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Critical Reviews in Biotechnology
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Progress in physicochemical parameters of
microalgae cultivation for biofuel production
Nazia Hossain & Teuku Meurah Indra Mahlia
To cite this article: Nazia Hossain & Teuku Meurah Indra Mahlia (2019): Progress in
physicochemical parameters of microalgae cultivation for biofuel production, Critical Reviews in
Biotechnology, DOI: 10.1080/07388551.2019.1624945
To link to this article: https://doi.org/10.1080/07388551.2019.1624945
Published online: 11 Jun 2019.
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REVIEW ARTICLE
Progress in physicochemical parameters of microalgae cultivation for
biofuel production
Nazia Hossain
a
and Teuku Meurah Indra Mahlia
b
a
Department of Civil and Infrastructure Engineering, School of Engineering, RMIT University, Melbourne, VIC, Australia;
b
School of
Information, Systems and Modelling, Faculty of Engineering and Information Technology, University of Technology Sydney, Sydney,
NSW, Australia
ABSTRACT
Microalgae have been exploited for biofuel generation in the current era due to its enormous
energy content, fast cellular growth rate, inexpensive culture approaches, accumulation of inor-
ganic compounds, and CO
2
sequestration. Currently, research is ongoing towards the advance-
ment of the microalgae cultivation parameters to enhance the biomass yield. The main objective
of this study was to delineate the progress of physicochemical parameters for microalgae cultiva-
tion such as gaseous transfer, mixing, light demand, temperature, pH, nutrients and the culture
period. This review demonstrates the latest research trends on mass transfer coefficient of differ-
ent microalgae culturing reactors, gas velocity optimization, light intensity, retention time, and
radiance effects on microalgae cellular growth, temperature impact on chlorophyll production,
and nutrient dosage ratios for cellulosic metabolism to avoid nutrient deprivation. Besides that,
cultivation approaches for microalgae associated with mathematical modeling for different
parameters, mechanisms of microalgal growth rate and doubling time have been elaborately
described. Along with that, this review also documents potential lipid-carbohydrate-protein
enriched microalgae candidates for biofuel, biomass productivity, and different cultivation condi-
tions including open-pond cultivation, closed-loop cultivation, and photobioreactors. Various
photobioreactor types, the microalgae strain, productivity, advantages, and limitations were tabu-
lated. In line with microalgae cultivation, this study also outlines in detail numerous biofuels
from microalgae.
ARTICLE HISTORY
Received 20 December 2018
Revised 21 April 2019
Accepted 1 May 2019
KEYWORDS
Physicochemical
parameters; cultivation
approaches; microalgae;
microalgal biofuel
Introduction
Microalgae are referred as single or multicellular micro-
organisms that can be found abundantly, in the pres-
ence of moisture all over the world. Previous studies
indicated that the favorable microalgae cultivation
sources could be fresh water, saline water, or waste-
water. Over the last few decades, microalgae have
encouraged substantial attention for biofuel production
due to their high content of cellular carbohydrates,
lipids, and proteins [1,2]. According to the microalgae
cellular study, lipid content is the main driving factor to
produce biodiesel, while proteins and carbohydrates
are the key components for bio-oil and bioethanol
production, respectively [3–6]. Table 1 tabulates several
microalgae species that contain adequate amounts
of carbohydrates, lipids, and proteins for bio-
fuel production.
Driven by the necessity to find alternative sources
for biofuel generation, the microalgae cultivation
approach is being emphasized over energy crops owing
to higher energy-efficiency, cost-effectiveness, and
lower land-water footprints [16,17]. Cultivation of
microalgae is a unique type of cultivation approach
based on the different criteria of land availability, oper-
ational methods, the variety of microalgae, water type,
climate, geography, sunlight availability, cultivation
cost, and other features to manufacture biofuel [18]. To
address the domination of microalgae over other
energy crops for biofuel production, it should be men-
tioned that microalgae provide higher biomass yield
and lower spatial demand than typical energy crops. In
addition, microalgae represent some other positive
aspects such as: (i) microalgae are deemed as superior
because of their sunlight uptake capability and value
added components production via photosynthesis
CONTACT Nazia Hossain bristy808.nh@gmail.com Department of Civil and Infrastructure Engineering, School of Engineering, RMIT University, GPO
Box 2476, La Trobe St., Melbourne VIC 3001, Australia
ß2019 Informa UK Limited, trading as Taylor & Francis Group
CRITICAL REVIEWS IN BIOTECHNOLOGY
https://doi.org/10.1080/07388551.2019.1624945
respiration, (ii) the total batch of microalgal biomass is
easily utilized since they contain simple reproductive
organs and no vascular bundles, (iii) along with the
massive oil production, they offer many other value
added co-products for pharmaceutical, medicinal and
agricultural purposes, (iv) microalgae strain selection,
sequencing, and isolation can easily be experimented
upon due to their simple cell structure and division
methods, (v) microalgae biomass is cultivated, even
with limited inorganic supplementation contents while
other crops rarely survive because of protein lacking in
soil, and (vi) simple technologies can be implemented
for scaling up microalgae generation at both pilot or
industrial levels without modifying a cultivation sys-
tem [19,20].
The progress of both physical and biochemical
parameters in the cultivation systems have been
reviewed in this study. Various types of microalgae cul-
tivation systems have recently been applied for diverse
purposes such as water purification, biofuel production,
nutritional and medicinal compounds extraction and
others. Therefore, an elaborated review was needed to
comprehensively discuss the cultivation system and
determine the system variation in terms of geography,
climate, timeliness, purposes, land usages, efficiency,
cost-effectiveness and others. Hence, during microalgae
cultivation conditions, physicochemical factors of culti-
vation, mostly experiment cultivation approaches, their
applications, advantages and disadvantages which are
discussed in this review in order to draw detailed
insight into microalgae production for manufacturing
commercial products [18–22]. According to a previous
study, microalgae commercialization manifested eco-
nomic and environmental conveniences with negligible
mechanical drawbacks [23]. It should be noted that
large-scale microalgae cultivation has been initiated
from the early 1960s in Japan with Chlorella species.
Later, during the 1980s, Mexico and Thailand have
established plant-scale of microalgae cultivation using
Spirulina and Chlorella species, respectively.
Subsequently, Japan, Australia, Israel, USA, and Brazil
started to industrialize the microalgae cultivation with
miscellaneous species such as Dunaliella salina,
Chlorella sp,Spirogyra,Cyanobacteria, and others [20].
Several commercial microalgae species productivity,
microalgae-based biofuel yield and processing technol-
ogies have been tabulated in Table 2.
The main objectives of this review are the biomass
productivity determination of potential microalgae for
biofuel, representation of the progress of physicochemi-
cal parameters for the cultivation, and comprehensive
elaboration of suitable biofuels from microalgae. This
study has emphasized the major cultivation conditions:
photoautotrophic, heterotrophic, mixotrophic, and pho-
toheterotrophic. In line with that, both conventional
(e.g. open-pond, closed-loop) and advanced-techno-
logical microalgae cultivation methods (e.g. big-bag,
flat-plate photobioreactor), positive and negative
aspects of each approach, suitability, and plant imple-
mentation based on their geographical position, cli-
mate, ecology, and environment have been presented
and discussed in this review. The fundamental parame-
ters for microalgae cultivation are gaseous transfers,
mixing, light demand, temperature, pH, nutrients, and
the culture period have been delineated inclusively. It
should be noted that physicochemical characteristics
are considered as the key determinants for microalgae
conversion into productive products. Therefore, micro-
algae conversion into a biofuel can be projected as a
beneficial project for basic cultivators and industries as
well as in abandoned ponds and lakes which can be
planned for suitable microalgae cultivation. The effica-
cies of cultural parameters also play a significant role
on the optimizing of overall yield for divergent microal-
gal biofuels: bioethanol, biodiesel, biobutanol, bio-oil,
biochar, syngas and bioelectricity, microbial fuel
cells and biogas (biomethane and biohydrogen),
numerous biomass conversion technologies and proc-
esses [48–56].
Cultivation approaches of microalgae
Experimental studies on microalgae cultivation
approaches in Brazil reported that each microalgae life-
cycle was determined by distinct geometries and func-
tional parameters and they were determined and
Table 1. Carbohydrates-lipids-proteins that enrich microalgae species.
Microalgae species Carbohydrates (%) Lipid (%) Protein (%) References
Chlorella sp. 12–17 14–22 51–65.2 [7,8]
Spirulina platensis 8–63 4–950–65 [7,9]
Dunaliella sp. 4–40.2 6–18.2 49–57 [7,10]
Scenedesmus sp. 10–52 12–26.7 50–56 [7,11]
Spirulina maxima 13–16 0.9–7 56.6–71 [7,12]
Chlamydomonas sp. 2.9–17 14–65.8 43–56 [7,13]
Spirogyra 33–64 5.23–21 12–24.4 [7,14,15]
2 N. HOSSAIN AND T. M. I. MAHLIA
analyzed by Equation (1) and [42,57].
PA¼PV:V
A(1)
PV¼lx(2)
where P
A
defines regional microalgae biomass yield
(based on biomass cultivation area) (kgm
2
d
1
), P
V
is
volumetric yield or productivity (kgm
3
d
1
), Vis for
total volume (m
3
), Ais for the total plowed area (m
2
),
lis for the specific growth rate (with the stationary
state d
1
), xis for the microalgae biomass concentra-
tion (kgm
3
).
Since microalgae demand an adequate nutrition to
enhance the energy component, and sufficient nutrient
supply is considered to be the sole factor for continu-
ous microalgal growth. Organic carbon sources (such as
carbohydrate, fat, protein) and water are referred to as
the major surviving substances for microalgae produc-
tion. Other macronutrients such as phosphorus, nitro-
gen are also vital constituents for growth optimization
[58]. Based on a constant environment with adequate
nutrient supply and internal cellular nutrient contents,
the dynamics of the microalgae growth rate can be
accurately determined by the Droop model while
growth rate on the basis of external supplement con-
centration to culture media can be simulated through
the Monod model. The Droop model and Monod model
have been expressed with Equations (3) and (5),
respectively and Equation (4) represents maximum
microalgae cell growth rate by the Droop model [59].
lD¼lDmax 1qmin
q
(3)
lDmax ¼lmqm
qmlmqmin
(4)
lm¼lmax N
½
KNþN
½ (5)
where l
D
¼specific growth rate by the Droop model
(s
1
), l
D
max
¼maximum growth rate by the Droop
model (s
1
), q
min
¼minimum cell quota, q¼cell quota,
l
m
(s
1
), q
m
¼maximum rate of nutrient uptake per
cell, K
N
¼half-saturation constant for nutrients
(mgL
1
), [N] ¼nutrient concentration (mgL
1
).
Previous experimental studies revealed that add-
itional supplementation, especially salts and vitamins,
boosted biofuel generation to the highest productiv-
ity through fermentation [60]. In line with that, physi-
cochemical factors such as O
2
gas removal, CO
2
uptake, quality and quantity of light penetration to
the culture medium, temperature and pH range, cul-
ture period, gas transfer coefficient, presence of toxic
chemical, salinity percentage, invasion of other harm-
ful microbes, biotic factors and others are also simul-
taneously correlated with microalgae growth curves.
Mixing and dilution rates, turbulence, medium depth,
bicarbonate (HCO
3
–
) addition, harvesting frequency,
Table 2. Microalgae biomass productivity, microalgae-based biofuel yield and processing technologies.
Microalgae species Biomass productivity Biofuels types Biofuel productivity
Biofuel processing
technologies References
Chlorella vulgaris,
Scenedesmus sp.,
Chlorococcum sp.
0.28 gL
–1
d
–1
Bioethanol 12.20 mgL
–1
Simultaneous
saccharification and
fermentation (SSF)
[16,24]
C. vulgaris 103 gm
–2
Biobutanol 3.86 gL
–1
ABE fermentation [25,26]
Chlorella protothecoides 2.0–7.7 gL
–1
d
–1
Biodiesel 1214 mgL
–1
d
–1
Direct transesterification [16]
Arthrospira platensis 48.5–153.3 gL
–1
d
–1
Bio-oil 29.5% w/w
dry-biomass
Hydrothermal
liquefaction
[27,28]
Nannochloropsis gaditana 12.0–53.0 gL
–1
d
–1
Bio-oil 40% w/w
dry-biomass
Pyrolysis [16,29]
Spirulina sp. 0.22 gL
–1
d
–1
Bio-oil 45.6% w/w
dry-biomass
Co-liquefaction [30,31]
Chlorella pyrenoidosa 2.95 gL
–1
Bio-oil 87.5% w/w
dry-biomass
Catalytic hydropyrolysis [32,33]
Chlamydomonas
reinhardtii
0.185–0.352 gL
–1
Bio-oil 26 ± 2% w/w
dry-biomass
Solvent extraction [34,35]
Chlamydomonas
reinhardtii
0.185–0.352 gL
–1
Bio-char 44 ± 1% w/w
dry-biomass
Pyrolysis in fixed-bed
reactor (FBR)
[34,35]
Spirulina sp.
Scenedesmus sp.
C. vulgaris
0.22 gL
–1
d
–1
Bioelectricity from syngas 8.67 ± 0.10 Wm
–3
Anode substrate at
microbial fuel cell
(MFC) technology
[31,36]
C. vulgaris 2.11 g
DCW
L
–1
d
–1DCW:Dry-
Cell-Weight
Bioelectricity from syngas 19.80–20.25 mWm
–2
Cathode substrate at
microbial fuel cell
(MFC) technology
[37,38]
Spirulina platensis 60 gm
–2
d
–1
Bioelectricity from syngas 2018–4433 mWm
–2
Electrode substrate at
microbial fuel cell
(MFC) technology
[39,40]
C. vulgaris ESP6 2.72 gL
–1
Biohydrogen 240 mLL
–1
h
–1
Dark fermentation [41,42]
C. reinhardtii 0.185-0.352 gL
–1
Biohydrogen 2.92 H
2
mgL
–1
h
–1
Photo fermentation [35,43]
Scenedesmus obliquus 1.41 gL
–1
Biohydrogen 56.8 mLg
VS
–1VS:volatile solids
Dark fermentation [44,45]
C. vulgaris
Scenedesmus sp.
2.0 gL
–1
d
–1
Biomethane 154–252 Lkg
VS
–1
Anaerobic digestion [46,47]
CRITICAL REVIEWS IN BIOTECHNOLOGY 3
and others carry an important significance as func-
tional parameters [58].
According to the investigation of cellular kinetics,
the cell number of microalgae biomass doubled within
24 h under suitable culture conditions and parameters.
The cell growth process comprised five stages of the
life cycle: lag phase, log phase (exponential), linear
growth phase, stationary phase, and death or decline
phase. Amongst these five phases, exponential or log
phase was considered as the most productive stage
where the microalgae doubled the total population
number within approximately 3.5 h incubation and the
time required to double the cell population is known as
the doubling time. Doubling time is closely correlated
with nutrient concentration and cell growth speed.
Growth rate and doubling time were determined using
Equations (6) and (7) [61–63].
Growth rate K0
ðÞ
¼ln n2=n1
t2t1
(6)
Doubling time t0
ðÞ
¼1
K0=ln 2
ðÞ (7)
where K’defines the microalgae growth rate (h
1
), t
1
is
an initial stage of the culture period (h), t
2
is the final
stage of the culture period (h), n
1
and n
2
are the total
population of microalgae biomass at t
1
and t
2
,
respectively.
The average growth rate for the total life cycle span
was determined on a cell concentration basis at initial
and final stages of the whole life cycle. During the cell
growth period, nutrient addition, especially nitrogen
and phosphorus compounds, should be sufficiently per-
petuated to avoid nutrient deprivation [62,64]. With the
rise in growth rate across different phases, the nutrient
concentration deceased gradually due to limiting
nutrients [60]. Figure 1 represents the correlation
among cultural period/time, growth rate and nutrient
concentration.
Cultivation conditions
The most fundamental factors behind microalgae culti-
vation are the cultivation conditions, chemical compos-
ition, light intensity, and other growth characteristics of
microalgae. It has been reported that culture conditions
are classified into four main groups: photoautotrophi-
cally, heterotrophically, mixotrophically, and photohe-
terotrophically [65]. The chemical component
requirement and growth characteristics are shown in
detail in Table 3. Photoautotrophical culture is mainly a
light based system where the culture medium utilizes
light as the core energy derived from photosynthetic
reactions [69]. To cultivate various species of microalgae
in aquacultural industries worldwide, this approach (e.g.
the big-bag method) is preferred by maintaining micro-
algae broth in a big bag as a closed system approach.
Several studies indicated that blue-green algae, e.g.
Haematococcus pluvial,Spirulina,Chlorella, and some
distinct marine algae are suitable candidates for photo-
autotrophically or heteroautotrophically. The major
challenge of this process was a highly expensive invest-
ment to develop the cultivation. Extra labor costs, artifi-
cial light supply, temperature control of the whole
process, and recurrent culture crush were needed. A
poor standard culture mixture plays a role behind this
additional cost investment [22,61,69].
The mixotrophic approach could be either auto-
trophic or heterotrophic. Both CO
2
and essential com-
pounds (organic, inorganic both) are prerequites for the
process with the presence of the photosynthetic
respiratory system [70]. For instance, Chlorella vulgaris
and Nannochloropsis sp. exhibited 4.0 gL
1
and
1.2 gL
1
biomass productivity, respectively at mixotro-
phic approaches [66]. A suitable concentration of essen-
tial compounds or sufficient light can maintain a
healthy environment for the microalgal cultural broth.
Amphitrophy is a popular and well-established example
for the mixotrophical algae cultivation method. On the
other hand, through the photoelectrotrophic condition,
the combined application of light and organic com-
pounds acts as food sources for cellular metabolism
[22]. This process is also familiar with other terms such
as photoassimilation, photoorganotrophy and photo-
metabolism. Stoichiometry of growth formulation,
metabolite products and system performance may vary
with different cultures. For example, Scenedesmus acu-
tus and Selenastrum capricornutum are two suitable
strain types that can adapt with different culture meth-
ods such as heterotrophcally, photoautotrophically, or
photoelectrotrophically. Nutrients, vitamins, salts,
and minerals also play a role as prerequisites for cell
growth optimization. Shrinkage of the light paths,
Figure 1. Correlation between microalgae growth rate, nutri-
ent concentration, and culture time for each life-cycle
span [28].
4 N. HOSSAIN AND T. M. I. MAHLIA
enhancement of the light availability per cell, and gas
transfer optimization are considered to be key princi-
ples to the design model culturing methods. This
approach also maintains a genuine and contamination
free process resulting in rigorous growth, a small culti-
vation area, excellent light utilization per cell, higher
productivity, temperature control choices (either
indoor or outdoor), generation of high quality and con-
sistent final product. Nevertheless, it is also worth men-
tioning that challenges to reduce operational costs
remain [71,72].
Open pond cultivation approach
The open pond cultivation approach is considered to
be the most popular, cost-effective, and well-estab-
lished method for microalgae cultivation. Through
open cultivation systems, microalgae grow in ponds,
lakes, oceans, seas, lakes, dumped wet areas, drainage
ditches, and wastewater ponds randomly employed for
the natural metabolic process. This approach has been
practiced for the last few decades in order to treat
wastewater for CO
2
sequestration. Recently, this
approach has been implemented for commercial pur-
poses due to the cost-effective plant set up and the
maintenance for both pilot and large-scale production.
Large scale investigations have revealed that the aver-
age pond depth range remains to be between 5 and
1000 cm. Figure 2 presents the open pond microalgae
cultivation process. On the other hand, raceway pond
systems are the most commercial and popular open
pond culture procedures where the pond shape is built
with the raceway and is circulated by paddle wheels for
proper aeration and mixing of the culture [57,71]. The
building materials could be of concrete or mud com-
position with a plastic or PVC liner. To elaborate the
specifications, an experimental study stated that
“Raceway open ponds with 22.7 units occupying the
total area of 22,700 m
2
with 100 m length, 10m width
and 35 cm depth and the raceway construction material
was a concrete cavity covered with 2 mm of thick poly-
vinyl chloride plastic (PVC)”[57]. The advantages are
low cost for temperature and light maintenance, simple
and cheap established methods, and culture mainten-
ance through light and temperature suitability driven
by the climate, season, and geography. A study con-
cluded that only 0.34–0.45 US$ per kg was invested in
producing microalgae commercially by the raceway
pond system [74]. During the summer, the water evap-
oration rate climbs high, but the light remains suffi-
cient. The cooling effect of high water evaporation
rates reduces the temperature and that helps to main-
tain the microalgae growth media temperature.
However, the addition of an extra water supply to
the pond in summer is required sometimes to balance
water amounts, though it is deemed as an engineering
challenge [75]. In contrast, water loss is negligible dur-
ing the winter or rainy season while the light is insuffi-
cient. The shortcomings of open pond cultivation are
low with an inconsistent cell density ranging between
Table 3. Different microalgae cultivation conditions have been characterized [22,65–68].
Cultivation conditions Photoautotrophic Heterotrophic Mixotrophic Photoheterotrophic
Energy sources NaHCO
3
/CO
2
and solar or
artificial light
Organic compound:
glucose (without light)
Organic compounds:
glucose, CO
2
and
light both
Organic compound:
glucose (with light)
Carbon sources Inorganic compounds Organic compounds such
as glucose, acetate etc.
Both organic and
inorganic compounds
Organic compounds such
as glucose, acetate etc.
Reactor Suitability Open pond,
photobioreactor
Conventional fermenter Photobioreactor Photobioreactor
Cost Cheap Moderate Expensive Expensive
Large scale
application issues
Less concentration of cell
number, expensive to
condense cell broth
Chance of contamination,
expensive substrate
Probability of
contamination, High
equipment, substrate
and maintenance cost
Contamination probability,
expensive equipment,
substrate and
maintenance required
Biomass
productivity (gL
–1
)
0.22–1.00 0.17–5.30 0.45–4.27 0.23–2.70
Highly productive
microalgae species
Chlorella protothecoides,
Chlamydomonas sp.
BTA9032,Chlorella sp.
BTA 9031 and others.
Chlorella protothecoides,
Chlorella vulgaris
and others
Chlorella protothecoides,
Chlorella vulgaris,
Chlorella vulgaris ESP-31
and others
Chlorella protothecoides,N.
Oleoabundans UTEX 1185
and others
Figure 2. Open pond (circular) microalgae cultivation [20,73].
CRITICAL REVIEWS IN BIOTECHNOLOGY 5
0.1 and 1.5 gL
1
owing to incompatible CO
2
uptake,
gas transfer rate, culture medium mixture, turbulence,
cellular hydrodynamic stress, and contamination caused
by predators. Previous studies projected 10 years as the
average lifetime expectancy for open pond cultivations
[57,73,76]. However, stirred circular-tank ponds, and
unstirred shallow ponds are also popular models of the
open pond system. Likewise, in the raceway open
pond, general functions and challenges are almost simi-
lar but a comparatively larger area is required for culti-
vation [76].
Closed-loop system approach
Though the closed-loop system exhibits some structural
resemblance with the open pond system, it is not
exposed to the open environment with free aeration.
Figure 3 presented the closed loop cultivation system
structure. The system regulates to be more contamin-
ation free, demands a sterilized CO
2
source and light
maintenance mechanically that precludes high costs for
plant set up and maintenance [61]. Under this
approach, microalgae can be raised in several ways
such as photoautotrophically, heterotrophically, mixo-
trophically, and photoelectrotrophically. Organic com-
pounds such as glucose, and acetate are considered to
be carbon sources for the heterotrophic cultivation sys-
tem and these compounds are supposed to be fed and
well-maintained during the culture period [71]. Previous
investigations recommended Chlorella,Crypthecodinium
cohnii,Dinoflagellate,Tetraselmis, and others as suitable
algal candidates. Japanese industries have produced
approximately 550 ton of Chlorella sp. commercially.
This culture system is well established for commercial
purposes. Designs and operational technologies are
also well understood. High cell growth and cell popula-
tion densities, low operation-harvesting-handling cost
are the positive aspects of this culture method
although it is not perfectly suitable to cater for all types
of microalgae. It should be mentioned that with this
system, the chemical composition of the growth
medium might accelerate under heterotrophic condi-
tions [61,77]. An experimental study has demonstrated
that large-scale closed-loop microalgae cultivation sys-
tems is sited inside temperature controlled green-house
for biodiesel production [75]. The CO
2
uptake efficiency
of this system is roughly 50–80% in most cases. For
higher productivity of microalgae, several studies have
presented 65–73 lmolL
1
pure CO
2
uptake by biomass
[78]. The major advantage of the closed-loop system is
the robustness of the system, well-maintenance of
monoalgae culture, greater efficiency of CO
2
fixation,
and low contamination risks that results in high value
products. The constraint of this process is the require-
ment for additional costs for construction and mainten-
ance which might not be economically viable for
biofuel extraction [58]. A study has presented [74] that
large-scale closed-loop microalgae cultivation system
require 4.2 US$kg
1
while 10.15US$kg
1
has been
invested for the laboratory scale [74]. An experimental
study to cultivate microalgae in the closed-loop system
in the green house is not economically favorable for
biodiesel production [75].
Another closed system is a big bag which can be
maintained with heterotrophic conditions. The “big”
word indicates large-scale production of microalgae in
huge bags. Thousand liters of liquid or above is fitted
with big bag culture applications with proper aeration
conditions. Big bag (Figure 4) contained a huge sterile
strong plastic bag that can hold the whole medium and
it was connected to a nutrient dose pump, light sour-
ces, gas exit, and biomass exit pathways. Batch mode
and semicontinuous systems were recommended for a
commercial big bag approach. Spirulina,Dunaliella, and
Chlorella sp. are potential species to be cultivated in
such a big bag [65,73].
Photobioreactor (PBR)
The photobioreactor (PBR) is a special type of high-tech
microalgae culturing reactor used for growing
Figure 3. Closed-loop microalgae cultivation [57,73].
6 N. HOSSAIN AND T. M. I. MAHLIA
microalgae in diverse conditions attributed to suitable
microalgae strains utilizing light intensity. The light
source could be solar, artificial (by fluorescent lamp or
other type of light source) or a mixed light utilization
system [79,80]. Flat-plate PBR (Figure 5), horizontal/
inclined tubular PBR can be used for an outdoor
approach utilizing sunlight where temperature control
was challenging. On the other hand, airlift PBR, bubble
column, helical, a-shaped can be administered by artifi-
cial light via fluorescent lamp. These PBR yielded very
high biomass productivity because of the excellent
temperature control and proper light availability, albeit
they were very expensive [83–87]. Research & develop-
ment (R&D) units in many places worldwide are experi-
menting on thermocouple water jacket integration with
the double-wall plant design so that heating and cool-
ing water circuits can control temperature while it
would be necessary for cultural growth [86]. Several
famous PBR descriptions, algae strains, advantages, and
limitations were presented in Table 4.
Physicochemical parameters of cultivation
Physicochemical parameters are referred as the driving
factors for desired microalgae biomass yield for each
type of reactor. A uniform supply of CO
2
and light to
the culture functions as macronutrients for the cells.
Other demands such as adequate mixing, retaining of
the certain culture period, a decent pH, and tempera-
ture maintenance, together with consistent nutrient dis-
semination were also counted as significant factors to
conduct healthy microalgae culture techniques [55,90].
Gaseous transfer
Carbon sources are the major elements for efficient
microalgae cell growth. To grow microalgae in an aque-
ous environment, carbon source exists in several chem-
ical forms such as carbon dioxide [CO
2
(aq)], carbonic
acid [H
2
CO
3
], bicarbonate ion [HCO
3
–
], and carbonate
ion [CO
32–
]. Carbon sources were converted into
soluble food substances via chemical reactions in the
presence of certain temperatures and pH. Amongst
various chemical forms of carbon sources, CO
2
was the
most utilized forms due to the availability, easy uptake,
cost-effectiveness, and environment friendly character-
istics. Accordingly, CO
2
gas-enriched air was greatly pre-
ferred on an industrial scale to be utilized as a natural
carbon source [90]. In addition, during CO
2
gas injection
the CO
2
/O
2
ratio should be balanced in order to acquire
a high photosynthesis rate. Consequently, the major
carboxylating enzyme of the algae, rubisco can easily
handle CO
2
for the Calvin cycle avoiding photorespir-
ation caused by O
2
.ACO
2
/O
2
ratio balance can be
maintained via outward air bubbling through the cul-
ture with proper pH monitoring [94]. CO
2
fixation was
taken place via attaching CO
2
molecules with five car-
bon sugar, ribulose-1,5-biphosphate [95]. The overall
equation of the Calvin Cycle (light independent) is
shown in Rc. 1:
3CO2þ9ATP þ6NADPH þ6Hþ!C3H6O3þ9ATP
þ8P þ6NADPþþ3H2O (Rc. 1)
where C
3
H
6
O
3
is three combined molecules of carbohy-
drate (CH
2
O), ATP is adenosine triphosphate, the NADP
is nicotinamide adenine dinucleotide phosphate, and
ADP is adenosine diphosphate.
Additionally, the prime food source of microalgae,
CO
2
gas supply, also improved the mass transfer coeffi-
cient, recovered CO
2
deficiency, adjusted the level ofFigure 5. Flat-plate photobioreactor [81,82].
Figure 4. Big bag microalgae cultivation method [73].
CRITICAL REVIEWS IN BIOTECHNOLOGY 7
dissolved O
2
toxicity, lowered nutrient gradients, cell
sedimentation, clumping, fouling, and the dead zone as
well as optimized the photosynthesis rate. However,
occasionally the high aeration rate might cause cell
damage by mechanical shear forces and lead to a high
running expense [96]. Whenever CO
2
was injected to
the microalgae culture, gas transfer occurred with a
two-phase system: gas and liquid phases through sev-
eral sequential steps until the metabolic uptake
occurred by algae cells. The CO
2
gas transportation sys-
tem has been presented in detail in Figure 6.
During CO
2
transfer, a resistance appeared through
the fluid film interface. It dominated the overall gas
transfer rate and minimized the CO
2
gas convey
momentum. Hence, the rate of CO
2
(NCO
2
) gas transfer
was approximately measured by the Equation (8) [90].
NCO2¼kLaC
CO2LCCO2L
(8)
where NCO
2
is the gas transfer rate (kgm
3
s
1
), k
L
ais
the liquid phase mass transfer coefficient (s
1
) over a
specific area, C
CO
2
L
is the CO
2
concentration of the cul-
ture medium (kgm
3
) to equilibrate its actual pressure
Table 4. Various photobioreactor types, microalgae strain, productivity, advantages and limitations [77,80–82,86,88–93].
Type of photobioreactor Description
Microalgae candidates with
productivity (gL
–1
d
–1
) Advantages Limitations
Flat-plate Rectangular boxes
consisting of translucent
glass or plastic (PVC)
Nannochloropsis sp. (0.85),
S. platensis (2.15),
Synechocystis
aquatilis,Spirulina
Excellent light utilization,
outdoor culture, high
temperature capture
efficiency, excellent
algae immobilization,
easy to scale up, high
biomass productivity,
economically efficient,
less oxygen builds up,
easy cleaning system.
Large surface area needed,
scale up requires high
cost, control difficulty of
temperature and wall
growth, possibility of
hydrodynamic
shear strength.
Vertical-column Either draft tubes or split
cylinders (Diameter up
to 19 m)
P. cruentum (0.5) Low shear stress, easy to
sterilize and scale up,
excellent mass transfer
and mixing rate, easily
tempered and algae
immobilization, lower
photo-inhibition and
photo-oxidation,
moderate control of
temperature
and growth.
High shear stress, low
illumination surface
area, expensive and
sophisticated
construction
materials needed.
Horizontal or inclined tubular A number of clear
transparent horizontal
tubes (diameter
10 cm) consisting of
glass or plastic material.
S. platensis (0.25), Chlorella
sorokiniana, Spirulina,P.
Cruentum (0.36),
Nannochlopsis sp. (0.70),
Isochrysisgalbana (0.32)
Outdoor culture, good high
utilization, moderate
biomass productivity,
scalability, large surface
area, cost-effective.
Large land space needed,
fouling, pH gradient,
dissolved gases along
the tubes, possibility of
wall growth.
Internally Illuminated Internally illuminated with
fluorescent lamps,
impellers for agitation,
and spargers
for aeration.
Chlorella pyrenoidosa Easily heat-sterilized under
pressure, low possibility
of contamination, high
control on light
utilization, Continuous
algae growth in day and
night, excellent mixing.
Artificial light requirement,
extra technical efforts
are needed for outdoor
cultivation.
Airlift Simple vertical cylinder
with an air inlet at
bottom constructed with
glass or plastic.
Phaeodactylum tricornutum,
Dunaliella tertiolecta
Excellent mass transfer rate
and superficial velocity,
high volume, excellent
aeration and gas
exchange, high
light gradient.
High pressure requirement,
high energy
consumption, foaming
and fouling, lot of
bubble formation.
Helical Consisted of homologous
translucent tubes
bounded by
cylindrical surface.
S. plantensis (0.4) Good light utilization, good
control of temperature
and biomass growth,
very small cultivation
area requirement,
excellent to scale up.
Expensive to scale up due
to the very
unique shape.
ashaped 300 L a-shaped tubular
reactor with transparent
tubes (2.5 cm 25 m)
constructed with PVC.
Chlorella vulgaris Excellent light utilization,
good control of
temperature and
growth, high fluid flow
rate with low air
supply rate.
Large land area
requirement, very
difficult to scale up
8 N. HOSSAIN AND T. M. I. MAHLIA
on the gas side and C
CO
2
L
is CO
2
concentration (kgm
3
)
in the bulk of the medium.
Even though CO
2
is the macroelement for microal-
gae growth, the gas transfer rate played a significant
impact on growth, and high aeration rate is not recom-
mended for larger scaled PBRs due to the economic
aspect. An elevated aeration speed increased the han-
dling cost and this led the project to be very expensive.
According to an experimental investigation, 0.05 vvm
(vessel volumes per minute) was the favorable aeration
rate for efficient PBR optimization [97]. k
L
ais known as
a volumetric mass transfer coefficient and the utmost
significant factor in order to design and scale-up the
reactor and operate a biomass culture medium
approach [98]. k
L
a(CO
2
) characterized the CO
2
mass
transfer capacity between the gas and liquid phases of
the reactors, and determined the reactor’s ability for
perpetuating the specific microalgae cell growth rate.
Determination of k
L
a(CO
2
) can be modified by the
Equation (9) [98,99]. The k
L
aeffect of various microal-
gae culture media in different reactors is shown in
Table 5.
kLaCO2
ðÞ
¼ffiffiffiffiffiffiffiffiffiffi
DO2
DCO2
s:kLaO2
ðÞ (9)
where k
L
a(CO
2
) presents the volumetric mass transfer
coefficient for CO
2
(s
1
), k
L
a(O
2
) is the O
2
mass transfer
coefficient (s
1
), D
O
2
and D
CO
2
are the O
2
and CO
2
diffu-
sion coefficient (m
2
s
1
), respectively.
Liquid properties, liquid flow rate, the gas injection
approach, system set up, and geometry were the deter-
mining factors of k
L
a(CO
2
)[101]. Hence, k
L
avalue
increased simultaneously with an increasing rate of tan-
gential flow (liquid flow). It is noteworthy that the
tangential flow rate represented a thin boundary layer.
Nevertheless, the main constraints behind k
L
aimprove-
ments are bubble effects and the surface tension of the
medium caused by cultural metabolites. Currently,
much research is ongoing in order to minimize the obs-
tacle effects [90].
Mixing
Mixing of the culture medium into the vessel should be
handled effectively after supplying the main food sub-
stance, which is CO
2
to the algae cells. Good mixing is
one of the most important factors for maximum micro-
algal growth. Healthy mixing is required to prevent
microalgae sedimentation, develop nutritional CO
2
and
O
2
gradients as well as moved cells into light gradients
for maximum light utilization [102]. Therefore, mixing
should be maintained to a certain extent. For instance,
a very low mixing rate promoted settling that might
create dead zones where anaerobic conditions pre-
vailed, leading to culture deterioration. Moreover, inad-
equate mixing might cause clumping to develop in the
three solid-liquid-gas phases in the reactor. Conversely,
with high intensities of mixing, cell damage could occur
due to fluid mechanical stress [103]. Several types of
mixing approaches were being applied on the commer-
cial scale such as: pumping, mechanical stirring, CO
2
gas injection with specific gas velocity or a combination
of all these methods concurrently [90]. As a result of
microalgae cultivation research, superficial gas velocity
of Chlorella vulgaris cultivation was optimized at 8.333
10
4
m.s
1
[104].
The mixing turbulence in the PBR can be measured
either by using the Reynolds number (Re) or Swirl num-
ber (Sn). While the geometry of PBR was special and
the liquid properties (e.g. viscosity) did not play a sig-
nificant impact on the mixing rate, swirl number can be
applied in those cases and the average turbulence
inside the PBR can be calculated using the Equation
(10) [105–106].
Snv¼Ð
L
0ÐÐ
S
S
UVrdS dz
Ð
L
0ÐÐ
S
S
U2rdS dz
(10)
where Uis the average rotation (axis based) speed com-
ponent (ms
1
), Vis the average mixing rate (s
1
), ris
the radius from z coordinate (m), Sis the mixing turbu-
lence intensity, Lis the photobioractor length (m).
Dispersing across the gas layer vicinity
Transfer CO2 through gas-liquid interface
CO2 dispersal along the adjoining fluid layer
Transport CO2 from the thin liquid film to the bulk of liquid
Transfer from the bulk fluid layer to the lean fluid layer at the ultimate area of algal
cell wall
CO2 dispersion along the exposed fluid layer
Metabolic uptake by the cell wall
Transport CO2 gases to the flimsy gaseous state at ultimate interfacial area
Cell wall uptake CO2 to run metabolic process
Figure 6. CO
2
gas transfer system to algae cells through
sequential steps [90].
CRITICAL REVIEWS IN BIOTECHNOLOGY 9
However, while the liquid properties (e.g. viscosity)
may change with time variation, Reynolds number (Re)
was highly recommended for that scenerio to measure
the mixing turbulence. Mixing turbulence was meas-
ured by the Equation (11) [106].
Re ¼qVL
l(11)
where qis the density of the medium liquid (kgm
3
), V
is velocity of the liquid (ms
1
), Lis the length of photo-
bioractor (m), and lis medium liquid viscosity
(kgm
1
s
1
). It was evident that the other factors
related to the culture medium such as gas injection,
light, and nutrients would be functional while they
were combined with good mixture and turmoil. Re
3300 (transition flow) was counted as an ideal num-
ber for good turbulence with optimum condi-
tions [105].
Light demand
Light carries prior significance in order to generate
energy in microalgae cells via photosynthesis.
Accordingly, the effectiveness of light harvesting was
crucial factor for photobioreactor engineering during
microalgae culture [90,107]. The light based reactions
are comprised of both photochemical and redox reac-
tions. Chlorophyll II pigments (e.g. chlorophyll-a) are
capable of converting light energy (photon) into elec-
trons through a noncyclic reaction pathway of photo-
system II. The overall light-based reaction has been
presented in Rc. 2. To note, that this reaction takes
place into the thylakoid membrane of cellular chloro-
plast [108].
2H2Oþ2NADPþþ3ADP þ3P þlight !2NADPH
þ2Hþþ3ATP þO2(Rc. 2)
With the suitable PBR design and operational charac-
teristics, light demand for microalgae growth can be
enhanced by the prolonged light path in PBR. The
favorable length of the light path in PBR has been
projected as approximately above 0.10 m. This length
resulted positive economically for photobioreactor con-
struction, energy and higher liquid consumption per
area [109,110]. Dimensions of the inner section of PBR
were dependent on some particular characteristics such
as the concentration of biomass, the intensity of light
utilization, geometry as well as hydrodynamics for rigor-
ous light utilization. Sufficient light penetration of the
PBR was demonstrated by the modified Evers Model
(Figure 7) via Equation (12) and [22,105].
PDF s
ðÞ¼PDFin
Ð
1:5p
0:5p
cos hþp
ðÞ
dh
ð
1:5p
0:5p
cos hþp
ðÞ
exp achlachla
ðÞ
b
dh
2
6
43
7
5(12)
b¼rs
ðÞ
coshþr2rs
ðÞ
2sin h2
hi
0:5
(13)
Table 5. Mass transfer coefficient (k
L
a) in different types of reactors with various volumes for certain microalgal spe-
cies [86,87,100].
Reactor type Subtype reactor Volume (L) k
L
a(s
–1
) Microalgal species
Flat-plate –3 0.002 Synechocystis aquatilis
Internally-illuminated column –3 0.020 Chlorella pyrenoidosa
Airlift Concentric tube airlift column 12 0.020 Phaeodactylum tricornutum
Airlift tubular horizontal 200 0.014 Porphyridium cruentum
External loop airlift column 200 0.006 P. tricornutum
Split-cylinder internal-loop airlift 2 0.009 Haematococcus pluvialis
Bubble –13 0.002–0.005 Porphyridium sp.
2 0.020–0.025 Phaeodactylum sp.
Tubular –200 0.006 Phaeodactylum sp.
75 0.004 Phaeodactylum sp.
Figure 7. Light penetration pathway in PBR determined by
Evers model [105].
10 N. HOSSAIN AND T. M. I. MAHLIA
a,b,r, and sare light penetration pathways in a photo-
bioreactor (PBR) shown in Figure 7.
where PFD (s) is expressed for photon flux density
that saturates the photosystem, PFD
in
is the photon
flux density of light intensity occurrences on PBR exter-
ior, a
chl-a
is certain wavelength based Chl-a absorption
coefficient, Chl-a is the chlorophyll concentration (with
the presence of light in Photosystem II), ris the radius, s
is the theoretical interspaces between the exterior light
saturation point at a specific PFD.
Previous research studies have revealed that roughly
45% of the photosynthetically active radiance (PAR)
were scattered at a wavelength ranging 400–700 nm
though different strains varied with light irradiances
[90,111]. Divergent microalgae species such as
Isochrysis sp.,Microcystis aeruginosa,Chlorococcum littor-
ale,Dunaliella salina,Chlorella vulgaris, and
Pseudokirchneriella subcapitata exhibited average opti-
mum regular light irradiances at 80, 140, 170, 178, 208,
258 lEm
2
s
1
, respectively [112–114]. According to an
experimental study, the exponential growth rate and
the polysaccharide production of Porphyridium cruen-
tum was improved in the presence of blue light at the
wavelength of 400–500 nm. The light energy containing
each microalgae cell was closely correlated with some
specific factors: surface area of the culture in a photo-
bioreactor (A
v
), the total light incident radiance (I
o
), the
maximum growth yield of light (Y
G
), and the fraction of
photosynthetically available light (ɸ). Although it was
very challenging to determine the highest microalgae
growth in photobioreactors with a specific formula, it
was projected with the highest usage to factor opti-
mization by the Equations (14) and (15) [92].
lx¼uI0YG2r0
pr2(14)
where l
x
defines the biomass output rate. Most of the
time, I
o
and A
v
are constant for the reactor, Y
G
and ɸ
are variables.
AV¼2ro
pr2(15)
where rand r
o
are defined as interior and exterior radii
of either tubes or PBR, respectively.
Light utilization of PBR varied with two different
approaches: sunlight mode and artificial light mode
[114]. Sunlight mode PBR was cheaper to setup, pos-
sessed a high PBR surface and volume ratio required for
maximum utilization, controlling light intensity was dif-
ficult through this system and microalgae growth kinet-
ics was weather dependent. Arthrospira platensis
microalgae species grown in the Yamuna river in India
exhibited maximum lx;0.45d
1
at an energy flux per
unit of power, ɸ6.05 10
21
sm
2
with biomass prod-
uctivity, 16.77 mgL
1
h
1
[115]. Comparatively, an arti-
ficial light mode PBR was expensive to setup and
maintain albeit controlling light intensity was easy,
small surface area was required, PBR design was flexible
and optimum growth was constant [116]. In some PBR
setups, both models were applied with a combination
mode and the output exhibited maximum yield. The
method to estimate the efficiency of conversion in both
modes can be determined by the Equation (16) [117].
ELRF ¼ðPk
ðÞ
dk(16)
where ELRF defines emitted light radiant flux, Pis the
photon flux, and kis the wavelength of light (m).
However, with high light intensity, some strains
tended to deplete rapidly until the death phase due to
chlorophyll II degradation, low amount of cellular pro-
tein and carbohydrate, and higher carbon allocation
into the lipid that blocks almost the whole intracellular
spaces [118]. The effect of light intensity on the micro-
algae species, Chlorella sp. and Monoraphidium dybow-
skii were delineated at Figure 8.
Temperature
The microalgae growth rate maintained an exponential
correlation with the temperature rise like many other
microorganisms. The cell growth rate was elevated with
a temperature extension until reaching the optimum
temperature. At the optimum temperature, microalgae
cell growth decreased during the stationary phase.
According to experimental approaches, the optimum
temperature spectrum needed for microalgae culture
medium was between 20 and 30 C regardless and they
can survive with the temperature range 16–35 C.
Above 35 C, cells were reluctant to grow especially
during dark times, albeit below 16 C, cells condoned
somehow with low productivity until the water turned
into ice formation [71,112,119,120]. Psychrophilic (e.g.
Astrionella formosa), thermophilic (e.g. Aspergillus nidu-
lans) and mesophilic (Chlorella species such as Chlorella
vulgaris,Chlorella fusca,Chlorella kessleri,Chlorella pro-
thotecoids) strains were cultivated at optimum tempera-
tures with 17 C, 40 C, and 20–25 C, respectively [121].
Previous experimental studies demonstrated that the
carbohydrate and lipid content of Nannochloropsis ocu-
lata and C. vulgaris were doubled while the tempera-
ture was increased from 20 Cto25
C. Simultaneously,
an 8.81% lower energy content was observed with a
temperature rise from 25 Cto30
C[122]. The growth
rate of some strains started to deplete below the opti-
mal temperature. Cell growth of Thalassiosira
CRITICAL REVIEWS IN BIOTECHNOLOGY 11
pseudonana,Odontella aurita,Nannochloropsis oculata,
Isochrysis galbana, and Dunaliella tertiolecta was
depleted at 10 C from the 1st day of cultivation [123].
However, in some cases, some microalgae species
exhibited higher productivity in terms of growth and
lipid content with a temperature stressed environment
within a short period and these strains were projected
as preferred candidates for industrial biofuel sources.
For instance, Scenedesmus quadricauda exhibited
23.2 mgL
1
productivity and 33.5% lipid content at
40 C within 1 day cultivation [124].
pH
pH is another key factor to obtain maximum algae
growth rate. Hence, pH should be maintained from
time-to-time throughout the whole culture period. The
pH should be in the range of 7.0–9.0 for favorable
growth conditions whereas the optimum value was
forecasted to be 8.2–8.7. One of the most experimented
microalgae species, Chlorella sp., exhibited the max-
imum growth rate at pH 8.0. Lack of regular pH main-
tenance can collapse the system and an unexpected
cell death condition may arise [59,125–127]. An alkaline
environment is much more favorable compared to an
acidic medium due to the higher concentration of nitro-
gen (N) and phosphorus (P) that comes from the water.
Furthermore, pH played a significant role to control
the protein and pigment content of microalgae
[59,127,128]. According to microalgae cultivation
research, at pH 7.0, two separate sets of Chlorella pyre-
noidosa were grown in a semi batch mode: with proper
maintenance and without maintenance. With proper
pH supervision, cellular growth productivity was 3.64
times higher than without supervision [129].
Nutrients
Nutrients are considered to be the enhancing factors to
acquire maximum biomass yield for pilot or industrial
scale scenarios. Microalgae demand inorganic nutrients
including macronutrients (N & P mainly with 16 N:1P
ratio), vitamins (B
6
,B
12
, and others) and trace elements
(salt of iron, nickel, cobalt, manganese, zinc and selen-
ium) for cellular growth [21,80,127]. For instance,
Chlorella sp. presented the maximum growth rate at an
N/P ratio of 15–30 [127]. Limited nitrogen produced
20–53% higher lipid accumulation in microalgae and it
was a suitable condition for commercial biodiesel pro-
duction albeit limited nitrogen reduced the overall
growth rate of Chlorella vulgaris ESP-31 [68]. On the
other hand, the photosynthetic electron transport for
Haematococcus pluvialis was vandalized because of
nitrogen deprivation that caused massive loss of the
cytochrome b6/f complex. Cellular metabolism of blue-
green microalgae (cyanobacteria), Aspergillus nidulans,
Microcystis aeruginosa,Coelastrella rubescens, and
Spirulina platensis was remarkably affected due to a
nitrogen deficiency [97]. Silicon was the most signifi-
cant nutrient for the cellular growth of diatoms such as
Cyclotella cryptica. For this scenario, silicon-deficiency
presented a negative correlation with the cellular
metabolism. Hence, extra nutrient addition has been
recommended for the culture of microalgae culture in
order to avoid nutrient limitation risk factors [48,130].
Culture period
Culture periods for microalgae cultivation should
be very specific for each algal species and strain.
Culture time varied from 5 days to 25 days based on
the algae strain and the climate conditions. Culture
periods for each batch is known as the hydraulic
Figure 8. Effect of light intensity on photosynthetic activity (F
v
/F
m
) microalgae cell growth within 0–14 days cultivation period
where LL represents low light, ML for medium light, and HL for high light [118].
12 N. HOSSAIN AND T. M. I. MAHLIA
retention time (HRT) and it is a significant key aspect
for the set-up of well-configured open pond or closed
systems [19]. According to the previous microalgae
study, effective HRT for Chlorophyceae species was
2–3 days for the highest biomass productivity. Another
study has discussed that C. vulgaris demanded a 5 days
culturing period in coffee manufacturing industrial
wastewater whereas 25 days is needed for leather
manufacturing industrial wastewater to remove heavy
pollutants. After removal of heavy pollutants, the C. vul-
garis growth cycle reached the death phase that
resulted many dead cells and the microalgae color
turned into yellowish/brownish color from green
[131,132]. In contrast, fresh water microalgae species
were grown in a batch reactor, Thalassiosira pseudo-
nana and Odontella aurita exhibited doubling time
within 5–10 days incubation while the cell number of I.
galbana and Chromulina chromonoides strains doubled
at 6–12 days and Dunaliella tertiolecta doubled in mass
within 1–5 days incubation [123].
Biofuels from microalgae
Bioethanol, bioacetone, biobutanol, and
biomethanol
Microalgae bioethanol (Alganol) derived from cellulosic
carbohydrates via a fermentation process are being
applied worldwide as a transportation fuel (such as E10,
E20, E85, etc.) and is blended with diesel and gasoline.
Alganol is also being used as intermediates for solid
oxide fuel cells in some industries [133–135]. Currently,
microalgae are being considered as a potential feed-
stock for commercial bioethanol production in many
regions all over the world. Due to the presence of a
remarkable amount of cellular carbohydrates in
microalgae such as cellulosic polysaccharides and
starch and a zero lignin component, microalgae have
been proven to be a great source of bioethanol produc-
tion. For bioethanol generation, microalgae biomass
has been pretreated through hydrolysis by alkalis, acids,
catalysts (e.g. solid carbon acid catalyst), nanocatalysts
(e.g. CeO
2
, ZnO, CaO, SiO
2
, and others) or enzymes (e.g.
cellulase), then the hydrolysates were fermented by
yeast (Saccharomyces cerevisiae). The distillation pro-
cess, a prominent separation method, was imple-
mented to separate bioethanol from the fermentation
medium. Later, pure bioethanol can be obtained
through dehydration by evaporating steams incorpo-
rated with the raw bioethanol [136–143]. Figure 9 rep-
resents the overview of bioethanol production from
microalgae. A carbohydrate-enriched microalgae spe-
cies, Kappaphycus alvarezii yielded approximately 27%
and 31% (w/w) potential reducing sugar content for
laboratory and pilot scales, respectively excluding sugar
loss of the residue. Subsequently, 80% of this reducing
sugar was converted into pure bioethanol with select-
ive quantity and quality [145]. Other experimented
microalgae species, Scenedesmus sp., yielded 93% sugar
and 86% bioethanol after yeast fermentation [146].
Previous studies also reported that popular
microalgae species, Chlorella vulgaris and Spirulina pla-
tensis contained 55% and 60–65% carbohydrates,
respectively and yielded 87.6%–92.3% and 53.5%–57%
bioethanol, respectively [3,147,148]. Tribonema sp. and
Porphyridium cruemtum produced 56.1% and
65.4%–70% bioethanol, respectively [149,150].
Microalgae butanol can be employed as both a solv-
ent and a biofuel due to long hydrocarbon chain and
high calorific values (29.2 MJdm
3
). Biobutanol has
been produced via anaerobic fermentation in a
Microalgae
Biomass
Starch Hydrolysis (Pre-treated with
alkali/acid/enzymes/catalysts/nano-
catalysts
Unfermented sugars,
Protein, Lipids, Fatty
acids
Continuous Fermentation
(Immobilized yeast cells)
pH:5.0, temp: 25°C-32°C
Distillation
Dehydration
Bioethanol
CO2
Figure 9. Bioethanol (C
2
H
5
OH) production from microalgae [136–140,144].
CRITICAL REVIEWS IN BIOTECHNOLOGY 13
membrane reactor in the presence of Clostridium bac-
teria such as Clostridium acetobutylicum, Clostridium sac-
charobutylicum. Pervaporation methods have been
applied to separate biobutanol, bioacetone and bioe-
thanol from the fermentation system [151–153].
Experimental studies investigated that 3.86 gL
1
biobu-
tanol was achieved from the microalgae biodiesel resi-
dues and 3.37 gL
1
biobutanol has been obtained
from fresh C. vulgaris via ABE fermentation [25,154].
Microalgae biobutanol (known as Solalgal) has been
marketed commercially as an encapsulated lubricant
and oil using solar energy by Bunge Global Innovation
in countries like Peoria, Galva and Brazil. Along with
that, biobutanol is also being generated as co-product
from bioethanol industries [152,153].
Biodiesel
Biodiesel has been applied widely as one of the most
popular, affordable, eco-friendly and alternative green
fuel. The fuel performance of biodiesel has been proved
to be almost similar to diesel for transportation fuel
purposes without any engine modification [4,155–157].
Microalgae have attracted a great deal of attention
recently for biodiesel production since microalgae con-
tains a large amount of cellular lipid component, the
main element for biodiesel production [2,158,159]. An
experimental investigation demonstrated that a micro-
algal-biodiesel based energy approach eliminated
45.77% greenhouse gases from the environment com-
pared to fossil fuel application [160]. Biodiesel was pro-
duced from the cellular lipid content of microalgae
with an alcohol producing fatty acid esters via thermo-
chemical and hydrothermal reactions by the effect of
acids, alkalis, catalysts and nanocatalyst [4,161,162].
Figure 10 has presented an overview of biodiesel pro-
duction from dry and wet microalgae. The cellular tri-
glycerides (lipid content), incorporated with methanol,
produced glycerol and biodiesel in the presence of
alkalis e.g. KOH, NaOH. Rc. 3, have shown the reaction
[163]. Biodiesel from microalgae can be processed
through either one step or two steps extraction
approaches. Both wet and dry microalgal biomass
showed the capability to produce biodiesel through
esterification or transesterification processes.
CH2COOR1CHCOOR2CH2COOR3TriglycerideðÞ
þ3CH3OH Methanol
ðÞ
KOHKOHCH2OH
CHOH CH2OH Glycerol
ðÞ
þCH3COOR1
CH3COOR2CH3COOR3Biodiesel
ðÞ
(Rc. 3)
Microalgae species, Chlamydomonas reinhardtii pro-
duced 15 ± 2% w/w
dry-biomass
bio-diesel [34,163].
Chlorella prototheocoids yielded 45.6% total lipid and
85.8% FAME (fatty acid methyl ester) [166]. Other micro-
algae species like Spirulina maxima, Spirulina sp.,
Tetraselmis elliptica produced 86.10%, 99.32%, 37% bio-
diesel, respectively [34,155,163,167]. The lipid conver-
sion efficiency of Chlorella pyrenoidosa to biodiesel was
investigated as 95.1% through a transesterification pro-
cess in the presence of a solid acid catalyst,
graphene oxide (nanomaterial). Euglena sanguine
yielded an almost 98% lipid content due to the effect
of calcinated CaO [164,168]. Nannochlorum sp.,
Nannochloropsis oceanica,Haematococcus pluvialis,
Anabaena,Chlorococcum sp.,Botryosphaerella sudetica
have been shown to produce a reasonable amount of
biodiesel [165,166,169].
Bio-oil and bio-char
Microalgal bio-oil and bio-char are assorted as a biofuel
while bio-char can be stored underground as a carbon
sink for hundreds of years for the next generation.
Microalgae bio-oil and bio-char are carbon-rich inherent
products from thermochemical (e.g. fast pyrolysis,
slow pyrolysis, torrefaction, catalytic hydropyrolysis,
hydrogenation, hydrodeoxygenation, and others) and
hydrothermal (e.g. carbonization, liquefaction, wet
impregnation, and others) technologies. These biofuels
were co-combusted with fossil coals employing a mer-
cantile application to produce heat for electricity gener-
ation industries [2,170,171]. Figures 11 and 12 presents
the bio-oil and bio-char production processes
from microalgae, respectively. Microalgae species,
Chlamydomonas reinhardtii produced 26 ± 2% w/w
dry-
Pre-treated Microalgae Biomass
Drying Lipid Extraction
Lipid Hydrolysis Lipid Extraction
Esterification Transesterification
Methanol with catalyst/Nano-catalyst
Biodiesel
Free Fatty Acid
Glycerol (mixed with Alcohol/Catalysts/nano-catalyst)
Figure 10. Biodiesel production overview from microal-
gae [155,163–165].
14 N. HOSSAIN AND T. M. I. MAHLIA
biomass
bio-oil and 44 ± 1% w/w
dry-biomass
bio-char [34].
An experimental investigation with Chlamydomonas
reinhardtii presented that bio-char from this microalgae
was able to produce 24.63–30.11 MJkg
1
heat [177].
Moreover, hydrocarbon enriched colonial green micro-
algae: Botryococcus braunii,Cyanobacteria sp. and
Spirulina sp. yielded 68%, 21.10%, 34.30% bio-oil,
respectively [5,178]. C. vulgaris,Scenedemus sp.,
C. sorokiniana,Galdieria sulphuraria,S. obliquus,
Nannochloropsis oculata,Anthrospira platensis,
Dunaliella tertiolecta all produced remarkable amounts
of bio-oil [179–183]. Microalgae were co-pyrolysed and
co-combusted with other forms of biomass to produce
bio-oil and bio-char, respectively. The equations relating
to bio-oil and bio-char yields are shown by Equations
(17) and (18) [30]. With other lignocellulosic biomass
types, Spirulina sp. was co-pyrolysed and 45.63% w/w
bio-oil was obtained containing 34 MJkg
1
calorific
value [30]. The highest energy recovery of bio-oil was
87.5% with a calorific value of 39.3 MJkg
1
from C. pyr-
enoidosa via catalytic hydropyrolysis with the presence
of Mo
2
C catalyst [32]. Another nano-catalyst, Pd/C
revealed an outstanding heating value of 42.9 MJkg
1
from Nannochloropsis oculata bio-oil via hydro-deoxy-
genation and hydrogenation process [172]. Apart from
this, bio-bitumen was also produced from microalgal
bio-oil via orthogonal experimental methods using free
radical polymerization. Bio-gasoline was produced from
bio-oil through hydrogenation processes [184]. They
can be used as an excellent alternative fuel addition of
petroleum bitumen [185].
Bio-oil yield wt%
ðÞ
¼mbio-oil
mbiomass
100% (17)
Bio-char yield ¼mbio-char
mbiomass
100% (18)
An energy consumption ratio of combined bio-char
and bio-oil with Spirulina sp. was obtained as 0.49, aver-
age [170]. Bio-char from microalgae was analyzed
through elemental analysis with a CHNO analyzer and
energy dispersive X-ray (EDX). Based on previous
experimental studies, bio-char from mixed species of
Chlamydomonas reinhardtii,Stigonematales sp., and
Spirulina sp., grown in wastewater, has presented a
30%–46% carbon content, a small amount of oxygen
content, other inorganic elements such as Mg, K, Ca, Al,
Si and high amounts of nitrogen [2,34]. Moreover,
C. vulgaris,Chlamydomonas reinhardti,S. obliquus,
Nannochloropsis sp.,Anthrospira platensis,Spirogyra sp.,
and Lacustrine algae produced 26–38%, 44%, 28–50%,
24.8%–33.5%, 25%–31%, 28%, 41.3%–48.3% bio-char,
respectively [174,175].
Syngas and electricity
Synthesis gas (syngas) is deemed to be a high potential
fuel source for the production of heat, electricity as well
as a transportation fuel through fuel cells for industrial
purposes. Due to excessive flue gas exhaust caused by
fossil fuels, syngas from biomass combustion has
resulted in remarkable current attention. Experimental
studies have shown that microalgae are capable of gen-
erating a large amount of syngas from different types
of species and strains as a result of combustion [186].
Figure 13 presents stepwise syngas and electricity pro-
duction from microalgae. Syngas was generated via
various conversion processes such as direct combus-
tion, supercritical water gasification, hydrothermal gas-
ification etc. The major syngas components generated
from microalgae have been reported to be: CO, CH
4
,
CO
2
and H
2
with an average heating value of
17–24 MJkg
1dry-feed
[186,187]. Through pyrolysis,
green microalgae such as Spirulina sp,Chlorella vulgaris,
Scenedesmus almeriensis produced 50%–84% syngas
while Chlorella bulgari produced 89.21% syngas in the
presence of activated carbon. The presence of activated
Wet Microalgae
Biomass
Drying Torrefaction/ Hydrothermal
Carbonization/ Microwave
assisted Pyrolysis (200°C-750°C)
Hydrothermal
Liquefaction
Liquid-liquid
Extraction
Aqueous Co-
products
Microalgal Biochar
(Solid)
Gaseous
Products
Bio-oil
Recovery
Heat
Generation
Figure 12. Biochar production from microalgae [174–176].
Low
Concentrated
Microalgae
Condensation Microwave
Heating
Evaporation
Extraction with
solvent/nano-catalyst
Bio-CH4
Formation
Power
Generation Bio-oil
Heat CO2
Figure 11. Bio-oil production flowchart from microal-
gae [32,34,172,173].
CRITICAL REVIEWS IN BIOTECHNOLOGY 15
carbon and ZnCl
2
also revealed very low carbon mon-
oxide (CO) emissions and enhanced biohydrogen pro-
duction. During the pyrolysis of microalgae,
endothermic reactions took place and syngas has been
produced by Rc.4–Rc.7 [188–190].
CH4þH2O$CO þ3H2(Rc. 4)
CH4þCO2$2CO þ2H2(Rc. 5)
CþCO2$2CO (Rc. 6)
CþH2O$CO þH2(Rc. 7)
The gaseous elements (syngas) with liquid (bio-oil
and tar) products from microalgae were determined by
Equation (19) [188].
Yt¼Mt
Mm
100% (19)
where Y
t
¼yield of total gaseous and liquid product, M
t
¼mass of total gaseous and liquid product, M
m
¼mass
of microalgae.
The syngas has been applied as a raw material for
electricity production through microbial fuel cells
(MFC). Dried microalgae powder has been implemented
as an anode substrate in MFC. Mixed species of
Spirulina sp.,Scenedesmus sp. and C. vulgaris have pro-
duced a maximum power density 8.67 ± 0.10Wm
3
(1926 ± 21.40 mWm
2
) through MFC at R
ext
¼100 X
[36]. As a cathode substrate, C. vulgaris grown in saline
water produced a power density of
19.80–20.25 mWm
2
[37]. As the electrode substrate,
Spirulina platensis produced a power density of
2018–4433 mWm
2
[39].
Biohydrogen
Biohydrogen (BioH
2
) is one of the promising bioenergy
sources to mitigate fossil fuel utilization. Nowadays,
carbohydrate-rich microalgae are being studied for
commercial biohydrogen production [193]. Microalgae
were pretreated and degraded, subsequently the
treated biomass was run through several methods to
generate biohydrogen such as dark fermentation, pho-
tofermentation (directly under the light during cultiva-
tion), biophotolysis, solid-state anaerobic digestion, and
others. Among pretreatment processes, the thermal-
acid application has been proved as the most efficient
approach. This process was capable to recover 100%
carbohydrate as reducing sugars for fermentation.
According to laboratory experiments, different types of
tropical microalgal species such as C. vulgaris,S. obli-
quus, wastewater consortium, Chlorella sp., algae bloom
in lake, tropical native freshwater (lake) consortium
induced 81, 113.1, 46.8, 7.1, 47.1, 45.4 mL H
2
/g
VS
Microbial Fuel
Cell(MFC)
Electricity
Generation
Heat
Generation
Wet Microalgae
Biomass
Drying (300°C) Pyrolysis
(300°C-350°C)
Combustion
(500°C-750°C)
Steam/O2
Methanol
Synthesis
(850°C-1000°C)
Tar,
Ash
Syngas (H2,
CO, CH4)
Pressurized Liquid
Extraction
Supercritical
Hydrothermal
Gasification
CO2
CO2
Figure 13. Syngas and electricity production from microalgae [36,37,39,187–192].
16 N. HOSSAIN AND T. M. I. MAHLIA
biohydrogen (volatile solids), respectively [194,196].
Based on a previous experimental study, a mixotropic
culture of C. vulgaris ESP6 isolated strain, produced
240 mLL
1
h
1
biohydrogen [41]. Another study
delineated that a well-known tropical microalgae spe-
cies, C. reinhardtii, was capable to generate
2.92 mgL
1
h
1
H
2
with outdoor environmental condi-
tions [43] as well as C. reinhardtii strains such as C. rein-
hardtii 137c, C. reinhardtii ATCC 824, C. reinhardtii UTEX
90, C. reinhardtii WT 11/32B, C. reinhardtii L159I-N230Y,
C. reinhardtii CC-124, C. reinhardtii Dang 137 were cap-
able of generating 1.40 mLL
1
h
1
,6mLL
1
h
1
,
225 mLL
1
, 0.66 mLL
1
h
1
, 5.77 mLL
1
h
1
,
2.09 mLL
1
h
1
, 215 mLL
1
bioH
2
, respectively [196].
Another set of tests exhibited that S. obliquus, grown in
urban wastewater in Portugal, yielded 56.80 mLg
VS1
bioH
2
[44]. Dark fermentation of immobilized blue-
green mixed cultured microalgae with Bacillus cereus
and Brevumdimonas naejangsanensis demonstrated
cumulative bioH
2
production 1.50 mol H
2
per degraded
glucose from degraded cells of these species [197].
Based on the study of photobiological H
2
production
from freshwater (pond) green microalgae strain, C. vul-
garis MSU-AGM 14 and C. vulgaris MSU 01 processed
2 g bioH
2
L
1
h
1
and 26 mL bioH
2
L
1
d
1
, respectively
[196,198]. Besides these microalgal species and strains,
Chlorella fusca,Chlorococcum littorale,Platymonas sub-
cordiformis, other blue-green microalgae (cyanobac-
teria) such as Nostoc,Anabaena,Calothrix,Oscillatoria,
Synechocystis,Synechococcus,Gleobacter were efficiently
eligible to generate biohydrogen via divergent proc-
esses [196]. Nannochloropsis sp. microalgae was also co-
pyrolyzed with scum and yielded biohydrogen index
above 0.70 [173]. The mechanism of biohydrogen pro-
duction from microalgae via dark and photo fermenta-
tion is shown in Figure 14.
Biomethane
Biomethane (BioCH
4
), the most significant component
of biogas, and trials are being carried out on the
laboratory and pilot scale worldwide associated with
microalgal ponds since microalgae turned out to be a
new hope to produce good quality biofuels. Amongst
production methods, anaerobic digestion is the most
popular technological method for biomethane gener-
ation from microalgae. Figures 15 and 16 depict the
overall and the two-stage process for bio-methane pro-
duction from microalgae, respectively. Based on the
algal oil studies, Acutodesmus obliquus,C. vulgaris, and
Chlorella emersonii yielded 323 Lkg
1
, 406 Lkg
1
, and
308 Lkg
1
biomethane, respectively [203–205]. In add-
ition, mixed microalgae species from primary treated
wastewater and sewage yielded 0.15 L CH
4
.g
VS1
and
0.52 L CH
4
.g
VS1
on average, respectively. N. Salina,
C. reinhardtii,I. galbama,C. kessleri,Nannochloropsis sp.,
Phaeodactylum tricornutum,Spirulina maxima,
Nannochloropsis gaditana,Scenedesmus spp.,Chlorella
sorokiniana, native tropical microalgae consortium dis-
played 0.430 Lg
VS1
, 0.242 Lg
COD1
, 0.019 Lg
1
,
0.276 Lg
VS1
, 0.357 Lg
VS1
, 0.557 Lg
VS1
, 0.299 Lg
VS1
,
0.190g
VS1
, 0.390 Lg
VS1
, 0.206 Lg
VS1
, 0.200 Lg
VS1
,
Microalga
l Biomass
C6H12O6
2H2O + CH4
Hydrolysis Acidogenesis
Acetogenesis
Methanogenesis
4H2
CO2 + CH4
2CH3COOH + CH4
2C2H5OH + 2CO2
Figure 15. Biomethane production mechanism from microal-
gal biomass [200,201].
Figure 16. Two-stage process biomethane production by
microalgae (drawn by authors based on the literature [202]).
Hydrolysis
Microalgae C6H12O6+2H2OPyruvate + NADP
2CH3COOH+2CO2+4H2
(Dark Fermentation)
8H2+4CO2
(Photo Fermentation)
Glycolysis
4H2O
Figure 14. A biohydrogen production overview from microal-
gae [199].
CRITICAL REVIEWS IN BIOTECHNOLOGY 17
0.432 Lg
VS1
biomethane in average, respectively
[194,200,203,206–208]. Another study demonstrated
that a mixed culture of C. vulgaris and Scenedesmus spp.
yielded the highest rate of biomethane production
0.252 Lg
VS1
[46]. Moreover, the photosynthetic biogas
production method was efficient and yield
97.2%–97.4% biomethane when connected in a pond
with a high concentration of algal biomass [209].
Conclusions
Biofuel production by microalgae has been projected to
be a successful and realistic approach for alternative
fuel technology processes by biofuel experts all over
the world. A comprehensive microalgae cultivation ana-
lysis with maximum productivity was the pre-requisite
for the desired amount of biofuel production. The latest
research focusses on advancements in physicochemical
parameters in this review and presents revolutionary
enhancements in microalgae biomass yield. This review
also highlights significant research and development
(R&D) for commercial microalgae cultivation as a source
of biofuels. R&D has uncovered several new ways to
optimize the biomass yield in terms of various types of
bioreactor, different cultivation conditions and factors
based on various algal strains and their geography.
Along with microalgae cultivation and biofuel gener-
ation, integration of value added co-product extraction
will be an additional advantage for the economic scen-
ario. This review encourages the biofuel R&D sector to
convert unused, abandoned waste water sources, and
wet barren lands into microalgae culturing farms as a
significant source of biofuel production. It is strongly
recommended to examine approaches for large scale
process development, economical feasibility with value
added co-products generation and optimization stipula-
tions for future commercial purposes. Moreover, it is
also recommended to determine the foremost effective
conversion key vehicles for microalgal biofuel produc-
tion industrially.
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
Nazia Hossain http://orcid.org/0000-0001-7925-0894
Teuku Meurah Indra Mahlia http://orcid.org/0000-0002-
6985-929X
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