSF process. After that, SSF process was se-
lected to optimize the bioethanol yield through repeated-batches by
using immobilized yeast cells. A total energy recovery of 85.81% was
achieved from the microalgal biomass in the form of bioethanol. In
another study, enzymatic and dilute acid pretreatments were used to
convert the carbohydrates of the residual biomass Chlorella sp. KR-1
into bioethanol [89]. After lipid extraction, the residual biomass from
Chlorella sp. KR-1 was reused for saccharification through simple en-
zymatic with Pectinex at pH 5.5 and 45 °C then chemical pretreatment
with 0.3 N HCl at 121 °C for 15 min through SHF process. 0.16 g
ethanol/g residual biomass was produced through this process.
Furthermore, another combined pretreatment study was conducted
to examine the enzymatic hydrolysis of Chlorococum humicola with the
used of cellulase from Trichoderma reesei, ATCC 26,921 after cell dis-
ruption by ultra-sonication [57]. Ultrasonication was used to destroy
the cell wall of the microalgae followed by the hydrolysis at different
range of temperatures (28–60 °C), pH within the range of 2.5–7.5 for
72 h and substrate concentrations with constant enzyme dosage (20 mg
cellulase). The highest glucose yield in this study was 64.2% (w/w) at
40 °C, pH 4.8 and a substrate concentration of 10 g/L of microalgal
biomass. Moreover, Karatay et al. [72] had employed different com-
bined pretreatments such as sonication, temperature with pressure, acid
hydrolysis with and without autoclaving in order to find out the most
appropriate pretreatment process to optimize the cell disruption on
halophilic microalgae Dunaliella sp. for bioethanol production. The
most effective combined method was 1% H2SO4 with autoclaved which
produced 0.91 ± 0.05 g/L bioethanol after 72 h fermentation with
30 g/L microalgae loading.
Keris-Sen & Gurol [74] pretreated mixed microalgal cultures
86
Energy Conversion and Management 173 (2018) 81–94
Bioethanol
Fermentation Distillation
predominantly Chlorococcum sp. at 0.25–2.0 g O3 g/L dry weight bio-
mass (DWB) with different ozone doses and different enzyme con-
centrations. The results obtained showed the highest glucose yield of
80.6% (w/w) of total carbohydrates under the optimum conditions of
2.5 g/L biomass concentration after 4 h hydrolysis time with 0.5 g O3 g/
L DWB ozone dose and 1.2 mL−1 g−1 DWB enzyme concentration. This
study shows that ozone pretreatment with enzymes saccharification
attain a much higher glucose yield compare to the use of enzymes only.
In addition, hydrothermal fractionation combined with enzymatic
method by Kim et al. [78] resulted in a production of 11.8 g ethanol/L.
Eighteen identified microalgae strains collected from coastal water
Pearl River Delta were selected for further biomass and ethanol pro-
duction [54]. The study indicated that dilute acid and cellulase from
Trichoderma resei treated S. abundans PKUAC 12 biomass was the su-
perior feedstock for bioethanol production resulted 0.103 g ethanol/ g
dry weight algae [54]. On the other hand, study of microalgae Scene-
desmus bijugatus residues which consisted 26% of carbohydrates content
performed the pretreatment of autoclave and acid hydrolysis (H2SO4)
with various concentrations 1–3% (v/v), at 130 °C for 45 min had ob-
tained an ethanol production of 0.158 g ethanol/g residual biomass
[148].
In addition, another study showed liquid hot water (LHW) com-
bined with enzymatic hydrolysis was a reliable approach to destroy the
cell structures to produce bioethanol from microalgae [110,160].
Deionized water was added to 0.8 g of lyophilized microalgae (Scene-
desmus sp. WZKMT) according to various solid-to-liquid ratios (w/v)
1:5, 1:10, 1:15, and 1:20. Then, the mixtures were maintained at dif-
ferent temperatures (100, 120, 140, 160, 180 and 200 °C) for different
timings of 20, 40, 60, and 80 min [160]. After LHW pretreatment, en-
zymatic hydrolysis was continued to pretreat the mixtures which
achieved a result in concentration and recovery of glucose of 89.32%
under an optimal condition at 147 °C, 41 min with solid-to-liquid ratio
at 1:13 (w/v).
4. Bioethanol production through fermentation process
Fermentation is a metabolic conversion activity of monosaccharides
to bioethanol and other by-products in the presence of fermentative
microorganism in supporting conditions namely temperature and pH
range [44]. An introduction of specific fermentation agent such as yeast
C.K. Phwan et al. Energy Conversion and Management 173 (2018) 81–94

or bacteria is commonly used in the fermentation process. These fer-

References

(J. K. [78]
mentative microorganisms including S. cerevisiae, P. stipitis, Kluyver-

[148]
[160]
[44]

[89]

[57]

[72]

[74]

[54]
omyces fragilis, K. marxianus, E. coli, Klebsiella oxytoca, and Z. mobilis.
Bioethanol derived from microalgae can be produced through micro-

Highest glucose yield was 64.2% (w/

Highest glucose yield was 80.6% (w/

0.91 ± 0.05 g/L ethanol production
algal photosynthesis and intracellular anaerobic fermentation [38].

0.158 g ethanol/ g residual biomass

0.16 g ethanol/g residual biomass
Some of the starch-accumulating microalgae such as C. reinhardtii,
ii. 9.64 g/L ethanol production

Concentration and recovery of

i. 10.5 g/L ethanol production

glucose 14.223 g L−1, 89.32%

0.103 g of ethanol/g biomass
Chlamydomonas moewusii, C. vulgaris, Chlorococcum littorale, and Spir-

w) of total carbohydrates
ulina sp. that are capable expelling ethanol through the cell by means of
intracellular process under anaerobic condition in the absence of light
[21,143]. Theoretically, 1 kg of glucose and xylose can produce 0.49 kg

11.8 g ethanol/L
of CO2 with ethanol yield of 0.51 kg [4]. Microorganisms E. coli, Z.
mobilis and S. cerevisiae have promising ethanol production with high
Results

efficiency and widely utilization in microalgae fermentation, especially

Saccharomyces [21]. Some of the research studies conducted with dif-
w)

ferent fermentative microorganisms during fermentation process from

72 h, 120 rpm repeated batch fermentation for 7
ii. S. cerevisiae with Ca-alginate, SSF, pH 5, 30 °C,
Sonication: frequency 40 kHz, 2.2Kw, 15 min and enzymatic: ≥1 i. S. cerevisiae, SSF, pH 5, 30 °C, 72 h, 120 rpm

microalgae are stated in Table 4.

Enzymatic: 0.8 mL enzyme/g residual biomass, pH 5.5, 45 °C and S. cerevisiae, SHF, pH 6, 30 °C, 20 h, 180 rpm

4.1. Fermentative microorganisms for bioethanol production from

microalgae
E. coli, SSF, 37 °C, 72 h, 150 rpm

4.1.1. Yeast
Yeast can be defined as ascomycetous or basidiomycetous fungi
S. cerevisiae, pH 6, 72 h
Fermentation process

whose asexual growth by budding or fission reproduction which do not

30 °C, 48 h, 200 rpm
S. cerevisiae, pH 5.5,

form their sexual states within or upon a fruiting body [86,104]. Yeasts
such as Saccharomyces cerevisiae and Schizosaccharomyces pombe are
S. cerevisiae

well known as crabtree-positive yeast because they could produce

cycles

ethanol in the presence of oxygen [117]. There are some other yeasts
strains such as Candida shehatae, Kluyveromyces marxianus, Kluyver-
–

4 h for 0.5 g O3 g/L dry weight biomass (DWB) ozone dose and –

LHW: 1:13 (w/v),147 °C, 40 min and enzymatic: 0.125% (w/v), –

omyces fragilis, Pichia stipitis, Pachysolen tannophilus are use in fermen-

115.5 °C, 46.7 min reaction time, 25% (w/w) solid loading and
Ultrasonication: frequency at 40 kHz, 130 W, for 25 min and

tation process [42,104,105,119]. In contrast to glucose fermentation,

Autoclave and chemical (H2SO4) Autoclave: 121 °C, 15 min and chemical: 1% H2SO4, 121 °C,

Chemical: 3% H2SO4, 110 °C, 30 min and enzymatic: 10 mg

native S. cerevisiae cannot ferment pentoses but xylose-fermenting

enzymatic: 14,000 α-amylase units (AAU)/g and 350

yeasts such as P. stipites is able to ferment xylose, glucose, mannose,

Autoclave and chemical (H2SO4) Autoclave and chemical: 2% H2SO4, 130 °C, 45 min.
αmylase and 10 mg glucoamylase, pH 5.5, 30 min.

galactose and cellobiose with high ethanol yield [84]. However, the
1.2 mL−1 g−1 DWB, pH 4.8, 50 °C, 4 h, 160 rpm

overall ethanol yields and productivity from xylose-fermenting yeasts

Different types of combined pretreatment from microalgae and the results obtained from various studies.

are much lower than glucose fermentation by S. cerevisiae [33]. In ad-

0.02 g enzyme/g substrate, pH 4.5,40 °C
unit per mg of solid, pH 5.0, 1 h, 50 °C

dition, the evolution of the metabolic engineering and current re-

chemical: Dilute HCl, 121 °C, 15 min

combinant DNA techniques had overcome this xylose obstacle situation

in S. cerevisiae [33,104,126]. To date, there are still limited literatures
pH 4.5, 37 °C, 48 h, 150 rpm
glucoamylase units (GAU)/g

related to the production of bioethanol from microalgae with other

yeasts strains except for S. cerevisiae.
S. cerevisiae has been generally recognized as safe (GRAS) to use by
FDA and nonpathogenic for human [43,108]. It is widely used in in-
Hydrothermal:

dustrial activity due to its ability to convert hexoses into bioethanol,

enzymatic:

enzymatic:
Condition

high tolerance to bioethanol and other unwanted inhibitory compounds

15 min

[11]. Kim et al. [77] conducted a study between seawater Porphyridium

cruentum (SPC) and freshwater Porphyridium cruentum (FPC) with dry
yeast S. cerevisiae via separate hydrolysis and fermentation (SHF) and
Enzymatic and chemical (HCI)

Ultrasonication and enzymatic

Pretreatment/Saccharification

Hydrothermal and enzymatic

simultaneous saccharification and fermentation (SSF). The result of the

Liquid hot water (LHW) and
Sonication and enzymatic

study revealed that SSF process was more efficient than SHF process for
Chemical (H2SO4) and
Ozone and enzymatic

bioethanol production from both SPC and FPC with the yielding of 2.77
and 2.98 mg/mL, respectively after an 9 h fermentation process [77].
On the contrary, mixed culture Chlorella vulgaris YSL001 and Uronema
belkae YSL010 were pretreated with ultra-sonication, heat and enzy-
enzymatic

enzymatic

matic hydrolysis. Then, fermentative bacterial and Dekkera bruxellensis

ATCC34447 yeast were added in different fermenters. The outcomes of
the fermentation process shows a higher ethanol yield by using yeast as
fermentative microorganism when comparing to mixed bacterial cul-
Scenedesmus abundans PKUAC 12
(dominated by Chlrococcum

ture [66]. The microalga Scenedesmus obliquus was used for fermenta-
tion with three yeast strains: Kluyveromyces marxianus IGC 2671, Sac-
Chlamydomonas mexicana

Mixed microalgal culture

Scenedesmus sp. WZKMT

charomyces carlsbergensis ATCC 6269 and Saccharomyces bayanus [103].

Chlorococum humicola

Scenedesmus bijugatus

The lowest ethanol concentration produced by S. bayanus (9 g/L) while

Schizocytrium sp.

K. marxianus and Saccharomyces carlsbergensis achieved higher ethanol

Dunaliella sp.
Chlorella sp.
Microalgae

yields, 11.7 g/L and 11.2 g/L, respectively.

KR-1

sp.)

Karatay et al. [72] reported microbial growth and fermentation

Table 3

rates were greatly influenced by pH value of growth medium and it

indirectly affected the bioethanol productivity. Furthermore, four

87
C.K. Phwan et al. Energy Conversion and Management 173 (2018) 81–94

Table 4
Comparison of ethanol yield from various microalgae by using different fermentative microorganisms.
Microalgae Pretreatment Fermentative Fermentation condition Maximum ethanol production References
microorganism

Chlorococum sp. Supercritical fluid S. bayanus 30 °C, 60 h, 200 rpm 3.83 g/L [58]
Chlorococcum infusionum Chemical (NaOH) S. cerevisiae Process: SHF, 72 h, 0.26 g ethanol/g algae [59]
200 rpm
Chlamydomonas reinhardtii UTEX Enzymatic S. cerevisiae S288C Process: SSF 235 mg ethanol/g algae [31]
90 30 °C, 40 h, 160 rpm
Chlorella Chemical (HCI and S. cerevisiae Y01 30 °C, 48 h, 200 rpm 22.60 g/L [163]
MgCI2)
Chlorella variabilis Viral and enzymatic E. coli KO11 pH 6.5 0.326 g/g carbohydrate consumed [27]
35 °C, 3 days, 150 rpm
Chlorella vulgaris Chemical (H2SO4) E. coli SJL2526 Process: SHF 0.4 g ethanol/g algae [90]
pH 7.0, 37 °C, 170 rpm
Chlorella vulgaris FSP-E Chemical (H2SO4) Z. mobilis Process: SHF 11.66 g−1 [64]
pH 5–6,
30 °C within 12 h
Porphyridium cruemtum Enzymatic S. cerevisiae KCTC 7906 Process: SSF 2.77 mg/mL (seawater) and 2.98 mg/mL [77]
pH 4.8, 37 °C, 9 h (freshwater)
Scenedesmus obliquus CNW-N Chemical (H2SO4) Z. mobilis ATCC29191 Process: SHF 8.55 g/L [64]
pH 6,
30 °C within 4 h

different types of microorganisms (Saccharomyces cerevisiae, Brettano-
myces custersainus, Pichia stipitis, and Klebsiella oxytoca) were studied to
compare the bioethanol production from microalgae Microcystis aeru-
ginosa in a shaking incubator under 25 °C at 150 rpm for 30 h. Results
showed the highest ethanol content was obtained by S. cerevisiae in the
fermented samples [75]. Even though S. cerevisiae was able to convert
many fermentable sugars into ethanol efficiently but it was not able to
ferment pentoses. In contrary, Brettanomyces custersainus was capable to
convert pentoses and Pichia stipitis was able to convert xylose to ethanol
faster than any other microorganisms [75,84].
4.1.2. Bacteria
The most promising ethanologenic bacteria for industrial exploita-
tion are Escherichia coli, Klebsiella oxytoca and Zymomonas mobilis [11].
Table 5 shows comparison of the advantages and disadvantages of E.
coli, K. oxytoca and Z. mobilis in bioethanol production. E. coli has
several advantages in biofuels production due to its high growth rates,
ability to grow in low-cost mineral media in anaerobic condition, which
significantly reduces the production cost, and ability to use different
carbons sources such as carbohydrates, polyoils and fatty acids
[51,152]. Three different algae species: Undaria pinnatifid, Chlorella
vulgaris, and Chlamydomonas reinhardtii were used to study the sac-
charification conditions at several temperatures, acid concentrations,
pH value, and durations with some ethanolic E. coli W3110 strains.
Maximum ethanol yield of 0.4 g ethanol/g biomass was obtained by
pretreated C. vulgaris with E. coli SJL2526 [90]. Although E. coli was
capable to ferment a wider range of sugars than S. cerevisiae and Z.
mobilis strains but the ethanol yield was lower due to formation of other
by-products in higher rates and final concentration [155]. On the other
hand, K. oxytoca is a Gram-negative bacterium which can survive at pH
as low as 5.0 and commonly found in paper and pulp streams [11].
Recombinant strains of K. oxytoca containing Z. mobilis pdc (pyruvate
decarboxylase) and adhB (alcohol dehydrogenase) genes have been
created to enable the direct metabolism of pyruvate ethanol but it im-
poses an additional costs on the ethanol production process [120]. Most
of the studies with the utilization of K. oxytoca to produce bioethanol
are related to lignocellulosic biomass instead of microalgae biomass.
However, genetic modification in natural bacteria such as E. coli KO11
and K. oxytoca have been studied to generate new strains to ferment
both xylose and glucose for a higher ethanol production [161].
Z. mobilis is well known for its ability to produce bioethanol rapidly
from glucose-based feedstock and able to achieve 5% higher yields and
up to five-fold higher volumetric productivity when comparing with
traditional yeast fermentation in comparative performance trials [11].
Furthermore, Z. mobilis is recognized as a safe status ethanologenic
bacteria that has many industrial biocatalyst characteristics and the
investigation of DNA restriction-modification systems in Z. mobilis also
helps to increase the transformation proficiency for more control strain
development [157]. Pre-cultured Z. mobilis cells were inoculated in the
solution of Scenedesmus obliquus CNW-N after acid hydrolysis via se-
parate hydrolysis and fermentation (SHF) showed an ethanol con-
centration of 8.55 g/L which represented theoretical yield of nearly
99.8% [65]. A comparison study between Z. mobilis and S. cerevisiae
from algae Spirogyra exhibited that Z. mobilis was more effective than S.
cerevisiae to produce bioethanol which were 9.70% ethanol (v/v) and
4.09% ethanol (v/v), respectively after a 96 h fermentation process
[136].
4.2. Separate hydrolysis and fermentation (SHF)
SHF is a fermentation process based on separation of hydrolysis to
degrade the feedstock into monosaccharides continuously by fermen-
tation process with fermentative microorganism that converts fermen-
table sugars into ethanol [68]. The advantages of this process are low
cost of chemicals, short residence time and simple equipment system
[92]. SHF can produce ethanol at optimum temperature independently
but the accumulation of glucose and cellobiose during hydrolysis have
inhibitory effects that result in the formation of end-product inhibition
as well as high probability of unpreventable contamination
[68,96,155]. Furthermore, inhibition of cellulose and ß-glucosidase
enzyme by glucose during hydrolysis led to lower solids loadings and
higher enzyme loadings to obtain a reasonable yields [9]. Additional
neutralizing and purification steps need to be done to prevent the for-
mation of unfavorable by-products during hydrolysis process [87].
Therefore, the disadvantage of SHF result in the development of Si-
multaneous Saccharification and Fermentation (SSF) process.
4.3. Simultaneous saccharification and fermentation (SSF)
In contrast with SHF, SSF conducts both hydrolysis and fermenta-
tion simultaneously in single step in a single reactor. The feedstocks,
enzyme and yeast were added together in an orderly manner to release
the fermentable sugars rapidly and convert them into bioethanol [68].
This process is always effective when combined with dilute acid or high
temperature water pretreatment. In SSF, carbohydrate polymers are
convert to fermentable sugars by cellulases and xylanases [9]. SSF

88
C.K. Phwan et al.
Table 5
Comparison of the advantages and disadvantages of E. coli, K. oxytoca and Z. mobilis in bioethanol production.
Species Advantages
Escherichia coli – capability to ferment a wider range of sugars (pentose and glucose) than S. cerevisiae
and Z. mobilis strains [155].
– amenability for genetic modification to generate more ethanol yield [161].
Klebsiella oxytoca – tolerant to pH range as low as 5.0 [11].
– amenability for genetic modification to generate more ethanol yield [161].
Zymomonas mobilis – 5% higher ethanol yields and up to five-fold higher volumetric productivity of ethanol
than traditional yeast fermentation [11].
– High tolerance of ethanol yield which up to 14% (v/v) [3].
process requires a compatible condition with similar pH, temperature
and optimum substrate concentration [12]. From a comparative study
between SHF and SSF conducted by El-Dalatony et al. [44], SSF showed
a greater efficiency of fermentation process than SHF for bioethanol
production from microalgae Chlamydomonas Mexicana with production
of 10.5 g/L ethanol and 8.48 g/L of ethanol, respectively. Moreover,
Kim et al. [77] reported the red microalgae Porphyridium cruentum
showed that SSF was more efficient than SHF for bioethanol production
from both seawater P. cruentum and freshwater P. cruentum with
ethanol conversion of 65.4% and 70.3%, respectively. Many studies
stated SSF was more preferable than SHF due to its ability to reduce the
cost (required only a small amount of enzyme), processing time, less
contamination, low inhibitory effects and high ethanol production rate
[4,36,68,133].
4.4. Simultaneous saccharification and co-fermentation (SSCF)
SSCF involves processes for both hemicellulose sugars (pentose) and
cellulose sugars (hexose) in which hydrolysis and fermentation occur
simultaneously in a single reactor [60]. This process is recommended
when significant involvement of the C5 sugars (pentoses) are found
after hydrolysis [155]. Genetically engineered fermentative micro-
organisms S. cerevisiae and Z. mobilis are generally used to co-ferment
both glucose and xylose in SSCF process [39]. Based on evaluation from
simulation technical routes on experimental information of the software
ASPEN PLUS 7.1, the simulation results showed SSCF route achieved
the highest bioethanol yield of 23.6% in comparison to SSF route and
SHF route which provided an ethanol yield of 20.1% and 18.5%, re-
spectively [115]. Thus, SSCF is able to perform the hydrolysis and co-
fermentation of pentose and hexose sugars in one vessel without lim-
iting ethanol production from cellulosic biomass [92]. SSCF are able to
ferment glucose and pentoses in same reactor while the fermentation of
SSF is separated from pentoses but both processes have a short overall
process of enzymatic hydrolysis, low cost and higher ethanol yield than
SHF [104].
4.5. Dark fermentation
Dark fermentation is generally used to convert organic substrates
into biohydrogen [21]. Complex organic polymers presence in the fer-
mentative and hydrolytic microorganisms will be breakdown into
monomers through hydrolysis, then converted into a mixture of organic
acids with low molecular weight and alcohols, mainly acetic and
ethanol [21]. The degradation of intracellular starch into pyruvate is
accomplished through a metabolic pathway called Embden-Meyerhof-
Parnas and pentose phosphate pathway by using pyruvate decarbox-
ylase and alcohol dehydrogenase [34]. There are many microalgae and
cyanobacteria that are able to expel ethanol through the cell wall
during intracellular process without light. Examples included C. re-
inhardtii, Chlamydomonas moewusii, C. vulgaris, Oscillatoria limnetica,
Oscillatoria limosa, Gleocapsa alpícola, Cyanothece sp., Chlorococcum
89
Energy Conversion and Management 173 (2018) 81–94
Disadvantages
– ethanol yield was lower due to formation of other
fermentation products [155].
– narrow pH and temperature growth range [3].
– produce organic acids and low tolerance to ethanol [144].
– lower ethanol yield than E. coli [40].
– cannot ferment xylose sugars and low tolerance to inhibitors
[50].
littorale, Spirulina sp. and Synechococcus sp. [21,111,143]. Algal cells
that have high concentration of carbohydrates content are more sui-
table for dark fermentation process fall under the classes of Chlor-
ophyceae (include Chlamydomonas and Chlorella), Prasinophyceae,
Cryptophyceae and Cyanophyceae [71]. An early study done by Hirano
et al. [62] obtained an ethanol yield approximately 1% (w/w) from
dark fermentation of Chlamydomonas reinhardii with optimal condition
of pH 7–8 at a temperature of 25–30 °C. In another study of ethanol
production by dark fermentation from marine green algae Chlorococcum
littorale was reported by Ueno et al. [143]. From the observation, cel-
lular starch was decomposed at higher temperatures after dark anae-
robic incubation with maximum ethanol yield of 450 µmol/g of dry
weight at 30 °C. In addition, it was stated ethanol productivity can be
increased by addition of methyl viologen but drastically decreased of
hydrogen formation [143]. However, dark fermentation to produce
ethanol from microalgae is still not as popular as other fermentation
methods which can produce higher ethanol yield. In fact, dark fer-
mentation to produce ethanol is less efficient due to influence of light
and oxygen [71].
4.6. Photofermentation
The “photofermentative” is a natural mechanism to convert sunlight
into fermentation product through a metabolic pathway of ethanol
synthesis that can be summarized as the conversion of phosphoglyce-
rate to pyruvate by two enzymes [21]. Pyruvate decarboxylase (PDC)
and alcohol dehydrogenase (ADH) are the two enzymes which help to
produce acetaldehyde as intermediate then converted into ethanol after
inorganic carbon fixation by Calvin cycle [21]. The study of the influ-
enced hybrid system (in which the effluent of C. reinhardtii containing
organic acid was used as substrate to R. capsulatus) and co-culture (C.
reinhardtii and R. capsulatus) on the photofermentation showed the
maximum of ethanol content obtained was 19.94 ± 2.67 g/L
(0.17 ± 0.02 g/L h) [34].
4.7. Consolidated bioprocessing (CBP) for ethanol production
Consolidated bioprocessing is an integration process which com-
bines production of saccharolytic enzymes, pretreatment, saccharifica-
tion and fermentation steps in a single reactor and mediates by a single
microorganism or microbial consortium [145,150,161]. This poten-
tially reduces the operational costs and achieve a higher process effi-
ciency [39,46]. Some fungi such as Fusarium oxysporum, Neurospora
crassa and Paecilomyces sp are said to be practicable for CBP [4].
However, no natural microorganism has been reported that can sin-
gularly cover all the features desired for CBP [145,156]. Therefore, this
is important to develop an organism to compromise all the features
during process [61]. Thermophilic bacteria such as Clostridium ther-
mocellum has an optimal growth temperature at 65 °C are believed
practicable as they have both cellulolytic and ethanologenic char-
acteristics to survive under high temperature condition [46,61].
C.K. Phwan et al.
According to Xu et al. [156], C. thermocellum JYT01, a gram-positives
anaerobic and thermophilic bacterium had reached its optimum con-
dition of growth at temperature 65 °C with pH ranging 6.5–7.4 yielded
an ethanol concentration of 491 mM. This finding demonstrate the
potential advantages of cellulolytic enzymes in bacteria and cellulo-
some activity in bioethanol production. CBP technique is not highly
recommended due to intolerant of high ethanol yields in the typical
microorganism employed [145]. Thus, further research focus on re-
combinant strategies on cellulolytic and solventogenic microorganism
through microbial engineering to improve the applicability to this
technique is strongly recommended [1,150]. CBP technique is still a
infancy stage of establishment. It is popular to use for bioethanol pro-
duction from lignocellulosic biomass instead of microalgae biomass,
which no study has been reported to use this techniques for ethanol
conversion from microalgae [120].
5. Economic and environmental impacts of bioethanol produced
from microalgae
Microalgae have driven the economic impacts in biotech, food,
pharmaceutical, agriculture and cosmetic industries [22]. Bioethanol
production from microalgae require less energy consumption compared
with biodiesel production and the ethanol yields are more comparable
to those from sugary or lignocellulosic substrates [7].
5.1. Economic sustainability among the industries
Microalgae are getting more popular among the industries espe-
cially biofuels. A systematic supply chain is essential to maximize the
profitability of algae biofuel [15]. Bioethanol obtained from microalgae
may offer a suitable alternative way to replace petroleum-based fuels in
the future [134]. Biofuels such as diesel, gasoline and bioethanol pro-
duce from microalgae can be used for transportation or directly trans-
ported for electricity generation [102]. Bioethanol has more efficiency
advantages over gasoline such as higher octane number (1 0 8), wider
flammability limits, greater flame speeds and higher heat of vaporiza-
tion that allow for a higher compression ratio and shorter burning time
[10]. In U.S. and Australia, Algae Biofuels operated by PetroSun was set
up in June 2006 to investigate about the production of biodiesel,
ethanol, methanol methane and hydrogen from microalgae [133]. The
company also provided feedstock, half or up to 150 million gallons per
year to another company BioAlternatives. Furthermore, a total of 8000
gallons of liquid biofuel per acre per year were generated by Algenol in
U.S. which is a company started bioethanol production from algae in
2006 by using algae feedstocks, sunlight, CO2 and saltwater [15,133].
In California U.S., Sapphire Energy, Inc was founded in 2007 with an
investment greater than $100 million to produce 100,000 gallons/year
of ethanol in fuel-grade standard [15]. The development of the algae
biofuel market was forecasted to increase by year 2030 and will dom-
inate 75% of the market share [49,151].
5.2. Environmental influences of bioethanol from microalgae
Scarcity and inconsistent price of fossil hydrocarbons are emerging
issues. Production of biofuels from renewable supplies especially from
algae is an ingenious notion that can be thought in relation to economic
viability and ecological sustainability. Biofuel produced from micro-
algae has a high performance to serve as a bioresource due to its ability
to carry out plant type photosynthesis to convert solar energy into
chemical energy through utilization of CO2 [56]. The reduction of CO2
level provides energy facilities with zero or almost zero emissions on
both air contaminants and greenhouse gasses [15]. In addition, a study
evaluated the emissions of bioethanol in different spark ignition (SI)
engines found out the carbon monoxide (CO) emissions decreased when
using blended ethanol fuel in SI engine [140]. Bioethanol-blended fuel
for automobiles can reduce the reliance on petroleum and cut down the
90
Energy Conversion and Management 173 (2018) 81–94
greenhouse gas emission from vehicles [11]. Bioethanol has been con-
sidered as eco-friendly biofuel because it can reduce the interfacial
tension of petrol with reverence to water that allows the ethanol-ga-
soline non aqueous phase liquid (NAPL) to pass through minor pore
spaces and penetrate easily from vadose zone to water table under-
ground [15].
5.3. Challenges and future outlook of algae bioethanol
Bioethanol has garnered popularity in both developing and devel-
oped countries since the utilization of fossil petrol caused harm to the
global environment and oil reserves [4]. However, one major barrier is
the inconsistency of technologies to commercialize the bioethanol
production required further investigation in this direction [8]. Fur-
thermore, bioethanol production from microalgae are also facing some
hurdles such as high capital cost of facilities, lack of implementation of
relevant policies and insufficient government support in the commer-
cialization process [15,68]. Researchers are still focusing on the im-
provement of algae bioethanol technologies and looking for a better
transgenic algae strain to obtain reproducible results [132].
A concept known as “fourth-generation algal biofuels” or “photo-
synthetic biofuels” which make use of synthetic biology of algae and
cyanobacteria. Most of the fourth-generation biofuels research made
are focused on the photobiological solar fuel production depends on
unicellular algae and cyanobacteria [2]. It involves in a direct appli-
cation of photosynthesis for the generation of fuels and chemicals
through a metabolic engineering process where a single photosynthetic
microorganism can be acted as catalysts and processors to synthesize
and secretes ready to use products with high photosynthesis efficiency
[101]. The basis of the fourth-generation biofuel production of algae
metabolic engineering uses recombinant DNA and bioengineering
methods to directly modified the cellular metabolism through in-
troduction, deletion, and/or modification of algae metabolic networks
to enhance the biofuel production [94]. This significant approach
provides high biofuel productivity and more economic sustainability by
cutting the biomass separation and processing costs as compared to
traditional approach [37]. Nonetheless, fourth-generation biofuel is
claimed to be carbon negative rather than carbon neutral as it captures
more waste CO2 than it produces [2]. The study of fourth-generation
algal biofuels is relatively scarce and the metabolic engineering in-
formation of its technical performance is very inadequate [93].
6. Conclusions
Microalgae appear to be one of the prominent feedstock to produce
biofuels recently. The pretreatment techniques to enhance biodegrad-
ability to simple sugars has been the subject of intensive research cur-
rently with a focus on increasing bioethanol yield at the lowest eco-
nomic and environmental costs. Some challenges in chemical
pretreatments include recovery waste from reactions, environmental
pollutions from solvents and by products. Even though application of
enzymes in pretreatment can produce higher yield of ethanol compared
to other pretreatments but the main obstacle for this method is the
production cost of ethanol will be increased due to the high cost of
enzymes. The process to generate this green energy still exhibited some
limitations such as the selection of the microalgae strains, optimization
of pretreatment to disrupt the cell, selection of fermentative micro-
organism and the cost of scale up the processes. In order to select the
desirable algae strains for commercial biofuel production, high biomass
yield with high carbohydrates and lipid contents are taken into con-
sideration [63]. Addressing this problem might require intensive re-
search in biotechnology, genetic modification and metabolic en-
gineering based on evolutionary engineering to enhance the lipids and
cellulose synthesis pathway in microalgae and the tolerance of fer-
mentative microorganisms to increase ethanol productivity [69]. Lack
of efficient establishment of technologies and findings is one of the
C.K. Phwan et al.
obstacle to expand the commercial value of algae bioethanol. Both
economic and sustainability barriers impede the commercial produc-
tion of bioethanol from microalgae for fuel market application. Al-
though the potential to develop bioethanol production from microalgae
is bright and valid, but it will be more important to develop sustainable
and eco-friendly biofuels in a more economically viable and compatible
worldwide way.
Acknowledgement
This study is supported by the University of Malaya under MRSA-
FRGS (MO014-2016), Fundamental Research Grant Scheme (Malaysia,
FRGS/1/2015/SG05/UNIM/03/1), the Prototype Research Grant
Scheme (PRGS/2/2015/SG05/UNIM/03/1) and SATU Joint Research
Scheme (RU018J-2016, RU018L-2016, RU018O-2016 and RU018C-
2016).
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