Journal Pre-proofs

Insights into upstream processing of microalgae: A review

Ehsan Daneshvar, Yong Sik Ok, Samad Tavakoli, Binoy Sarkar, Sabry M.
Shaheen, Hui Hong, Yongkang Luo, Jörg Rinklebe, Hocheol Song, Amit
Bhatnagar

PII: S0960-8524(21)00209-1
DOI: https://doi.org/10.1016/j.biortech.2021.124870
Reference: BITE 124870

To appear in: Bioresource Technology

Received Date: 1 January 2021

Revised Date: 10 February 2021
Accepted Date: 12 February 2021

Please cite this article as: Daneshvar, E., Sik Ok, Y., Tavakoli, S., Sarkar, B., Shaheen, S.M., Hong, H., Luo, Y.,
Rinklebe, J., Song, H., Bhatnagar, A., Insights into upstream processing of microalgae: A review, Bioresource
Technology (2021), doi: https://doi.org/10.1016/j.biortech.2021.124870

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2021 Elsevier Ltd. All rights reserved.

Insights into upstream processing of microalgae: A review

Ehsan Daneshvar a,1, Yong Sik Ok b,1, Samad Tavakoli c, Binoy Sarkar d, Sabry M.

Shaheen e, f, g, Hui Hong c,h, Yongkang Luo c,h, Jörg Rinklebe e, i, Hocheol Song j, Amit

Bhatnagar a, *

aDepartment of Separation Science, LUT School of Engineering Science, LUT University,

Sammonkatu 12, FI-50130, Mikkeli, Finland
b Korea Biochar Research Center, APRU Sustainable Waste Management Program and Division of
Environmental Science and Ecological Engineering, Korea University, Anam-ro 145, Seongbuk-gu,
Seoul 02841, Republic of Korea
cCollege of Food Science and Nutritional Engineering, China Agricultural University, Beijing,
100083, China
d Lancaster Environment Centre, Lancaster University, Lancaster, LA1 4YQ, United Kingdom
e University
of Wuppertal, School of Architecture and Civil Engineering, Institute of Foundation
Engineering, Water- and Waste-Management, Laboratory of Soil- and Groundwater-Management,
Pauluskirchstraße 7, 42285 Wuppertal, Germany
f King
Abdulaziz University, Faculty of Meteorology, Environment, and Arid Land Agriculture,
Department of Arid Land Agriculture, Jeddah 21589, Saudi Arabia
g Universityof Kafrelsheikh, Faculty of Agriculture, Department of Soil and Water Sciences, 33516
Kafr El-Sheikh, Egypt
hBeijing Higher Institution Engineering Research Center of Animal Product, China Agricultural
University, Beijing, 100083, China
i University
of Sejong, Department of Environment, Energy and Geoinformatics, 98 Gunja-Dong,
Guangjin-Gu, Seoul, Republic of Korea
jDepartment of Environment and Energy, Sejong University, 209 Neungdong-ro, Gwangjin-gu, Seoul,
05006, Republic of Korea

1These authors share co-first-authorship.

Corresponding author: Email address: [email protected]; [email protected] (A.

Bhatnagar)

Page 1 of 49
Abstract

The aim of this review is to provide insights into the upstream processing of microalgae, and

highlighting the advantages of each step. This review discusses the most important steps of

the upstream processing in microalgae research such as cultivation modes, photobioreactors

design, preparation of culture medium, control of environmental factors, supply of

microalgae seeds and monitoring of microalgae growth. An extensive list of bioreactors and

their working volumes used, elemental composition of some well-known formulated

cultivation media, different types of wastewater used for microalgae cultivation and

environmental variables studied in microalgae research has been compiled in this review

from the vast literature. This review also highlights existing challenges and knowledge gaps

in upstream processing of microalgae and future research needs are suggested.

Key words: Microalgae cultivation; Bioreactors; Culture media; Wastewater treatment;

Environmental factors; Upstream processing.

Page 2 of 49
1. Introduction

Microalgae are a diverse group of microorganisms that can be found in water, soil

(Subashchandrabose et al., 2011), air (Sahu and Tangutur, 2015), trees bark microhabitats (Wicker

and Bhatnagar, 2020), and in some cases, even on animals (Pauli et al., 2014). Microalgae are

eukaryotic microorganisms containing chlorophyll a found as individual cells or small

colonies (Phwan et al., 2018). As compared to other microorganisms and terrestrial plants,

microalgae have several unique advantages. As primary producers and capable of performing

photosynthesis, they absorb sunlight (photons) and assimilate carbon dioxide (CO2) from the

atmosphere for biomass production. On the other hand, the majority of bacteria and fungi

without photosynthetic apparatus have to feed on organic matters. Unlike plants, the growth

of microalgae is not limited to arable lands and fresh water (Sajjadi et al., 2018). Microalgae

can be cultivated on unproductive lands, such as infertile, arid, semi-arid lands, and polluted

soils that are not usable for conventional agriculture (Junying et al., 2013). Also, these

microorganisms can grow in saline water and even in nutrient-enriched wastewater.

Moreover, microalgae cultivation is not limited to seasons; it can be repeated year-around and

can be harvested daily (Gouveia and Oliveira, 2009).

Because of the above-mentioned benefits and their numerous applications, microalgae have

attracted the attention of researchers from various fields such as environmental sciences,

biology, genetics, chemistry, chemical engineering, medicine, polymer science, agriculture,

and aquaculture. Research on microalgae is diverse, from genomic investigation to

wastewater treatment, from pharmaceutical extraction to bioenergy production, from CO2

bio-mitigation to biofertilizer manufacturing, among others. Microalgae research mainly

comprises of upstream, midstream, and downstream processes. Upstream processing focuses

on microalgae cultivation and maximization of biomass production. Midstream processing

aims to harvest microalgae from cultivation media, dry the collected biomass, and rupture the

Page 3 of 49
microalgal cell walls before the extraction process. Downstream processing targets the

extraction and purification of the bioproduct(s) from microalgal biomass (Manirafasha et al.,

2016).

Upstream processes are considered as the baseline in microalgae research. These processes

are important, technically and economically, as they directly affect the quality and quantity of

the produced microalgal biomass. Upstream processes have several main steps (e.g.

bioreactor design, cultivation media preparation, CO2 supplementation, and adjustment and

control of environmental factors, etc.) that should be considered in microalgae research.

Owing to the importance of determining steps in upstream process, they have been reviewed

previously. For example, the design and operation of different types of photobioreactors for

microalgae cultivation has been reviewed (Vo et al., 2019). Li et al. (2019) evaluated the

application of industrial, agricultural, and municipal wastewaters for microalgae cultivation.

In another study, influence of different factors such as light, nutrients, pH, and CO2 on

microalgae growth were investigated (Junying et al., 2013). In previously published articles,

some important factors of upstream process along with midstream and downstream processes

were discussed. For instance, cultivation (upstream) and harvesting (midstream) of

microalgae for biodiesel and biofuel production (downstream) was reviewed by Yin et al.

(2020).

Despite the availability of several review articles related to different aspects of microalgae

cultivation, there is lack of a comprehensive review on the upstream processes in microalgae

research. Previously published review papers do not address all steps of microalgae

cultivation and usually, their content has focused on a specific research field such as

wastewater treatment, biodiesel production, agriculture, biomedicine etc. Thus, the aim of

this review is to provide insights into the upstream processing of microalgae and highlighting

the advantages of each step. For this purpose, the main pillars of upstream process in

Page 4 of 49
microalgae research are discussed, including different cultivation modes, photobioreactor

design, culture media preparation, microalgae supply, environmental factors, and microalgal

growth monitoring. Overall, this review attempts to present comprehensive recommendations

for microalgae upstream processing.

2. Microalgae cultivation modes

Like all living cells, microalgae also need a source of energy and starting materials to

maintain steady biosynthesis, growth, and cell division (Sun et al., 2018). Depending on the

sources of carbon and energy used, microalgae are categorized into photoautotrophic,

heterotrophic, mixotrophic, and photoheterotrophic (Fig. 1), as also reported elsewhere (Hu et

al., 2018). It should be noted that heterotrophic and mixotrophic microalgae have the ability of

photoautotrophic metabolism also. In fact, heterotrophic and mixotrophic as secondary

metabolic pathways might be observed in some photoautotrophic microalgae species.

Cultivation of microalgae occurs via four pathways namely photoautotrophic, heterotrophic,

mixotrophic, and photoheterotrophic modes. Cultivation modes directly affect the

requirements of the research and design, and the resulting growth of microalgae and

biochemical composition of biomass. Therefore, it is one of the very first factors that needs to

be determined in microalgae research.

2.1. Photoautotrophic cultivation of microalgae

The photoautotrophic process is the oldest and most common method of microalgae

cultivation (Chew et al., 2018). Photoautotrophic microalgae biosynthesize organic matter by

utilizing inorganic carbon as the source of carbon and light as a source of energy (Huang et al.,

2010), forming chemical energy via photosynthesis. Eq. 1 shows biofixation of carbon and

photosynthesis in organisms with chlorophyll a:

Light
CO2 + H2O C6H12O6 + O2 (Eq. 1)

Page 5 of 49
CO2 and bicarbonate (HCO3-) are the foremost sources of carbon for cell growth of

photoautotrophic microalgae (Kim et al., 2014). This implies that sequestration of CO2 occurs

via photoautotrophic cultivation mode. The ability of CO2 biofixation by phototrophic algae

has attracted the attention of researchers toward the development of carbon capture and

utilization (CCU) strategies. CCU, as a distinguishing feature of microalgae, has two major

benefits: it can assist in reducing greenhouse gas (GHG) emissions and, consequently,

contribute towards mitigating climate change. In addition, the carbon captured by microalgae

is fixed in their molecular structure, such as lipids, proteins, and carbohydrates, which can be

utilized to produce many value-added biobased products (Subhash et al., 2017). Lower

biological contamination risk is another advantage of the photoautotrophic cultivation mode.

This is due to the absence of organic carbon in the autotrophic cultivation, which protects the

medium against the heterotrophic bacteria. Hence, this cultivation mode is more appropriate

for the outdoor cultivation of microalgae than other cultivation modes (Chew et al., 2018). The

photoautotrophic mode is recommended for outdoor scale-up cultivation of microalgae, but

its application is limited by light-dependency. Large-scale outdoor photoautotrophic

cultivation with artificial light is expensive, therefore, finding a suitable location is essential

to optimize the process. Sunlight irradiation varies depending on the geographical region,

season, and climatic condition. The lower biomass productivity of microalgae cultivated

under the photoautotrophic mode as compared to the heterotrophic and mixotrophic

cultivation modes is another drawback. Lower biomass productivity in the photoautotrophic

cultivation mode is attributed to the self-shading effect on the microalgal vertical distribution

that prevents light availability for denser cultivation (Nitsos et al., 2020). Hence, light as a

single source of energy has a critical role in the successful implementation of

photoautotrophic microalgae cultivation mode.

2.2. Heterotrophic cultivation of microalgae

Page 6 of 49
In the heterotrophic cultivation mode, microalgae can grow in the absence of light.

Heterotrophic microalgae species can provide the required carbon and energy for cellular

metabolism through the consumption of organic carbon (Lam and Lee, 2012a). Heterotrophic

microalgae have unique features. Biomass productivity in the heterotrophic cultivation mode

is higher than that in the photoautotrophic cultivation mode. This is due to the light-

independency of heterotrophic microalgae that facilitate high cell density without

photoinhibition, a limiting factor in photoautotrophic mode (Chew et al., 2018). The feasibility

of microalgae cultivation under dark conditions reduces the requirement of high surface to

volume ratio, which eases the design of the heterotrophic microalgae bioreactor (Zhan et al.,

2017). Overall, high biomass production and light-independency of the heterotrophic

cultivation reduce production costs compared to the photoautotrophic cultivation.

Nonetheless, the heterotrophic cultivation has several disadvantages that need to be

considered. All microalgae can grow photoautotrophically, but few species can grow

heterotrophically. Heterotrophic microalgae cannot consume CO2, even though they generate

CO2 through the metabolism of organic carbon. Therefore, they are not useful in CO2

mitigation research (Hu et al., 2018). The high risk of biological contamination with competing

heterotrophic microorganisms, such as bacteria, yeast, and fungi, is another drawback of the

heterotrophic cultivation mode, which could negatively affect biomass production and quality

of the products of interest. Under heterotrophic conditions, other heterotrophic

microorganisms compete with heterotrophic microalgae for the same sugar-based organic

carbon substrates. Thus, the growth of microalgae is reduced in the presence of bacteria

having high growth rate and less doubling time (Hu et al., 2018). For the heterotrophic

cultivation, everything in contact with microalgae such as reactor, supplied gases, and the

medium, needs to be sterilized thoroughly to avoid culture contamination (Di Caprio et al.,

Page 7 of 49
2019). This problem is more significant for outdoor microalgae cultivation, including open

ponds and raceways due to uncontrolled conditions (Bilad et al., 2014).

2.3. Mixotrophic cultivation of microalgae

Some microalgae species grow under mixotrophic conditions by using inorganic carbon and

organic compounds simultaneously. Suitable microalgae for the mixotrophic cultivation have

cellular apparatus for the photoautotrophic and heterotrophic metabolism, and based on the

definition of mixotrophy, both inorganic and organic carbon are necessary for their growth.

Although during mixotrophic cultivation, microalgae can grow photoautotrophically or

heterotrophically, there is no apparent switch between heterotrophic and autotrophic

metabolisms (Grobbelaar, 2013). Hence, switching between these two modes should not be

confused with mixotrophy. The mixotrophic microalgae need illumination for biofixation of

CO2 through photosynthesis, and organic substrates for aerobic respiration while in total

darkness, the metabolism turns to heterotrophy (Perez-Garcia and Bashan, 2015). Mixotrophic

microalgae, possessing photoautotrophic and heterotrophic features, benefit from the

advantages of photo- and heterotrophic modes. The combined use of CO2, organic

compounds and light is the distinguishing property of mixotrophic microalgae. This ability

maximizes the usage of different resources to supply carbon and energy demands, and

supports the requirements of both photoautotrophic and heterotrophic metabolisms. In the

mixotrophic cultivation mode, the light requirement is lower than for photoautotrophic

growth, which eliminates the associated light limitation. Some compounds, such as pigments,

which are not produced in the heterotrophic cultivation mode due to light absence, are

produced in mixotrophic cultivation (Lee, 2003). Like photoautotrophic microalgae,

mixotrophic microalgae participate in CO2 reduction via photosynthesis. The released CO2

from respiration under heterotrophic metabolism is trapped and reused during

photoautotrophic growth (Gaignard et al., 2019), which overall decreases CO2 emissions

Page 8 of 49
compared to the heterotrophic cultivation mode. Ananthi et al. (2021) reported that biomass

productivity in the mixotrophic cultivation mode is higher than in the photoautotrophic and

heterotrophic cultivation modes. Li et al. (2014) found that the maximum dry weight of

Chlorella sorokiniana cultivated mixotrophically was 2.4 and 5.2 times that of the same

species cultivated heterotrophically and photoautotrophically, respectively. While the

mixotrophic cultivation mode holds the advantages of both heterotrophic and

photoautotrophic cultivation modes, microalgae cultivation via mixotrophic mode also

encounters various disadvantages. Similar to the heterotrophic cultivation mode, the

application of an organic substrate increases the cost of the mixotrophic cultivation.

Consequently, the maintenance of axenic cultures in a pure state is difficult because of the

sugar-based culture compounds, which increase the risk of contamination by unwanted

heterotrophic microorganisms. Although mixotrophy reduces light-dependency, light is still a

vital element for microalgae growth. Therefore, the mixotrophic cultivation requires organic

substrates and sterilization (to prevent contamination) in addition to the presence of light that

can increase the overall cost of the bioreactor design and operation. Moreover, only few

microalgae species grow mixotrophically, which diminishes the research opportunity that can

benefit from biodiversity. Details of several heterotrophic and mixotrophic microalgae

species can be found in a review published by Hu et al. (2018).

2.4. Photoheterotrophic cultivation of microalgae

Photoautotrophy is also known as photo-organotrophy, photo-metabolism, or photo-

assimilation (Chew et al., 2018). Photoheterotrophic microalgae like Chlorella vulgaris ESP-31

are a group of microalgae that require light as a source of energy, and organic carbon as a

source of carbon (Yeh et al., 2012). Unlike photoautotrophs and mixotrophs, photoheterotrophs

cannot metabolize CO2. In contrast to heterotrophs, photoheterotrophs cannot grow on

glucose without light. The photoheterotrophs use glucose as a building material, but not as

Page 9 of 49
the source of energy. In the light phase of photosynthesis, the energy of light is transformed

into chemical energy of nicotinamide adenine dinucleotide phosphate (NADPH) and

adenosine triphosphate (ATP). The synthesized ATP and NADPH are used in dark phase for

the assimilation of glucose to biomass. In contrast to mixotrophs and heterotrophs, the

generation of CO2 is negligible in photoheterotrophs as the Calvin cycle is not active

(Chojnacka and Marquez-Rocha, 2004). Photoheterotrophy is an expensive cultivation mode as

microalgae need both organic carbon and illumination for growth and special design of

photobioreactor is required for microalgae cultivation (Chew et al., 2018; Ananthi et al., 2021).

Considering the discussion presented above, it can be concluded that microalgae benefit from

a worthwhile metabolic diversity. The diverse metabolic pathways enable microalgae to adapt

to and use different sources of energy and carbon. Microalgae cultivation modes can be

flexible depending on the availability of light, CO2, and organic carbon (Fig. 1). Also,

photoautotrophic, heterotrophic, mixotrophic, and photoheterotrophic metabolisms fit for

different research goals and industrial applications of microalgae. Although photoautotrophy

is the dominant metabolic pathway, all other individual pathways have their own advantages

which make them suitable for a variety of applications. Clear identification of metabolic

pathways must be performed and required sources of energy and carbon should be provided

for microalgae growth for achieving target outcomes.

3. Bioreactors for microalgae cultivation

In microalgae research, the selection of an appropriate vessel or container for microalgae

cultivation is one of the important factors that need to be decided at an early stage. Typically,

the term bioreactor is applied to the containers which support the growth of microalgae for

biomass production and product formation. In this regard, photobioreactors refer to

bioreactors which supply light for phototrophic microalgae that need light as a source of

Page 10 of 49
energy. Bioreactors with different sizes, shapes and materials are available for microalgae

cultivation. The design can be as simple as handmade bioreactors to high-tech

photobioreactors. Handmade bioreactors are mainly transparent glassware or polymeric

materials such as polycarbonate, which can be assembled by researchers themselves or

manufactured and sold by local companies. For instance, Choi et al. (2019) designed a

polymer film-based photobioreactor for microalgae cultivation. They developed a

polypropylene-based bubble column photobioreactor (10 cm diameter and 120 cm height) for

the cultivation of several microalgae species. Erlenmeyer flasks, bottles, or jars with different

volumes have been widely used as bioreactors for microalgae cultivation. These are equipped

with a tube for aeration and mixing of medium and covered by a cap, cotton stopper,

aluminum foil, or parafilm to decrease evaporation and contamination (Aghaalipour et al.,

2020; Supraja et al., 2020). Fig. 2(A) shows a schematic of a typical handmade bioreactor for

microalgae cultivation designed by Daneshvar et al. (2019). The handmade bioreactor has one

inlet for the injection of air to supply CO2 and mixing power, one outlet for taking samples,

and one small hole for gas venting (Fig. 2(A)). A syringe connected to the outlet tube

facilitates the collection of samples necessary for the evaluation of microalgal growth,

biochemical analysis, and other specific measurements depending on the research objectives.

In addition, the sealing cap reduces the evaporation rate and protects the culture from

contamination better than other sealing options such as cotton, parafilm, and aluminum foils.

Although these simple bioreactors successfully assist in microalgal growth and biomass

production and can meet the requirements of many research topics, they are not always

appropriate. For example, in some experiments, pH level, CO2 and O2 concentrations,

temperature etc. should remain constant. In this case, commercially available advanced

bioreactors are required for controlling and monitoring sensitive parameters and to achieve

optimal system performance during the experiments. These high-tech bioreactors can regulate

Page 11 of 49
parameters such as temperature, pH, O2 and CO2 pressure, mass and heat transfer, nutrient

supply, hydraulic retention time, fluid velocity, shear stress, and cell growth (Mustafa et al.,

2018). Depending on the model and application, advanced bioreactors can control several of

the above-mentioned parameters. For instance, Li et al. (2003) used a stirred-tank fermenter

model BiofloIII, New Brunswick Scientific, Edison, NJ, as a bioreactor for microalgae

cultivation. They equipped the bioreactor with a pH sensor, pH meter, CO2 mass flow

controller, air mass flow controller, dissolved oxygen sensor, and oxygen meter. Typically,

this kind of high-tech photobioreactor has a data acquisition board and supervisory computer

for online monitoring of the parameters (Fig. 2(B)) (Naira et al., 2019). Handmade bioreactors

can also be upgraded with individual sensors based on the requirements of the experiment.

For example, Khichi et al. (2019) attached a pH and temperature probe, mass flow controller,

and heating/cooling coil system to a photobioreactor to control the pH and temperature of the

culture.

Microplates or multiwell plates have also been used for microalgae cultivation. Microplates

can be considered as miniature bioreactor that are appropriate for the experiments with high

numbers of treatments in tiny volume of microliters to milliliters. Fig. 2(C) shows a 96-well

microplate containing 12 column and 8 rows. Dao et al. (2018) cultivated microalgae in a

transparent 96-well microplate. Each well was filled with 100 μL of medium, and the

microplate was sealed with a breathable sealing film. In another study, Kim et al. (2019) used a

96-well microplate with 200 μL working volume for microalgae cultivation. To isolate the

experimental units, microplates were covered using a gas and light permeable membrane.

We would like to add to this discussion that the bioreactors which are used for microalgae

cultivation are extremely diverse in shape, volume, and materials. Table 1 presents a list of

bioreactors (and their working volumes) that have been used for the cultivation of different

microalgae strains in synthetic media and wastewater. The information presented in Table 1

Page 12 of 49
shows that the size of the bioreactors used in microalgae research varies from microliter (100

μL) to as large as thousands of liters (33,000 L). The volume of a research bioreactor does not

limit the selection of microalgae species, cultivation modes, and cultivation media (Table 1).

A microalgae research laboratory that is called phycolab, depending on the requirements of

the research, may include different types of bioreactors for microalgae cultivation. In

microalgae research, usually, preliminary studies are performed in small bioreactors such as

Erlenmeyer, bottles, or glass jars. As it has been illustrated in Fig. 3(A), these bioreactors can

be arranged in a shelf, and illuminated by artificial lights. The optimized experimental

conditions are used for scaling up the microalgae cultivation in larger bioreactors. Open

ponds, raceways, tubular photobioreactors, and flat plate photobioreactors are used for large-

scale microalgae cultivation. Open ponds and raceways are usually constructed using cement

and polyvinyl chloride materials. These ponds are shallow (around 30 cm) such that more

light can be absorbed maximizing the photosynthesis rate. A pedal is used to circulate and

mix the culture medium in raceway ponds. Tubular photobioreactors are long tubes, made

from glass or transparent materials. Usually, the diameter of the tubes is less than 10 cm for

appropriate light penetration. Tubular photobioreactors might have horizontal or vertical

forms. Horizontal tubes have panel-like system (tubes on the ground) and fence-like system

(tubes parallel up together). Vertical tubes are divided into bubble and airlift columns. Flat

plate photobioreactors with high surface area for light absorption are installed vertically or

inclined toward light sources. Due to short light-path and efficient light penetration,

rectangular photobioreactors are commonly used for microalgae cultivation. Similar to

vertical photobioreactors, an air sparger connected to air pump is used to mix and circulate

the medium in flat plate photobioreactors. Different types of photobioreactors and their

properties has been discussed in detail by Chew et al. (2018). Fig. 3 shows three types of

common bioreactors viz., flat plate (B), spiral (C), and bubble column (D) bioreactors, which

Page 13 of 49
are used frequently for pilot-scale cultivation of microalgae. Appropriate aeration,

illumination, medium circulation, and mass/heat transfer must be considered carefully in

designing of larger bioreactors.

4. Culture media and nutrients supplementation

Culture media are solutions containing essential nutrients that are needed by microalgae to

maintain a steady state, good health, and growth (Procházková et al., 2014; Grobbelaar, 2013).

Nutrients are categorized into macronutrients, micronutrients, and trace elements depending

on their required amount for optimal growth. The first group includes elements, such as

carbon (C), hydrogen (H), oxygen (O), nitrogen (N), and phosphorous (P), that microalgae

need in higher amount (g/L) in the cultivation media. Lower concentrations (mg/L or less) of

micronutrients such as cobalt (Co), zinc (Zn), manganese (Mn), and barium (Ba) in

cultivation media are sufficient for microalgae growth and biomass production (Grobbelaar,

2013).

Formulated media and different wastewaters (usually enriched in nitrogen and phosphorus

compounds) are frequently used as culture media to supply nutrients for microalgae growth

(Table 1). Formulated media are synthetic broth with recommended concentrations of micro-

and macro-nutrients. These media have been extensively tested for the cultivation of different

freshwater and marine microalgae species (e.g. f/2 medium developed by Guillard (1975)),

institutes (Culture collection of Algae at The University of Texas, UTEX,), or commercial

groups (The Culture Collection of Algae and Protozoa, CCAP). As stated in Table 2,

formulated media are popular by their abbreviates or commercial names such as BBM

(Bold’s Basal Medium), f/2 (Guillard), and BG11 (Blue-Green), in microalgae research

community. Each synthetic medium comes with specific instructions including the names of

Page 14 of 49
components (macro- and micronutrients) and their concentrations (mass concentration or

molarity), which describe the stepwise solution preparation (Polat et al., 2020).

Formulated media could be applicable as non-specific media, therefore, being useful for the

cultivation of many microalgae species or they might be designed for a specific group of

microalgae. For example, Bold’s Basal Medium (BBM) and Guillard (f/2) are two commonly

used formulated media for the cultivation of a diverse group of freshwater and marine

microalgae, respectively. Blue-Green (BG11) is an appropriate medium for the cultivation of

cyanobacteria, but it is also extensively used for the cultivation of microalgae (Enamala et al.,

2018). On the other hand, Zarrouk and f/2 + Silicon (Si) (Guillard’s medium for diatoms) are

specialized media for the cultivation of Spirulina sp. (cyanobacteria) and diatoms,

respectively (Araújo et al., 2013; Costa et al., 2018). A list of commonly used formulated media

for the cultivation of cyanobacteria, freshwater and marine microalgae, and their properties

have been reported by Geada et al. (2017). Procházková et al. (2014) introduced 30 elements as

sources of macro- and micronutrients for the autotrophic cultivation of microalgae. Chemical

compounds containing these elements and their concentrations in a few popular media are

presented in Table 2. It should be noted that supplying the essential elements is not limited to

the compounds presented in this table, and it depends on the recipe of the media. Based on

the information provided in Table 2, we can summarize that the concentrations of some

compounds such as vitamins are needed at very low concentrations (µg/L) in culture media.

Usually in microalgae laboratories, the culture media are prepared by diluting highly

concentrated solutions of individual compounds.

‘Modified media’ is another term that is commonly used when composition of medium is

changed slightly by increasing or decreasing the original concentrations of compound(s) in

culture media. Modified media are used to enhance microalgae biomass production, to

stimulate the production of special metabolites, or to study the effects of nutrient deficiency

Page 15 of 49
or deprivation based on the requirement of experimental design. For example, Anand et al.

(2019) used modified BG-11 media at various concentrations (10 – 100 mM) of NaCl,

MgCl2·6H2O, and CaCl2·2H2O for cultivation of Scenedesmus vacuolatus. They tested

salinity-driven stress as a biodiesel trigger to enhance lipid production in microalgae. The

same mixture of nutrients that has been designed for the autotrophic cultivation of microalgae

(Table 2) can be used for the heterotrophic, mixotrophic, and photoheterotrophic cultivation

modes after the addition of organic carbon sources such as glucose, acetate, or glycerol

(Perez-Garcia and Bashan, 2015). Smith et al. (2020) used original f/2 and modified f/2 (f/2

medium containing Si) for the photoautotrophic cultivation of green microalga and diatoms

(marine microalgae that need silicon to grow), respectively. Glucose, glycerol, and acetate as

organic carbon sources were added to f/2 medium for the heterotrophic cultivation of the

same microalgae.

Nutrient-enriched wastewaters are another low-cost and freely available medium that can

provide required macro- and micronutrients in addition to water for microalgal growth.

Contrary to formulated media, concentrations of nutrients in wastewater are unknown. When

evaluating the suitability of wastewater as microalgae cultivation media, the concentrations of

nutrients, specifically nitrogen (N), phosphorus (P), and carbon (C) must be quantified. The

threshold concentrations of these nutrients in wastewater should be considered to avoid

microalgal growth limitation. The elemental ratio of C:N:P in microalgal biomass are

approximately 106:16:1, which is known as the Redfield ratio (Grobbelaar, 2013). However,

the Redfield ratio is used to estimate the limitations of essential nutrients in microalgae

cultivation medium, but it cannot be generalized as an optimum value for all microalgal

species. Depending on the species, the ratio of N:P in wastewater can vary from 8 to 45

(Salama et al., 2017). In this regard, various types of domestic, agricultural, and industrial

wastewaters have been tested for the cultivation of numerous freshwater and marine

Page 16 of 49
microalgae species (Table 3). The characteristics of the wastewater used for microalgae

cultivation are usually provided in research articles. The main characteristics include physical

and chemical properties (e.g., pH, total suspended solid, color, and electrical conductivity)

and the concentrations of nutrients (e.g., NH4+, NO2−, NO3−, and PO43−). For example, Ansari

et al. (2019) measured several characteristics of municipal wastewater including pH, color,

odor, temperature, electrical conductivity, total dissolved solids, salinity, dissolved oxygen,

chemical oxygen demand, biochemical oxygen demand, alkalinity, NH4+, NO2−, NO3−, PO43−,

Fe, Zn, Na, and Mg. The utilization of wastewater as a medium for microalgae cultivation has

several advantages including i) low-cost production of microalgae biomass; ii) recovery of

nutrients from wastewater; and iii) treatment of wastewater for safe discharge (Salama et al.,

2017). Wastewater as cultivation medium has been utilized specifically for wastewater

treatment (Li et al., 2019), bioenergy production (Ananthi et al., 2021), and CO2 sequestration

(Razzak et al., 2017). It should be noted that microalgae biomass produced in wastewater

cannot be used for food, cosmetics, medicine production etc. for human consumption due to

hygienic point of view (the risk of contamination of biomass produced in wastewater with

organic and inorganic pollutants and microbes, which could be detrimental to human health).

Although the biomass produced in formulated media can be used for different applications

and human consumption, mass production in synthetic media is expensive compared to

wastewater. Therefore, formulated medium and wastewater as cultivation media have their

own advantages and disadvantages in microalgae research.

5. Adjustment of environmental factors governing microalgal growth

Several environmental factors, such as pH, temperature, irradiation, and aeration need to be

adjusted before cultivation of microalgae (Table 4). These parameters not only affect the

growth of microalgae, but also influence the biochemical composition of microalgal biomass.

Page 17 of 49
The individual and combined effects of these factors on microalgal growth have been

researched and reviewed extensively. After determining the cultivation mode, bioreactor

selection/design, and preparation of cultivation media, environmental factors must be

adjusted, before inoculation of microalgae. Below, we review and summarize the most

important environmental factors which affect microalgal growth and biochemical

composition.

5.1. Light

Microalgae have different types of pigments, such as chlorophyll a (all microalgae),

carotenoids, phycoerythrin (red microalgae), and phycocyanin (cyanobacteria) (Detweiler et

al., 2015). Photosynthetic microalgae can convert the energy of light to chemical energy using

these pigments. Therefore, except for the heterotrophic cultivation, all the photoautotrophic,

mixotrophic, and photoheterotrophic cultivation modes must have a light supply as the

primary energy source for microalgal growth. Natural sunlight, and fluorescent and LED

lights have been utilized as lighting system for microalgae cultures (Table 4). Ordinary

fluorescent lights, which are conventionally used in microalgae research, irradiate indivisible

growth-efficient and growth-inefficient wavelengths (Ra et al., 2016). LEDs produce a

narrower spectral range, which are more compatible with the absorption bands of microalgae

pigments (Hsieh-Lo et al., 2019). Despite stimulating microalgae cultures with fluorescent

lights or LEDs, the optimization of the lighting conditions is one of the key factors for

achieving the highest growth rate of microalgae. Light intensity, wavelength, and photoperiod

(lightning time) are three important characteristics of light that can significantly affect the

growth of microalgae in photosynthetic cultures. Light intensity is the amount of light

received on the surface per second (μmol m−2 s−1). Table 4 presents a wide range of light

intensities (from <100 μmol m−2 s−1 to >1000 μmol m−2 s−1) that have been tested in for

microalgal growth.

Page 18 of 49
The relationship between light intensity and photosynthesis rate is shown by photosynthetic

light-response curve. This curve has three phases, namely light-limitation, light-saturation,

and photoinhibition phase. Light limitation and photoinhibition are the two phases, capable of

decreasing or even terminating the microalgal growth. Light limitation occurs in cultures with

insufficient light intensity or high cell density. This could arise due to the self-shading effect

in reactors with a high biomass concentration that reduces the amount of light, penetrating

through the bioreactor, and negatively affects photon absorption and photosynthetic

efficiency (Holdmann et al., 2019). In the light-limitation phase, increase in the light intensity

enhances microalgal growth up to the area of light saturation. Further increase in light

intensity in this phase does not affect microalgal photosynthesis, while photoinhibition occurs

at light intensities higher than saturation point. Intensive irradiation at photoinhibition phase

damages photosystem II and decreases microalgal growth significantly, or collapses the

culture (Hsieh-Lo et al., 2019). The approximate light intensities of light-limitation, light-

saturation, and photoinhibition phases are up to 300 μmol m−2 s−1, 300-1600 μmol m−2 s−1,

and > 1600 μmol m−2 s−1, respectively (Straka and Rittmann, 2018).

The light spectrum of solar radiation consists of diverse wavelengths of energy, most of

which cannot be utilized by microalgae. Microalgae can use visible wavelengths from 400 to

700 nm through photosynthesis. This narrow spectrum is called photosynthetically active

radiation (PAR) range (Vadiveloo et al., 2015) and includes 400–500 nm and 600–700 nm

wavelengths (blue and red, respectively) which is the appropriate range for optimal

microalgal photosynthesis, and 500–600 nm and 700–800 nm (green-yellow and far-red,

respectively) are the transmitted or reflected wavelengths (Ramanna et al., 2018). Each

pigment has major absorption bands that can absorb specific wavelengths of PAR. For

example, the major absorption bands of chlorophyll a, chlorophyll b, and carotenoids are 450-

475 nm (blue of blue-green), 630-675 nm (red), and 500-600 nm, respectively (Teo et al.,

Page 19 of 49
2014). Zhao et al. (2013) reported the highest dry weight of Chlorella sp. as 412.93, 470.74,

518.43, and 560.79 mg/L under red light irradiation with intensities of 800, 1200, 1600, and

2000 μmol m−2 s−1, respectively. Fozer et al. (2019) found a higher photosynthetic efficiency

under mixed color irradiation than under monochromatic irradiation. They reported the

highest biomass productivity of 60.4, 50.0, 41.2, 40.3, 33.4, 31.7, and 29.86 mg/L/d under

purple (626 nm, 470 nm), blue-green (525 nm, 470 nm), yellow (626 nm, 525 nm), white

(626 nm, 525 nm, 470 nm), blue (470 nm), red (525 nm), and green (626 nm) illumination,

respectively.

In case of outdoor cultivation, microalgae receive a light based on natural day-night rhythm.

Under controlled laboratory conditions, the duration of lighting or photoperiod can vary from

0 (the heterotrophic cultivation mode) to 24 h. Microalgal growth has been evaluated under

different photoperiod cycles (e.g., 12:12, 14:10, 16:8, and 24:0 h light:dark) (Table 4). For

example, the highest cell density of the microalga Nannochloropsis sp. (3.0 × 107 cell/mL)

was observed in a 24:0 h photoperiod (Wahidin et al., 2013). Cell density decreased from 2.1 ×

107 to 1.3 × 107 cell/mL when the photoperiod decreased from 18:06 to 12:12 h. In another

study, the specific growth rates of Chlorella vulgaris were found to be 1.20, 1.8, and 1.7 /d at

photoperiods of 24:00, 16:08, and 12:12 h (light:dark), respectively (Atta et al., 2013). It

should be noted that 24 h lighting is not necessary for the continuous growth of all

microalgae species, and the optimum photoperiod depends on the light intensity and the

microalgae strains (Lam and Lee, 2012b).

5.2. The pH of cultivation medium

The pH of cultivation medium is another factor that significantly influences microalgae

metabolism and growth. The pH value controls the acid-base balance in the cultivation

medium, and affects the solubility and availability of different forms of inorganic carbon

(CO2, bicarbonate, and carbonate) and nutrients (phosphates and ammonium/ammonia) as

Page 20 of 49
well as their liquid-gas transfer phenomena (Rossi et al., 2020). For example, a high pH (>

9.75) is favorable for ammonia volatilization in which ammonium (NH4+) is converted to

ammonia (NH3) gas (Lu et al., 2019). In addition, changing the pH of the medium affects the

physiology and morphology of microalgae by activating the permeability of the membrane

cell for certain ions, and consequently affecting microalgae growth and biochemical

composition (Liang et al., 2011). Moreover, the pH of the cultivation medium is considered as a

tool for controlling biological contamination in wastewater. This is because a cultivation

medium with pH higher than 9 inhibits the growth of indigenous bacteria of wastewater (Lu et

al., 2019). Also, cultivation of microalgae, e.g., the green alga, Haematococcus pluvialis, at an

acidic pH of 4 has been recommended to avoid lethal fungal contamination of the culture

(Hwang et al., 2019).

Based on microalgal growth performance, the pH of cultivation medium can be classified as

fatal, tolerable, or optimal. Extremely low (acidic) and high (basic) pH values are fatal for

most microalgae species. For example, Sakarika and Kornaros (2016) studied the growth of C.

vulgaris at pH values in the range of 3 - 11. Lysis of microalgae cells was observed at highly

acidic (e.g., 3 and 4) and basic (e.g., 11) pH after two days of cultivation. Microalgae were

reported to grow in the pH range from 5 to 8, while based on growth parameters, the

optimum pH was found to be between 7.5 - 8. In another study, Bartley et al. (2014)

investigated the effects of pH, in the range from 5 to 10, on the growth and lipid

accumulation of microalgae, Nannochloropsis salina. Highest growth rates of 95.6 × 106 and

92.8 × 106 cells/mL were reported at optimum pH of 8 and 9, respectively. Therefore, most

microalgae species grow well in cultivation media with pH ranging between 7 to 9 (Aishvarya

et al., 2015), but such narrow range of pH cannot be applied for cultivation of all microalgae

species under controlled conditions. The optimum, acceptable, and lethal ranges of pH

depend on microalgae species and cultivation conditions. It has been observed that some

Page 21 of 49
microalgae species can tolerate extraordinary acidic or basic pH. For instance, Dunaliella

salina grows well in a pH close to 11.5, while the optimal pH of Dunaliella acidophila is

between 0.0 and 3.0 (Sakarika and Kornaros, 2016). Hence, the optimum pH range for

cultivation of the same commercial and well-known microalgae species can thus be found

from previous studies. For newly isolated microalgae strains having less available

information, the cultivation conditions need to be evaluated and optimized under controlled

laboratory conditions.

5.3. CO2 supplementation

Approximately 50% of the dry weight of microalgae biomass is composed of carbon, which

is mainly derived from CO2 (Bilad et al., 2014). Cultivation media with low concentrations of

CO2 negatively affects the synthesis of vital enzymes involved in carbon metabolism, such as

ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase

reactions (Chang et al., 2016). A carbon-limited environment also restricts the synthesis of

microalga pigments. In a study conducted by Miller and Holt (1977), the color of

Synechococcus lividus, cultivated under CO2 deprivation, changed to yellow after 96 h due to

the loss of pigments. The cells rapidly produced chlorophyll a and C-phycocyanin after

injecting CO2 into the culture. Therefore, it is necessary to supply CO2 for the healthy growth

of microalgae and to maximize biomass yield.

Atmospheric air with an approximate CO2 concentration of 0.04% is frequently used for

microalgae cultivation. Although aeration of the culture with ambient air can provide the

required CO2 for the growth of most microalgae species, some strains grow better at higher

CO2 concentration. In this regard, higher concentrations of CO2 can be provided by mixing

CO2 gas with atmospheric air. Zheng et al. (2012) applied a mixture of compressed air and

different concentrations of CO2 for the cultivation of C. vulgaris. Maximum biomass

Page 22 of 49
concentrations of 2.71, 3.32, 3.76, 2.59, and 0.65 g/L with 0.03 (ambient air), 1, 5, 10, and

15% of CO2, respectively were reported. However, microalgae growth was inhibited after 10

days in the medium with 0.03% CO2 as compared to the medium with 5% CO2, which could

be attributed to insufficient carbon. Higher concentrations of CO2 (above the optimum range)

can also be harmful to microalgae. Inhibition of growth of Chlorella sp. at 10 and 15% CO2

concentrations was observed by Chiu et al. (2008). This is due to the decrease in pH in the

cultivation medium with a high concentration of CO2, which can negatively affect the

activities of key photosynthetic enzymes such as ribulose 1,5-bisphosphate carboxylase-

oxygenase (Zheng et al., 2012). Pure CO2 has also been used as a carbon source for the

cultivation of microalgae (Wang et al., 2019). Pure CO2 can be supplied commercially via

high-pressure cylinders. The flow rate of CO2 can be adjusted using a flow meter, and the

pressure can be monitored online in case of high-tech bioreactors.

Flue gas is another source of CO2 that can provide the required carbon for microalgal growth.

Flue gas containing 6%−15% CO2 can also be used as a low-cost source of carbon for

microalgal cultivation (Abd Rahaman et al., 2011). Biomass productivity of Scenedesmus sp.

cultivated in media fed with ambient air containing 10% CO2 and flue gas containing 5.5%

CO2 was found to be 217.50 and 203 mg/L/d, respectively (Yoo et al., 2010). In another study

(Yadav et al., 2019), the highest biomass productivity of Chlorella sp. and Chlorococcum sp. in

cultivation media aerated with flue gas (containing 5% CO2) was found to be 208.93 and

105.42 mg/L/d, respectively, which were significantly higher than the ones obtained from

cultivation media aerated with ambient air (114.79 and 60.45 mg/L/d). Integration of

microalgae biomass production using flue gas not only provides CO2 for microalgal growth,

but also contributes towards controlling CO2 emissions and mitigating climate change. Flue

gas has high temperature that needs to be reduced before injecting to the microalgae

cultivation media. When replacing flue gas with atmospheric air and pure CO2, the effects of

Page 23 of 49
toxic gases and substances, such as CO, NOx, SOx, CxHy, heavy metals, and particulate matter

should be considered (Van Den Hende et al., 2012).

Due to its low solubility in water, CO2 easily escapes from the cultivation media through

aeration. Replacement of CO2 with other solid or liquid carbon sources has been extensively

investigated for the cultivation of microalgae. Besides CO2, bicarbonate-based compounds

with high water solubility (9.21% (w/w) at room temperature) are considered the main forms

of inorganic carbon for microalgae cultivation (Kim et al., 2019). Kim et al. (2019) showed that

sodium bicarbonate (NaHCO3) salt, extracted from flue gas by electrochemical CO2

mineralization, could support the growth of different species of freshwater, marine

microalgae, and cyanobacteria.

Based on the above discussion, it can be concluded that ambient air, enriched ambient air

with pure CO2, pure CO2, flue gas, and mineralized CO2 compounds are the main sources of

inorganic carbon that can be utilized for microalgae cultivation. Aeration of microalgae

culture using ambient air is more often used because it is inexpensive, easily accessible, and

available. However, low concentration of CO2 of atmospheric air and gas escaping from the

culture (due to low solubility) might limit the growth of microalgae. The advantages of other

carbon sources with higher concentrations of CO2 and solubility in water can enhance

microalgae growth. It is worth noting that apart from the carbon sources (air, pure CO2, flue

gas, or bicarbonate compounds), the pH of the cultivation medium strongly influences the

abundance of carbon species. CO2, bicarbonate, and carbonate are the dominant species at

pH<6, 7−10, and pH>10, respectively (Pedersen et al., 2013). Therefore, in addition to the

selection of an appropriate source of carbon, the adjustment of pH is also necessary to

maximize the growth of microalgae.

5.4. Aeration

Page 24 of 49
Aeration of the culture is another important factor that affects the growth of microalgae and

biomass yield. Usually, cultivation medium is aerated using an air compressor (Guo et al.,

2015), air pump (Supraja et al., 2020) or by agitation or shaking of the medium (Nedbal et al.,

2020). In cases of air injection, units such as L/min and vvm are used to denote the magnitude

of the aeration. In vvm, the first v represents the volume of air (L), the second v represents the

volume of medium (L), and m is minute (min). Different aeration rates from 0.1 to 10 L/min

have been tested on microalgae cultures (Table 4), however, optimum rates depend on

microalgae strains and the volume and shape of bioreactors (Barbosa et al., 2003). Features

such as weight, size, density of microalgae cells, and tolerance to shear stress influence the

optimum mixing and aeration rate. An aeration rate higher than the optimum value could

damage the microalgae cells due to shear force effects, increasing evaporation and the

operation costs (Guo et al., 2015). Han et al. (2015) investigated the effect of different aeration

rates (0.067 - 0.333 vvm) on microalgal growth. A maximum dry weight of microalga (1.24

g/L) was reported at 0.2 vvm aeration. The lowest and highest aeration rates were found to

negatively affect the growth of microalgae due to insufficient mixing and cell damage,

respectively.

As discussed in Section 5.3, aeration assists in proper supply of CO2 for microalgal growth.

In addition to carrying CO2 to the cultivation medium, aeration provides mixing power and

forms a turbulent flow in the culture and closed photobioreactor (Zhao et al., 2011). The

created turbulent flow and bubbles enhance mass transfer between the gas (CO2) and liquid

(culture medium) phases, which improves the diffusion of CO2 for photosynthesis (Zhao et al.,

2011). Appropriate mixing culture by optimized aeration also distributes microalgae cells

throughout the bioreactor homogeneously improving lighting conditions by exposing cells

from dark zones to illustrated zones (Zhao et al., 2011). Moreover, proper mixing of the

cultivation medium prevents nutrients, light, and temperature gradients as well as microalgae

Page 25 of 49
sedimentation in the culture broth (Guo et al., 2015). Therefore, aeration has a critical role in

microalgae growth, and it needs to be optimized in microalgae cultivation at different scales.

5.5. Temperature

Temperature is also a critical factor for microalgae cultivation. Temperature directly affects

the metabolism, nutrient uptake, CO2 biofixation, photosynthesis, and growth rate (Subhash et

al., 2014). In addition to growth, temperature also influences the physiology and biochemical

composition of microalgae including the quality and quantity of microalgal lipids (Teng et al.,

2020). Gonçalves et al. (2019) investigated the effect of temperature (between 20-36 °C) on the

biochemical composition of Pseudoneochloris marina (a green microalgae). Temperature

was found to significantly affect the amount of carbohydrates and saturated fatty acids of

microalgae biomass. Therefore, it is necessary to optimize the temperature of cultivation

medium for optimum growth of microalgae.

The adaptation and response of microalgae to different temperatures are closely related to the

origin of the microalgal species (Chokshi et al., 2015). Some species can tolerate extremely low

and high temperatures. For example, Chlamydomonas nivalis, known as snow algae, has been

isolated from the low-temperature environments of Antarctica (Fujii et al., 2010). Other

microalgae and cyanobacteria such as Cyanidium caldariu, Synechococcus elongatus, and

Chlorella sp. have shown maximum tolerance at 60 °C, 60 °C, and 45 °C, respectively

(Kumar et al., 2011). Cyanidiium caldarim, Galdieria partita, and Cyanidioschyzon melorae

were able to grow at 50 °C (Kurano et al., 1995). It should be noticed that most of the

microalgal species cannot tolerate extremely low or high temperatures. Cultivation of most

commercial and isolated microalgae species has been performed at temperatures between 20-

28 °C (Table 4). Although microalgae strains might grow in a wide range of temperature

conditions (Chokshi et al., 2015), the maximum growth rate of each microalgae species is

obtained at the optimum temperature. Higher and lower temperatures than the optimum can

Page 26 of 49
negatively affect the growth of microalgae and biomass production. It is also important to

know that microalgae tolerate lower temperatures better than higher temperatures.

Microalgae can sustain a decreased growth up to 15 °C below the optimum temperature,

however, only a few degrees higher than the optimum temperature can lead to microalgal cell

death (Enamala et al., 2018).

The optimum range of temperatures for cultivation of microalgae have been reported as 18 to

30 °C (Vuppaladadiyam et al., 2018), 15 to 26 °C (Hosseini et al., 2018), and 20 to 30 °C

(Enamala et al., 2018). Different optimum temperatures have been reported for the cultivation

of microalgae under different cultivation conditions such as indoor/outdoor cultivation,

open/closed systems, daytime, and light intensity. According to the information, presented in

Table 4, it can be pointed out that cultivation of most microalgae species has been

successfully performed at 25 °C. This implies that many microalgae species can grow well at

room temperature. However, temperature of culture needs to be adjusted and controlled in

case of species which are sensitive to temperature.

6. Microalgae supply

Providing microalgae seeds is the next step after the preparation of the bioreactor, media, and

adjustment of environmental factors in the microalgae cultivation process. Microalgae seeds

can be obtained from culture collections, or they can be isolated from natural water bodies

and wastewater drainages. Culture collections are resource centers which store living

microorganisms and their biological materials, such as cells. These centers, administered by

the government, universities, or companies, handle, preserve, and provide microalgae to

academics, and private and public industries to support their research and commercial

activities (de Oliveira Lourenço, 2020; DUYGU et al., 2017). Pure cultures of phytoplankton,

zooplankton, bacteria, fungi, and yeasts are available as axenic cultures for microalgae

Page 27 of 49
research (Table 5). These collections can supply a starter culture of different species of

microalgae, cyanobacteria, and diatoms either in liquid medium or on an agar slope. Algal

resource centers not only provide pure cultures as reference strains for research, but also

conserve microalgae species (de Oliveira Lourenço, 2020). Microalgae culture collections can

also provide useful information about the isolator, origin of isolation, appropriate cultivation

media, and optimum culture conditions that can facilitate the microalgae cultivation (Schulze

et al., 2019).

Microalgae can also be isolated from different environments, such as freshwater (lakes and

rivers), brackish and marine waters (seas and oceans), soil, and wastewater drainages (Table

5). Research and industrial applications of indigenous microalgae are highly recommended

due to the tolerance and compatibility of the latter with local geographical, climatic, and

ecological conditions (Duong et al., 2012). These species can grow under harsh conditions,

including hypersalinity, low or high temperatures, pH, and nutrient deficiency. For example,

de Morais and Costa (2007) isolated microalgae from ponds or lakes around coal or oil-fired

thermoelectric power plants. The combustion gases-adapted microalgae species were found

to grow efficiently under specific conditions prevalent in those areas.

In nature, microalgae cells are found together with other microorganisms or microalgae

strains. Several techniques including single-cell isolation, serial dilutions, medium

enrichment, micromanipulation, atomized cell spray, and fluorescence activated cell sorting

using flow cytometry have been introduced for the isolation of microalgae (Ghosh et al., 2016;

Pereira et al., 2011). Plating (streak, spread, and pour plate) is a common method for the

isolation of single colonies of microalgae from collected samples. Serial dilution is another

simple isolation technique that decreases the concentrations of unwanted microorganisms

(e.g., fungi and bacteria) and magnifies axenic cultures in higher dilution tubes (Barten et al.,

2020). It should be noted that pure cultures cannot be isolated by applying a single isolation

Page 28 of 49
method, however, a combination of isolation techniques could be more successful in isolating

pure microalgae. For example, the capillary method, micromanipulation, and UV radiation

might be needed after serial dilution to obtain axenic cultures (Ghosh et al., 2016; Pereira et al.,

2011; Andersen, 2005).

The isolated microalgae are identified using molecular and morphological techniques for

taxonomic classification and named using the binomial nomenclature system. Several

identification methods, as simple as optical microscopy, and high-tech methods such as

scanning electron microscopy (SEM), matrix-assisted laser desorption/ionization mass

spectrometry (MALDI-MS), reverse dot blot hybridization (RDBH), high-performance liquid

chromatography (HPLC), nuclear magnetic resonance spectroscopy (NMR), mass

spectrometry, infrared spectroscopy, and X-rays have been used to identify microalgae

(Ghosh et al., 2016). Among other techniques, 16s or 18s RNA sequence analysis is one of the

most reliable for the identification of newly isolated species/strains. The ribosomal genes are

the most conserved region of DNA in all cells and microalgae species, and are valuable tools

for determining the phylogeny of the species (Ghosh et al., 2016; Maid and Zetsche, 1991).

Culture collections provide certified microalgae species with accurate and safe information

for users. Once microalgae species are isolated, identified and maintained in culture

collection, these are available for immediate use. Therefore, culture collections provide faster

and easier access to microalgae.

For successful microalgal application, selection of suitable microalgal strain for a specific

cultivation purpose (e.g., reduction of CO2; and acquisition of microalgal biomass as a

feedstock for biofuels, materials, food, and feed) is another important consideration in the

upstream processes. A specific microalga species might be more appropriate for a specific

research because of its ability or feature related to high growth rate, unique metabolite

production, robustness, genetic manipulation, or composition of biomass. For instance,

Page 29 of 49
oleaginous microalgae such as Nannochloropsis sp. are more suitable for lipid extraction and

biodiesel production (Liu et al., 2017). Haematococcus sp. is well-known as a natural source

of astaxanthin. Spirulina sp. has been widely investigated for its use as a food ingredient

because of its high protein content. Likewise, diatoms (Thalassiosira pseudonana), source of

natural mesoporous silica, have shown ability as drug delivery tools in biomedicine research

(Delalat et al., 2015). Microalgae species such as Tetraselmis suecica and Isochrysis galbana

are widely used as biofeed in aquaculture research (Fitzer et al., 2019). Therefore, a careful

literature review can significantly help in the selection of more suitable microalgae species

while addressing specific research problems.

7. Microalgal growth monitoring

Evaluating microalgal growth can be considered the last step of upstream processes in

microalgae research. Analysis of microalgal growth can be performed at certain time

intervals or during the end of the experimental period. Evaluating microalgal growth is

important at least from two perspectives: (1) a direct index that monitors the performance of

cultivation systems toward selection of an appropriate microalgae species, cultivation media,

and optimized cultivation conditions; and (2) the amount of the produced microalgal biomass,

which is critical for the implementation of consequent mid- and downstream processes in

microalgae research. Measurement of microalgae dry mass, counting the number of

microalgae cells, and the value of optical density are frequently used for calculating

microalgal growth (Moheimani et al., 2013). Microalgae dry mass as a direct tool is the most

accurate method for measuring microalgal growth. In this method, the solid phase

(microalgae biomass) is separated from the liquid phase (cultivation media), and the weight

of biomass is measured after drying. Centrifugation and filtration are frequently used for the

separation of microalgae biomass from a certain volume of culture. The speed and frequency

Page 30 of 49
of rotation (revolutions per minute, rpm), and the mesh size are important factors that affect

the efficiency of cell separation and filtration, respectively. Centrifugation speed of 3000–

8000 rpm, and a revolution time of 5–8 min has been proposed for the centrifugation of

microalgal biomass (Liao et al., 2014; Daneshvar et al., 2018a). Pre-dried and pre-weighed

membrane filters (0.45 μm) or filter paper are used for the separation of microalgae cells (Li et

al., 2003). Subsequently, the collected biomass is dried in an oven or freeze-dried until the

microalgae weight becomes constant. Oven-drying is conducted in a temperature range of 60

°C to 110 °C for 2 to 24 h (Tang et al., 2012; Santana et al., 2017).

Counting the number of microalgae cells using a hemocytometer chamber is another

commonly used method for measuring microalgal growth. Hemocytometer counting

chambers are microscope-slide-sized base plates that were originally designed to count the

blood cells. Although there are various brands of hemocytometer chambers (i.e., Thoma,

Bürker, Bürker-Türk, and Fuchs-Rosenthal) with some differences in their design, the

principle of counting cells is the same. Usually, the counting chamber has a grid of specified

dimensions (1 mm × 1 mm squares). Each square is divided into smaller squares (0.05 mm ×

0.05 mm). The big (1 mm2) and small (0.0025 mm2) squares have the same depth of 0.1 mm,

therefore, the volume of each square can be calculated from its fixed dimension and depth.

To count the cells, a small drop of the solution containing microalgae is seeped into the

chamber underneath the coverslip, allowing the cell suspension to be drawn out by capillary

attraction. The microalgae cells can be counted with a light optical microscope at 10x to 40x

magnification. Finally, the concentration of cells can be presented as the number of cells per

unit volume of culture (µL or mL) (Moheimani et al., 2013). It should be noted that it is not

necessary to count all the 1 mm2 cells for a statistically significant count. Depending on the

concentration of microalgae cells in the sample, a subsample of type 2 (0.2 mm × 0.2 mm) or

type 3 (0.05 mm × 0.05 mm) can be selected to calculate the concentration of cells.

Page 31 of 49
Sometimes due to low volume of culture, high number of samples, or time limitation,

determination of dry biomass or counting the number of microalgae cells are not appropriate

methods for measuring microalgal growth. Measuring optical density (OD) is an alternative

indirect measurement when the volume of culture is low (e.g., µL to mL) for evaluating

microalgal growth (Santos-Ballardo et al., 2015). Absorbance of light by the microalgae

suspension can be related directly to dry mass or cell numbers using a suitable standard curve

with predetermined values at various wavelengths (650 - 750 nm) using spectrophotometer

(Wang et al., 2020; Wu et al., 2012; Almomani, 2020; Hosoglu et al., 2020; Fagerstone et al., 2011).

Microalgae OD in a small volume (µL) of culture, such as 96-well plates, can be performed

using a microplate reader (Abdelaziz et al., 2014). It is recommended to dilute highly dense

microalgae suspensions (wavelength > 1.00 nm) to avoid light absorption errors (Daneshvar et

al., 2018b).

From the above discussion, it can be concluded that the direct measurement of dry weight and

cell numbers are accurate and reliable to evaluate the growth of microalgae. However, these

methods might not be applicable in low volume of microplates or whenever the concentration

of cells is very low in culture. In such cases, microalgal growth can be indirectly calculated

by the measurement of optical density. However, measurement of optical density is less

accurate than the measurement of dry weight and cell numbers of microalgae.

8. Research needs and future directions

In recent years, the potential of microalgae has been increasingly exploited in numerous

research fields including environmental science, biology, genetics, chemistry, chemical

engineering, medicine, polymer science, agriculture, and aquaculture for diverse purposes.

Increased interests in microalgae market opportunities have led to fast-evolving scientific

research in the microalgae domain. Therefore, familiarization with the upstream processes is

Page 32 of 49
necessary. In this regard, all activities related to microalgae cultivation and biomass

production should be considered as a part of upstream processing. This review paper

discusses the main factors in microalgae cultivation including the cultivation modes,

bioreactors design, preparation of culture media, effect of environmental factors, supply of

microalgae seeds, and monitoring of microalgal growth. The detailed information provided in

this review, related to each of the above-mentioned determining factors can be beneficial in

exploring upstream processing in microalgae research. There are many research avenues that

can be explored further, for example, very little information is currently available on

photoheterotrophic cultivation of microalgae. A future research direction could be the

identification of specific requirements of photoheterotrophic cultivation mode. Research on

developing appropriate bioreactors for photoautotrophic, heterotrophic, mixotrophic, and

photoheterotrophic cultivation modes is necessary to facilitate cultivation of microalgae with

different metabolic pathways. Developing new culture media, suitable for little-known

microalgae species, can enhance the research opportunities. Additional investigations to solve

illumination problem are required as it is one of the main obstacles in microalgae research on

pilot-scale especially, in countries with less sunlight (e.g. Nordic and Scandinavian

countries). The main elements of upstream processes, as discussed in this review, should be

expanded by coherent studies to establish an organized protocol for microalgae cultivation.

Considering the high potential for microalgae research and its industrial applications, training

in microalgae cultivation should be promoted by relevant authorized research institutes. For

instance, researchers and students could be educated in the field of microalgal biotechnology

/ biorefineries through workshops or academic courses at universities and research institutes.

9. Conclusions

Page 33 of 49
 Careful assessment of underlying steps and aspects is critical to leverage the full potential of

microalgae during upstream processing. In this review, efforts have been made to highlight

the importance of vital steps of the upstream processing which are essential to make the

process more efficient. Selection of suitable strain of microalgae for specific purpose,

cultivation media, designing of bioreactors, environmental factors are some of the critical

aspects that influence the microalgal biomass production, and these have been thoroughly

discussed in this review. Additionally, this review has holistically addressed the technological

challenges that can influence the performance of upstream processing.

Acknowledgment

Some icons of Figures 2 and 3 are made using Pixel perfect from www.flaticon.com. Special

thanks to their creative team. We thank the Editor and three anonymous reviewers for their

constructive comments, which helped us to improve the quality of this paper.

References
1. Abd Rahaman, M.S., Cheng, L., Xu, X., Zhang, L., Chen, H., 2011. A review of carbon dioxide capture and
utilization by membrane integrated microalgal cultivation processes. Renew. Sust. Energ. Rev. 15, 4002-4012.
2. Abdelaziz, A.E., Leite, G.B., Belhaj, M.A., Hallenbeck, P.C., 2014. Screening microalgae native to Quebec for
wastewater treatment and biodiesel production. Bioresour. Technol. 157, 140-148.
3. Abid, A., Saidane, F., Hamdi, M., 2017. Feasibility of carbon dioxide sequestration by Spongiochloris sp
microalgae during petroleum wastewater treatment in airlift bioreactor. Bioresour. Technol. 234, 297-302.
4. Aghaalipour, E., Akbulut, A., Güllü, G., 2020. Carbon dioxide capture with microalgae species in continuous
gas-supplied closed cultivation systems. Biochem. Eng. J. 163, 107741.
5. Aishvarya, V., Jena, J., Pradhan, N., Panda, P., Sukla, L., 2015. Microalgae: cultivation and application,
Anonymous Environmental Microbial Biotechnology. Springer, pp. 289-311.
6. Almomani, F., 2020. Kinetic modeling of microalgae growth and CO2 bio-fixation using central composite
design statistical approach. Sci. Total Environ. 137594.
7. Álvarez-Díaz, P., Ruiz, J., Arbib, Z., Barragán, J., Garrido-Pérez, M., Perales, J., 2017. Freshwater microalgae
selection for simultaneous wastewater nutrient removal and lipid production. Algal Res. 24, 477-485.
8. Anand, V., Kashyap, M., Samadhiya, K., Ghosh, A., Kiran, B., 2019. Salinity driven stress to enhance lipid
production in Scenedesmus vacuolatus: A biodiesel trigger? Biomass Bioenergy 127, 105252.
9. Ananthi, V., Raja, R., Carvalho, I.S., Brindhadevi, K., Pugazhendhi, A., Arun, A., 2021. A realistic scenario on
microalgae based biodiesel production: Third generation biofuel. Fuel. 284, 118965.
10. Andersen, R.A., 2005. Algal culturing techniques. Elsevier.
11. Ansari, F.A., Ravindran, B., Gupta, S.K., Nasr, M., Rawat, I., Bux, F., 2019. Techno-economic estimation of
wastewater phycoremediation and environmental benefits using Scenedesmus obliquus microalgae. J. Environ.
Manage. 240, 293-302.
12. Araújo, C., Souza-Santos, L., 2013. Use of the microalgae Thalassiosira weissflogii to assess water toxicity in
the Suape industrial-port complex of Pernambuco. Brazil, Ecotoxicol. Environ. Saf. 89, 212-221.

Page 34 of 49
13. Atta, M., Idris, A., Bukhari, A., Wahidin, S., 2013. Intensity of blue LED light: a potential stimulus for biomass
and lipid content in fresh water microalgae Chlorella vulgaris. Bioresour. Technol. 148, 373-378.
14. Barbosa, M.J., Albrecht, M., Wijffels, R.H., 2003. Hydrodynamic stress and lethal events in sparged microalgae
cultures. Biotechnol. Bioeng. 83, 112-120.
15. Barten, R.J., Wijffels, R.H., Barbosa, M.J., 2020. Bioprospecting and characterization of temperature tolerant
microalgae from Bonaire. Algal Res. 50, 102008.
16. Bartley, M.L., Boeing, W.J., Dungan, B.N., Holguin, F.O., Schaub, T., 2014. pH effects on growth and lipid
accumulation of the biofuel microalgae Nannochloropsis salina and invading organisms. J. Appl. Phycol. 26,
1431-1437.
17. Bilad, M., Arafat, H.A., Vankelecom, I.F., 2014. Membrane technology in microalgae cultivation and
harvesting: a review. Biotechnol. Adv. 32, 1283-1300.
18. Blockx, J., Verfaillie, A., Thielemans, W., Muylaert, K., 2018. Unravelling the mechanism of chitosan-driven
flocculation of microalgae in seawater as a function of pH. ACS Sustain. Chem. Eng. 6, 11273-11279.
19. Chang, H., Huang, Y., Fu, Q., Liao, Q., Zhu, X., 2016. Kinetic characteristics and modeling of microalgae
Chlorella vulgaris growth and CO2 biofixation considering the coupled effects of light intensity and dissolved
inorganic carbon. Bioresour. Technol. 206, 231-238.
20. Chen, C., Kuo, E., Nagarajan, D., Ho, S., Dong, C., Lee, D., Chang, J., 2020. Cultivating Chlorella sorokiniana
AK-1 with swine wastewater for simultaneous wastewater treatment and algal biomass production. Bioresour.
Technol. 302, 122814.
21. Chew, K.W., Chia, S.R., Show, P.L., Yap, Y.J., Ling, T.C., Chang, J., 2018. Effects of water culture medium,
cultivation systems and growth modes for microalgae cultivation: a review. J. Taiwan Inst. Chem. Eng. 91, 332-
344.
22. Chiu, S., Kao, C., Chen, C., Kuan, T., Ong, S., Lin, C., 2008. Reduction of CO2 by a high-density culture of
Chlorella sp. in a semicontinuous photobioreactor. Bioresour. Technol. 99, 3389-3396.
23. Choi, H.I., Lee, J.S., Choi, J.W., Shin, Y.S., Sung, Y.J., Hong, M.E., Kwak, H.S., Kim, C.Y., Sim, S.J., 2019.
Performance and potential appraisal of various microalgae as direct combustion fuel. Bioresour. Technol. 273,
341-349.
24. Chojnacka, K., Marquez-Rocha, F.J., 2004. Kinetic and stoichiometric relationships of the energy and carbon
metabolism in the culture of microalgae. Biotechnology 3(1), 21-34.
25. Chokshi, K., Pancha, I., Trivedi, K., George, B., Maurya, R., Ghosh, A., Mishra, S., 2015. Biofuel potential of
the newly isolated microalgae Acutodesmus dimorphus under temperature induced oxidative stress conditions.
Bioresour. Technol. 180, 162-171.
26. Corcoran, A.A., Saunders, M.A., Hanley, A.P., Lee, P.A., Lopez, S., Ryan, R., Yohn, C.B., 2018. Iterative
screening of an evolutionary engineered Desmodesmus generates robust field strains with pesticide tolerance.
Algal Res. 31, 443-453.
27. Costa, S.S., Miranda, A.L., Andrade, B.B., de Jesus Assis, D., Souza, C.O., de Morais, M.G., Costa, J.A.V.,
Druzian, J.I., 2018. Influence of nitrogen on growth, biomass composition, production, and properties of
polyhydroxyalkanoates (PHAs) by microalgae. Int. J. Biol. Macromol. 116, 552-562.
28. Daneshvar, E., Antikainen, L., Koutra, E., Kornaros, M., Bhatnagar, A., 2018a. Investigation on the feasibility
of Chlorella vulgaris cultivation in a mixture of pulp and aquaculture effluents: treatment of wastewater and
lipid extraction. Bioresour. Technol. 255, 104-110.
29. Daneshvar, E., Santhosh, C., Antikainen, E., Bhatnagar, A., 2018b. Microalgal growth and nitrate removal
efficiency in different cultivation conditions: Effect of macro and micronutrients and salinity. J. Environ. Chem.
Eng. 6, 1848-1854.
30. Daneshvar, E., Zarrinmehr, M.J., Koutra, E., Kornaros, M., Farhadian, O., Bhatnagar, A., 2019. Sequential
cultivation of microalgae in raw and recycled dairy wastewater: microalgal growth, wastewater treatment and
biochemical composition. Bioresour. Technol. 273, 556-564.
31. Dao, G., Wu, G., Wang, X., Zhang, T., Zhan, X., Hu, H., 2018. Enhanced microalgae growth through stimulated
secretion of indole acetic acid by symbiotic bacteria. Algal Res. 33, 345-351.
32. de Morais, M.G., Costa, J.A.V., 2007. Isolation and selection of microalgae from coal fired thermoelectric
power plant for biofixation of carbon dioxide. Energy Convers. Manag. 48, 2169-2173.
33. de Oliveira Lourenço, S., 2020. Microalgae culture collections, strain maintenance, and propagation,
Anonymous Handbook of Microalgae-Based Processes and Products. Elsevier, pp. 49-84.
34. Delalat, B., Sheppard, V.C., Ghaemi, S.R., Rao, S., Prestidge, C.A., McPhee, G., Rogers, M., Donoghue, J.F.,
Pillay, V., Johns, T.G., 2015. Targeted drug delivery using genetically engineered diatom biosilica. Nat.
Commun. 6, 1-11.
35. Detweiler, A.M., Mioni, C.E., Hellier, K.L., Allen, J.J., Carter, S.A., Bebout, B.M., Fleming, E.E., Corrado, C.,
Prufert-Bebout, L.E., 2015. Evaluation of wavelength selective photovoltaic panels on microalgae growth and
photosynthetic efficiency. Algal Res. 9, 170-177.

Page 35 of 49
36. Di Caprio, F., Altimari, P., Iaquaniello, G., Toro, L., Pagnanelli, F., 2019. Heterotrophic cultivation of T.
obliquus under non-axenic conditions by uncoupled supply of nitrogen and glucose. Biochem. Eng. J. 145, 127-
136.
37. Dong, X., Zhao, Y., Li, T., Huang, L., Zhao, P., Xu, J., Ma, H., Yu, X., 2019. Enhancement of lipid production
and nutrient removal of Monoraphidium sp. FXY-10 by combined melatonin and molasses wastewater
treatment. J. Taiwan Inst. Chem. Eng. 99, 123-131.
38. Duong, V.T., Li, Y., Nowak, E., Schenk, P.M., 2012. Microalgae isolation and selection for prospective
biodiesel production. Energies. 5, 1835-1849.
39. DUYGU, D.Y., Udoh, A.U., Özer, T., Erkaya, İA., 2017. The Characteristics and Importance of Microalgae
Culture Collections. Süleyman Demirel Üniversitesi Eğirdir Su Ürünleri Fakültesi Dergisi. 13, 80-87.
40. Enamala, M.K., Enamala, S., Chavali, M., Donepudi, J., Yadavalli, R., Kolapalli, B., Aradhyula, T.V., Velpuri,
J., Kuppam, C., 2018. Production of biofuels from microalgae-A review on cultivation, harvesting, lipid
extraction, and numerous applications of microalgae. Renew. Sust. Energ. Rev. 94, 49-68.
41. Fagerstone, K.D., Quinn, J.C., Bradley, T.H., De Long, S.K., Marchese, A.J., 2011. Quantitative measurement
of direct nitrous oxide emissions from microalgae cultivation. Environ. Sci. Technol. 45, 9449-9456.
42. Fozer, D., Kiss, B., Lorincz, L., Szekely, E., Mizsey, P., Nemeth, A., 2019. Improvement of microalgae biomass
productivity and subsequent biogas yield of hydrothermal gasification via optimization of illumination. Renew.
Energy 138, 1262-1272.
43. Fitzer, S.C., Plancq, J., Floyd, C.J., Kemp, F.M., Toney, J.L., 2019. Increased pCO2 changes the lipid
production in important aquacultural feedstock algae Isochrysis galbana, but not in Tetraselmis suecica. Aquacu.
Fish. 4(4), 142-148.
44. Fret, J., Roef, L., Diels, L., Tavernier, S., Vyverman, W., Michiels, M., 2020. Combining medium recirculation
with alternating the microalga production strain: a laboratory and pilot scale cultivation test. Algal Res. 46,
101763.
45. Fujii, M., Takano, Y., Kojima, H., Hoshino, T., Tanaka, R., Fukui, M., 2010. Microbial community structure,
pigment composition, and nitrogen source of red snow in Antarctica. Microb. Ecol. 59, 466-475.
46. Gaignard, C., Gargouch, N., Dubessay, P., Delattre, C., Pierre, G., Laroche, C., Fendri, I., Abdelkafi, S.,
Michaud, P., 2019. New horizons in culture and valorization of red microalgae. Biotechnol. Adv. 37, 193-222.
47. Geada, P., Vasconcelos, V., Vicente, A., Fernandes, B., 2017. Microalgal biomass cultivation, Anonymous
Algal Green Chemistry. Elsevier, pp. 257-284.
48. Ghosh, A., Khanra, S., Mondal, M., Halder, G., Tiwari, O., Saini, S., Bhowmick, T.K., Gayen, K., 2016.
Progress toward isolation of strains and genetically engineered strains of microalgae for production of biofuel
and other value added chemicals: a review. Energy Convers. Manag. 113, 104-118.
49. Gonçalves, C.F., Menegol, T., Rech, R., 2019. Biochemical composition of green microalgae Pseudoneochloris
marina grown under different temperature and light conditions. Biocatal. Agric. Biotechnol. 18, 101032.
50. Gouveia, L., Oliveira, A.C., 2009. Microalgae as a raw material for biofuels production. J. Ind. Microbiol.
Biotechnol. 36, 269-274.
51. Grobbelaar, J.U., 2013. Inorganic algal nutrition, Handbook of microalgal culture: applied phycology and
biotechnology.John Wiley y Sons, Ltd.GBR. 123-133.
52. Guillard, R.R., 1975. Culture of phytoplankton for feeding marine invertebrates, Anonymous Culture of marine
invertebrate animals. Springer, pp. 29-60.
53. Guo, X., Yao, L., Huang, Q., 2015. Aeration and mass transfer optimization in a rectangular airlift loop
photobioreactor for the production of microalgae. Bioresour. Technol. 190, 189-195.
54. Han, F., Pei, H., Hu, W., Song, M., Ma, G., Pei, R., 2015. Optimization and lipid production enhancement of
microalgae culture by efficiently changing the conditions along with the growth-state. Energy Convers. Manag.
90, 315-322.
55. Holdmann, C., Schmid-Staiger, U., Hirth, T., 2019. Outdoor microalgae cultivation at different biomass
concentrations — Assessment of different daily and seasonal light scenarios by modeling. Algal Res. 38,
101405.
56. Hosoglu, M.I., Karagul-Yuceer, Y., Guneser, O., 2020. Aroma characterization of heterotrophic microalgae
Crypthecodinium cohnii using solid-phase microextraction and gas chromatography–mass
spectrometry/olfactometry during different growth phases. Algal Res. 101928.
57. Hosseini, N.S., Shang, H., Scott, J.A., 2018. Biosequestration of industrial off-gas CO2 for enhanced lipid
productivity in open microalgae cultivation systems. Renew. Sust. Energ. Rev. 92, 458-469.
58. Hsieh-Lo, M., Castillo, G., Ochoa-Becerra, M.A., Mojica, L., 2019. Phycocyanin and phycoerythrin: Strategies
to improve production yield and chemical stability. Algal Res. 42, 101600.
59. Hu, J., Nagarajan, D., Zhang, Q., Chang, J., Lee, D., 2018. Heterotrophic cultivation of microalgae for pigment
production: A review. Biotechnol. Adv. 36, 54-67.
60. Huang, G., Chen, F., Wei, D., Zhang, X., Chen, G., 2010. Biodiesel production by microalgal biotechnology.
Appl. Energy. 87, 38-46.

Page 36 of 49
61. Huo, S., Chen, J., Zhu, F., Zou, B., Chen, X., Basheer, S., Cui, F., Qian, J., 2019. Filamentous microalgae
Tribonema sp. cultivation in the anaerobic/oxic effluents of petrochemical wastewater for evaluating the
efficiency of recycling and treatment. Biochem. Eng. J. 145, 27-32.
62. Hwang, S., Choi, H.I., Sim, S.J., 2019. Acidic cultivation of Haematococcus pluvialis for improved astaxanthin
production in the presence of a lethal fungus. Bioresour. Technol. 278, 138-144.
63. Junying, Z., Junfeng, R., Baoning, Z., 2013. Factors in mass cultivation of microalgae for biodiesel. Chin. J.
Catal.34, 80-100.
64. Khichi, S.S., Rohith, S., Gehlot, K., Dutta, B., Ghosh, S., 2019. Online estimation of biomass, lipid and nitrate
dynamic profile using innovative light evolution kinetic model in flat panel airlift photobioreactor for
Botryococcus braunii under varying light conditions. Bioresour. Technol. Reports 6, 6-14.
65. Kim, G., Roh, K., Han, J., 2019. The use of bicarbonate for microalgae cultivation and its carbon footprint
analysis. Green Chem. 21, 5053-5062.
66. Kim, J., Lee, J., Lu, T., 2014. Effects of dissolved inorganic carbon and mixing on autotrophic growth of
Chlorella vulgaris. Biochem. Eng. J. 82, 34-40.
67. Kumar, K., Dasgupta, C.N., Nayak, B., Lindblad, P., Das, D., 2011. Development of suitable photobioreactors
for CO2 sequestration addressing global warming using green algae and cyanobacteria. Bioresour. Technol. 102,
4945-4953.
68. Kurano, N., Ikemoto, H., Miyashita, H., Hasegawa, T., Hata, H., Miyachi, S., 1995. Fixation and utilization of
carbon dioxide by microalgal photosynthesis. Energy Convers. Manag. 36, 689-692.
69. Lam, M.K., Lee, K.T., 2012a. Microalgae biofuels: a critical review of issues, problems and the way forward.
Biotechnol. Adv. 30, 673-690.
70. Lam, M.K., Lee, K.T., 2012b. Immobilization as a feasible method to simplify the separation of microalgae
from water for biodiesel production. Chem. Eng. J. 191, 263-268.
71. Lee, Y., 2003. Algal Nutrition‒Heterotrophic carbon nutrition. In: Richmond, A., (Ed.) Handbook of Microalgal
Culture: Biotechnology and Applied Phycology. John Wiley & Sons, 116-124.
72. Li, J., Xu, N.S., Su, W.W., 2003. Online estimation of stirred-tank microalgal photobioreactor cultures based on
dissolved oxygen measurement. Biochem. Eng. J. 14, 51-65.
73. Li, K., Liu, Q., Fang, F., Luo, R., Lu, Q., Zhou, W., Huo, S., Cheng, P., Liu, J., Addy, M., 2019. Microalgae-
based wastewater treatment for nutrients recovery: a review. Bioresour. Technol. 121934.
74. Li, T., Zheng, Y., Yu, L., Chen, S., 2014. Mixotrophic cultivation of a Chlorella sorokiniana strain for enhanced
biomass and lipid production. Biomass Bioenergy 66, 204-213.
75. Liang, G., Mo, Y., Tang, J., Zhou, Q., 2011. Improve lipid production by pH shifted-strategy in batch culture of
Chlorella protothecoides. Afr. J. Microbiol. Res. 5, 5030-5038.
76. Liao, Q., Li, L., Chen, R., Zhu, X., 2014. A novel photobioreactor generating the light/dark cycle to improve
microalgae cultivation. Bioresour. Technol. 161, 186-191.
77. Liu, J., Song, Y., Qiu, W., 2017. Oleaginous microalgae Nannochloropsis as a new model for biofuel
production: review & analysis. Renew. Sust. Energ. Rev. 72, 154-162.
78. Liu, X., Fujiwara, M., Kodera, T., Watanabe, K., Akizuki, S., Kishi, M., Koyama, M., Toda, T., Ban, S., 2020.
Conditions for continuous cultivation of Chlorella sorokiniana and nutrient removal from anaerobic digestion
effluent of aquatic macrophytes, Int. Biodeterior. Biodegrad. 149, 104923.
79. Lu, W., Alam, M.A., Liu, S., Xu, J., Saldivar, R.P., 2019. Critical processes and variables in microalgae
biomass production coupled with bioremediation of nutrients and CO2 from livestock farms: A review. Sci.
Total Environ. 135247.
80. Maid, U., Zetsche, K., 1991. Structural features of the plastid ribosomal RNA operons of two red algae:
Antithamnion sp. and Cyanidium caldarium. Plant Mol. Biol. 16, 537-546.
81. Manirafasha, E., Ndikubwimana, T., Zeng, X., Lu, Y., Jing, K., 2016. Phycobiliprotein: Potential microalgae
derived pharmaceutical and biological reagent. Biochem. Eng. J. 109, 282-296.
82. Miller, L.S., Holt, S.C., 1977. Effect of carbon dioxide on pigment and membrane content in Synechococcus
lividus. Arch. Microbiol. 115, 185-198.
83. Moheimani, N.R., Borowitzka, M.A., Isdepsky, A., Sing, S.F., 2013. Standard methods for measuring growth of
algae and their composition, Anonymous Algae for biofuels and energy. Springer, pp. 265-284.
84. Molitor, H.R., Moore, E.J., Schnoor, J.L., 2019. Maximum CO2 Utilization by Nutritious Microalgae. ACS
Sustain. Chem. Eng. 7 (10), 9474-9479.
85. Mustafa, M.G., Khan, M.G.M., Nguyen, D., Iqbal, S., 2018. Techniques in Biotechnology: Essential for
Industry, Anonymous Omics Technologies and Bio-Engineering. Elsevier, pp. 233-249.
86. Naira, V.R., Das, D., Maiti, S.K., 2019. Real time light intensity based carbon dioxide feeding for high cell-
density microalgae cultivation and biodiesel production in a bubble column photobioreactor under outdoor
natural sunlight. Bioresour. Technol. 284, 43-55.
87. Nedbal, J., Gao, L., Suhling, K., 2020. Bottom-Illuminated Orbital Shaker for Microalgae Cultivation. bioRxiv.
doi: https://doi.org/10.1101/2020.05.01.071878.

Page 37 of 49
88. Nguyen, T.D.P., Tran, T.N.T., Le, T.V.A., Phan, T.X.N., Show, P., Chia, S.R., 2019. Auto-flocculation through
cultivation of Chlorella vulgaris in seafood wastewater discharge: Influence of culture conditions on microalgae
growth and nutrient removal. J. Biosci. Bioeng. 127, 492-498.
89. Nitsos, C., Filali, R., Taidi, B., Lemaire, J., 2020. Current and novel approaches to downstream processing of
microalgae: A review. Biotechnol. Adv. 107650.
90. Oyebamiji, O.O., Boeing, W.J., Holguin, F.O., Ilori, O., Amund, O., 2019. Green microalgae cultured in textile
wastewater for biomass generation and biodetoxification of heavy metals and chromogenic substances.
Bioresour. Technol. Reports 7, 100247.
91. Patel, A.K., Huang, E.L., Low-DeÌcarie, E., Lefsrud, M.G., 2015. Comparative shotgun proteomic analysis of
wastewater-cultured microalgae: nitrogen sensing and carbon fixation for growth and nutrient removal in
Chlamydomonas reinhardtii. J. Proteome Res. 14, 3051-3067.
92. Pauli, J.N., Mendoza, J.E., Steffan, S.A., Carey, C.C., Weimer, P.J., Peery, M.Z., 2014. A syndrome of
mutualism reinforces the lifestyle of a sloth. Proc. Royal Soc. B 281, 20133006.
93. Pedersen, O., Colmer, T.D., Sand-Jensen, K., 2013. Underwater photosynthesis of submerged plants–recent
advances and methods. Front. Plant Sci. 4, 140.
94. Pena, A.C., Agustini, C.B., Trierweiler, L.F., Gutterres, M., 2020. Influence of period light on cultivation of
microalgae consortium for the treatment of tannery wastewaters from leather finishing stage. J. Clean. Prod.
121618.
95. Pereira, H., Barreira, L., Mozes, A., Florindo, C., Polo, C., Duarte, C.V., Custódio, L., Varela, J., 2011.
Microplate-based high throughput screening procedure for the isolation of lipid-rich marine microalgae.
Biotechnol. Biofuels 4, 61.
96. Perez-Garcia, O., Bashan, Y., 2015. Microalgal heterotrophic and mixotrophic culturing for bio-refining: from
metabolic routes to techno-economics, Anonymous Algal biorefineries. Springer, pp. 61-131.
97. Phwan, C.K., Ong, H.C., Chen, W., Ling, T.C., Ng, E.P., Show, P.L., 2018. Overview: comparison of
pretreatment technologies and fermentation processes of bioethanol from microalgae. Energy Convers. Manag.
173, 81-94.
98. Polat, E., Yüksel, E., Altınbaş, M., 2020. Mutual effect of sodium and magnesium on the cultivation of
microalgae Auxenochlorella protothecoides. Biomass Bioenergy 132, 105441.
99. Procházková, G., Brányiková, I., Zachleder, V., Brányik, T., 2014. Effect of nutrient supply status on biomass
composition of eukaryotic green microalgae. J. Appl. Phycol. 26, 1359-1377.
100. Qu, F., Jin, W., Zhou, X., Wang, M., Chen, C., Tu, R., Han, S., He, Z., Li, S., 2020. Nitrogen ion beam
implantation for enhanced lipid accumulation of Scenedesmus obliquus in municipal wastewater. Biomass
Bioenergy 134, 105483.
101. Ra, C.H., Kang, C., Jung, J., Jeong, G., Kim, S., 2016. Enhanced biomass production and lipid accumulation of
Picochlorum atomus using light-emitting diodes (LEDs). Bioresour. Technol. 218, 1279-1283.
102. Ramanna, L., Rawat, I., Zerrouki, D., Bux, F., 2018. A novel organic dye-based approach to increase photon
flux density for enhanced microalgal pigment production. J. Clean. Prod. 198, 187-194.
103. Razzak, S.A., Ali, S.A.M., Hossain, M.M., deLasa, H., 2017. Biological CO2 fixation with production of
microalgae in wastewater – a review. Renew. Sust. Energ. Rev. 76, 379-390.
104. Ren, H., Tuo, J., Addy, M.M., Zhang, R., Lu, Q., Anderson, E., Chen, P., Ruan, R., 2017. Cultivation of
Chlorella vulgaris in a pilot-scale photobioreactor using real centrate wastewater with waste glycerol for
improving microalgae biomass production and wastewater nutrients removal. Bioresour. Technol. 245, 1130-
1138.
105. Rossi, S., Casagli, F., Mantovani, M., Mezzanotte, V., Ficara, E., 2020. Selection of photosynthesis and
respiration models to assess the effect of environmental conditions on mixed microalgae consortia grown on
wastewater. Bioresour. Technol. 305, 122995.
106. Sá, M., Ferrer-Ledo, N., Wijffels, R., Crespo, J.G., Barbosa, M., Galinha, C.F., 2020. Monitoring of
eicosapentaenoic acid (EPA) production in the microalgae Nannochloropsis oceanica. Algal Res. 45, 101766.
107. Sahu, N., Tangutur, A.D., 2015. Airborne algae: overview of the current status and its implications on the
environment. Aerobiologia 31, 89-97.
108. Sajjadi, B., Chen, W., Raman, A.A.A., Ibrahim, S., 2018. Microalgae lipid and biomass for biofuel production:
A comprehensive review on lipid enhancement strategies and their effects on fatty acid composition. Renew.
Sust. Energ. Rev. 97, 200-232.
109. Sakarika, M., Kornaros, M., 2016. Effect of pH on growth and lipid accumulation kinetics of the microalga
Chlorella vulgaris grown heterotrophically under sulfur limitation. Bioresour. Technol. 219, 694-701.
110. Salama, E., Kurade, M.B., Abou-Shanab, R.A., El-Dalatony, M.M., Yang, I., Min, B., Jeon, B., 2017. Recent
progress in microalgal biomass production coupled with wastewater treatment for biofuel generation. Renew.
Sust. Energ. Rev. 79, 1189-1211.

Page 38 of 49
111. Santana, H., Cereijo, C.R., Teles, V.C., Nascimento, R.C., Fernandes, M.S., Brunale, P., Campanha, R.C.,
Soares, I.P., Silva, F.C., Sabaini, P.S., 2017. Microalgae cultivation in sugarcane vinasse: selection, growth and
biochemical characterization. Bioresour. Technol. 228, 133-140.
112. Santos-Ballardo, D.U., Rossi, S., Hernández, V., Gómez, R.V., del Carmen Rendón-Unceta, M., Caro-Corrales,
J., Valdez-Ortiz, A., 2015. A simple spectrophotometric method for biomass measurement of important
microalgae species in aquaculture. Aquaculture. 448, 87-92.
113. Schulze, P.S., Hulatt, C.J., Morales-Sánchez, D., Wijffels, R.H., Kiron, V., 2019. Fatty acids and proteins from
marine cold adapted microalgae for biotechnology. Algal Res. 42, 101604.
114. Shah, S.M.U., Radziah, C.C., Ibrahim, S., Latiff, F., Othman, M.F., Abdullah, M.A., 2014. Effects of
photoperiod, salinity and pH on cell growth and lipid content of Pavlova lutheri, Annals of microbiology. 64,
157-164.
115. Smith, J.P., Hughes, A., McEvoy, L., Day, J., 2020. Tailoring of the biochemical profiles of microalgae by
employing mixotrophic cultivation. Bioresour. Technol. Reports, 9, 100321.
116. Subashchandrabose, S.R., Ramakrishnan, B., Megharaj, M., Venkateswarlu, K., Naidu, R., 2011. Consortia of
cyanobacteria/microalgae and bacteria: biotechnological potential. Biotechnol. Adv. 29, 896-907.
117. Subhash, G.V., Rajvanshi, M., Kumar, B.N., Govindachary, S., Prasad, V., Dasgupta, S., 2017. Carbon
streaming in microalgae: extraction and analysis methods for high value compounds. Bioresour. Technol. 244,
1304-1316.
118. Subhash, G.V., Rohit, M., Devi, M.P., Swamy, Y., Mohan, S.V., 2014. Temperature induced stress influence on
biodiesel productivity during mixotrophic microalgae cultivation with wastewater. Bioresour. Technol. 169,
789-793.
119. Straka, L., Rittmann, B.E., 2018. Light-dependent kinetic model for microalgae experiencing photoacclimation,
photodamage, and photodamage repair. Algal Res. 31, 232-238.
120. Sun, H., Zhao, W., Mao, X., Li, Y., Wu, T., Chen, F., 2018. High-value biomass from microalgae production
platforms: strategies and progress based on carbon metabolism and energy conversion. Biotechnol. Biofuels 11,
227.
121. Supraja, K.V., Behera, B., Balasubramanian, P., 2020. Performance evaluation of hydroponic system for co-
cultivation of microalgae and tomato plant. J. Clean. Prod. 272, 122823.
122. Tang, H., Chen, M., Simon Ng, K., Salley, S.O., 2012. Continuous microalgae cultivation in a photobioreactor.
Biotechnol. Bioeng. 109, 2468-2474.
123. Teng, S.Y., Yew, G.Y., Sukačová, K., Show, P.L., Máša, V., Chang, J., 2020. Microalgae with artificial
intelligence: A digitalized perspective on genetics, systems and products. Biotechnol. Adv. 107631.
124. Teo, C.L., Atta, M., Bukhari, A., Taisir, M., Yusuf, A.M., Idris, A., 2014. Enhancing growth and lipid
production of marine microalgae for biodiesel production via the use of different LED wavelengths. Bioresour.
Technol. 162, 38-44.
125. Tran, D.T., Nguyen, H.Y., Van Do, T.C., Show, P.L., Le, T.G., 2020. Factors Affecting Pollutants Removal and
Biomass Production Capability of Chlorella variabilis TH03 in Domesic Wastewater. Mater. Sci. Energy
Technol. 3, 545-558.
126. Vadiveloo, A., Moheimani, N.R., Cosgrove, J.J., Bahri, P.A., Parlevliet, D., 2015. Effect of different light
spectra on the growth and productivity of acclimated Nannochloropsis sp.(Eustigmatophyceae). Algal Res. 8,
121-127.
127. Van Den Hende, S., Vervaeren, H., Boon, N., 2012. Flue gas compounds and microalgae:(Bio-) chemical
interactions leading to biotechnological opportunities. Biotechnol. Adv. 30, 1405-1424.
128. Vasconcelos Fernandes, T., Shrestha, R., Sui, Y., Papini, G., Zeeman, G., Vet, L.E., Wijffels, R.H., Lamers, P.,
2015. Closing domestic nutrient cycles using microalgae. Environ. Sci. Technol. 49, 12450-12456.
129. Viruela, A., Murgui, M., Gómez-Gil, T., Durán, F., Robles, A., Ruano, M.V., Ferrer, J., Seco, A., 2016. Water
resource recovery by means of microalgae cultivation in outdoor photobioreactors using the effluent from an
anaerobic membrane bioreactor fed with pre-treated sewage. Bioresour. Technol. 218, 447-454.
130. Vo, H.N.P., Ngo, H.H., Guo, W., Nguyen, T.M.H., Liu, Y., Liu, Y., Nguyen, D.D., Chang, S.W., 2019. A
critical review on designs and applications of microalgae-based photobioreactors for pollutants treatment. Sci.
Total Environ. 651, 1549-1568.
131. Vuppaladadiyam, A.K., Prinsen, P., Raheem, A., Luque, R., Zhao, M., 2018. Microalgae cultivation and
metabolites production: a comprehensive review. Biofuels Bioprod. Bioref. 12, 304-324.
132. Wahidin, S., Idris, A., Shaleh, S.R.M., 2013. The influence of light intensity and photoperiod on the growth and
lipid content of microalgae Nannochloropsis sp. Bioresour. Technol. 129, 7-11.
133. Wang, J., Cheng, W., Liu, W., Wang, H., Zhang, D., Qiao, Z., Jin, G., Liu, T., 2019. Field study on attached
cultivation of Arthrospira (Spirulina) with carbon dioxide as carbon source. Bioresour. Technol. 283, 270-276.
134. Wang, X., Zhang, T., Dao, G., Xu, Z., Wu, Y., Hu, H., 2020. Assessment and Mechanisms of Microalgae
Growth Inhibition by Phosphonates: Effects of Intrinsic Toxicity and Complexation. Water Res. 116333.

Page 39 of 49
135. Wicker, R., Bhatnagar, A., 2020. Application of Nordic microalgal-bacterial consortia for nutrient removal from
wastewater. Chem. Eng. J. 125567.
136. Wu, L.F., Chen, P.C., Huang, A.P., Lee, C.M., 2012. The feasibility of biodiesel production by microalgae
using industrial wastewater. Bioresour. Technol. 113, 14-18.
137. Xu, J., Li, T., Li, C., Zhu, S., Wang, Z., Zeng, E.Y., 2020. Lipid accumulation and eicosapentaenoic acid
distribution in response to nitrogen limitation in microalga Eustigmatos vischeri JHsu-01 (Eustigmatophyceae).
Algal Res. 48, 101910.
138. Yadav, G., Dash, S.K., Sen, R., 2019. A biorefinery for valorization of industrial waste-water and flue gas by
microalgae for waste mitigation, carbon-dioxide sequestration and algal biomass production. Sci. Total Environ.
688, 129-135.
139. Yan, C., Zhu, L., Wang, Y., 2016. Photosynthetic CO2 uptake by microalgae for biogas upgrading and
simultaneously biogas slurry decontamination by using of microalgae photobioreactor under various light
wavelengths, light intensities, and photoperiods. Appl. Energy 178, 9-18.
140. Yan, C., Wang, Z., Wu, X., Wen, S., Yu, J., Cong, W., 2020. Outdoor cultivation of Chlorella sp. in an
improved thin-film flat-plate photobioreactor in desertification areas. J. Biosci. Bioeng. 129(5), 619-623.
141. Yeh, K., Chen, C., Chang, J., 2012. pH-stat photoheterotrophic cultivation of indigenous Chlorella vulgaris
ESP-31 for biomass and lipid production using acetic acid as the carbon source. Biochem. Eng. J. 64, 1-7.
142. Yin, Z., Zhu, L., Li, S., Hu, T., Chu, R., Mo, F., Hu, D., Liu, C., Li, B., 2020. A comprehensive review on
cultivation and harvesting of microalgae for biodiesel production: Environmental pollution control and future
directions. Bioresour. Technol. 301, 122804.
143. Yoo, C., Jun, S., Lee, J., Ahn, C., Oh, H., 2010. Selection of microalgae for lipid production under high levels
carbon dioxide. Bioresour. Technol. 101, S71-S74.
144. Yoshimura, T., Okada, S., Honda, M., 2013. Culture of the hydrocarbon producing microalga Botryococcus
braunii strain Showa: Optimal CO2, salinity, temperature, and irradiance conditions. Bioresour. Technol. 133,
232-239.
145. Yuan, H., Zhang, X., Jiang, Z., Wang, X., Wang, Y., Cao, L., Zhang, X., 2020. Effect of light spectra on
microalgal biofilm: Cell growth, photosynthetic property, and main organic composition. Renew. Energy 157,
83-89.
146. Zhan, J., Rong, J., Wang, Q., 2017. Mixotrophic cultivation, a preferable microalgae cultivation mode for
biomass/bioenergy production, and bioremediation, advances and prospect. Int J Hydrogen Energy. 42, 8505-
8517.
147. Zhao, B., Zhang, Y., Xiong, K., Zhang, Z., Hao, X., Liu, T., 2011. Effect of cultivation mode on microalgal
growth and CO2 fixation. Chem. Eng. Res. Design. 89, 1758-1762.
148. Zhao, Y., Wang, J., Zhang, H., Yan, C., Zhang, Y., 2013. Effects of various LED light wavelengths and
intensities on microalgae-based simultaneous biogas upgrading and digestate nutrient reduction process.
Bioresour. Technol. 136, 461-468.
149. Zheng, H., Gao, Z., Yin, F., Ji, X., Huang, H., 2012. Effect of CO2 supply conditions on lipid production of
Chlorella vulgaris from enzymatic hydrolysates of lipid-extracted microalgal biomass residues. Bioresour.
Technol. 126, 24-30.
150. Zhou, W., Li, Y., Gao, Y., Zhao, H., 2017. Nutrients removal and recovery from saline wastewater by Spirulina
platensis. Bioresour. Technol. 245, 10-17.

Page 40 of 49
1 Table 1. A list of bioreactors and their working volumes used in microalgae research.
Bioreactor Working Microalgae species Cultivation medium Reference
volume
Microplates 100 μL Scenedesmus sp. Modified BG11 (Dao et al., 2018)
(96-well)
Microplates 150 μL Neochloris oleoabundans A seawater-type medium (Santos-Ballardo et
(24-well) al., 2015)
Microplates (96-well) 200 μL 8 green microalgae BG11 and f/2 (Kim et al., 2019)
Clear Multiwell Plate 4 mL 100 native microalgal strains Sterile municipal wastewater or BBM (Abdelaziz et al.,
2014)
Erlenmeyer flask 80 mL Auxenochlorella protothecoides Tris-acetate-phosphate (TAP) (Polat et al., 2020)
Erlenmeyer flask 100 mL Scenedesmus vacuolatus BG11 (Anand et al., 2019)
Transparent bottle 200 mL Chlorella vulgaris BBM medium (Daneshvar et al.,
2018b)
Flat-plate photobioreactors (transparent 1.6 L Chlorella vulgaris FACHB-31 BG11 (Chang et al., 2016)
polymethyl methacrylate)
Glass bottle photobioreactor 4 L Psammothidium sp. Allen Medium (Aghaalipour et al.,
2020)
Polymer film-based bubble column 5L Various microalgae species TAP, BG11, f/2 (Choi et al., 2019)
Bubble-driven column photobioreactor 9.6 L Chlorella sp. FC2 IITG BG11 (Naira et al., 2019)
Column photo-bioreactors 30 L Nannochloropsis oculate Wright’s cryptophyte (Blockx et al., 2018)
Quartz columns photobioreactor 50 L Chlorella vulgaris Secondary effluents samples (Almomani, 2020)
Flat-plate photobioreactor 550 L Scenedesmus sp. Nutrient-rich effluent from pretreated (Viruela et al., 2016)
sewage
Thin-film flat-plate photobioreactor 13000 L Chlorella sp. BG 11 (Yan et al., 2020)
(FPPBR)
Raceways 33000 L Desmodesmus armatus - (Corcoran et al.,
2018)

Page 41 of 49
3 Table 2. Elemental composition of four well-known formulated cultivation media of microalgae.
Element Compounds* BBM f/2 BG11 Zarrouk
C CO2, HCO3-, CO32- Aeration Aeration Aeration Aeration
O O2, H2O H2O H2O H2O H2O
H H2O H2O H2O H2O H2O
N NaNO3 0.25 g/L 0.075 g/L 1.5 g/L 2.5 g/L
Na NaCl 0.025 g/L - - 1 g/L
Na2CO3 - - 0.02 g/L -
NaHCO3 - - - 16.8 g/L
K KOH 0.031 g/L - - -
K2SO4 - - - 1 g/L
Ca CaCl2.2H2O 0.084 g/L - 0.036 g/L 0.08 g/L
P K2HPO4 0.75 g/L - 0.04 g/L 0.5 g/L
KH2PO4 0.175 g/L 5.65 µg/L - -
S MgSO4⋅7H2O 0.075 g/L - 0.075 g/L 0.2 g/L
Mg MgSO4.7H2O - - 0.075 -
Cl as Na+, K+, Ca2+ or NH4+ salts - - - -
Fe Fe-ammonium citrate - - 0.006 g/L -
FeSO4.7H2O 4.98 µg/L - - 0.01 g/L
FeCl3.6H2O - 3.15 µg/L - -
Zn ZnSO4.7H2O 8.82 µg/L 0.022 µg/L 0.222 µg/L 0.222 µg/L
Mn MnCl2.4H2O 1.44 µg/L 0.18 µg/L 1.81 µg/L 1.81 µg/L
B H3BO3 11.42 µg/L - 2.86 µg/L 2.86 µg/L
Mo Na2MoO4.2H2O - 0.006 µg/L 0.391 µg/L -
MoO3 0.71 µg/L - - 0.01 µg/L
Cu CuSO4.5H2O 1.57 µg/L 0.01 µg/L 0.079 µg/L 0.08 µg/L
Co Co(NO3)2.6H2O 0.49 µg/L - 0.0494 µg/L -
CoCl2.6H2O - 0.01 µg/L - -
Br as Na+, K+, Ca2+ or NH4+ salts Not applied in these media
Si Na3SiO3⋅9H2O Not applied in these media
V Na3VO4⋅16H2O Not applied in these media
Sr as sulfates or chlorides Not applied in these media
Al as sulfates or chlorides Not applied in these media
Rb as sulfates or chlorides Not applied in these media
Li as sulfates or chlorides Not applied in these media
I as Na+, K+, Ca2+ or NH4+ salts Not applied in these media
Se SeO32-, SeO42- Not applied in these media
Citric acid - - 0.006 g/L -
Vitamin B12 - 0.0005 µg/L - -
Vitamin B1 - 0.1 µg/L - -
Biotin - 0.0005 µg/L - -
EDTA 0.05 g/L 4.16 µg/L 0.001 g/L 0.01 g/L
pH - - 7.4 7.5
4

Page 42 of 49
10 Table 3. Different types of domestic, agricultural, and industrial wastewaters used for microalgae cultivation.
Wastewater N P C Microalgae Biomass References
production
Molasses wastewater 32.50 TN 2.42 TP 3770 COD Monoraphidium sp. 1.21 g/L (Dong et al., 2019)
Petrochemical wastewater 31.27 TN 1.95 TP 671.30 COD Tribonema sp. 4.4 g/L (Huo et al., 2019)
Swine wastewater 510 TN 76.10 TP 5200 COD Chlorella sorokiniana AK-1 8.08 g/L (Chen et al., 2020)
Domestic Wastewater 52 – 93 TN 13.40 – 28.50 TP 140 – 210 COD Chlorella variabilis 0.99 g/L (Tran et al., 2020)
Raw biogas slurry 271.45 TN 51.92 TP 997.23 DIC Chlorella sp. 0.53 g/L (Yan et al., 2016)
Municipal wastewater 52.20 NH4+ 8.47 PO43– 400 COD Acutodesmus obliquus 0.88 g/L (Ansari et al., 2019)
Mixture of black water and gray 95 TN 12 TP 700 COD Spirulina platensis 0.81 g/L (Zhou et al., 2017)
water
Centrate wastewater 64–289 TN 68–142 TP 1014–4611 Chlorella vulgaris 2.2 g/L (Ren et al., 2017)
COD
Hydrocarbon wastewater 63.50 TN 17 TP 285 COD Spongiochloris sp 8.51 g/L (Abid et al., 2017)
Secondarily treated urban wastewater 20.09 TN 1.55 TP 70 COD Scenedesmus obliquus 1.4 g/L (Álvarez-Díaz et al.,
2017)
Seafood wastewater 243.9 NH4+ 69.80 PO43– 610 HCO3– Chlorella vulgaris 0.49 g/L (Nguyen et al., 2019)
Tannery wastewater 103.80 TN 1.83 PO4-P 814 COD Tetraselmis sp. consortium 1.40 g/L (Pena et al., 2020)
Dairy wastewater 86.0 TN 8.75 PO43− 170.11 TOC   Tetraselmis suecica 0.58 g/L (Daneshvar et al., 2019)
Textile wastewater 373.6 NO3 78.70 Phosphate 42.44 Micractinium sp. 1.35 g/L (Oyebamiji et al., 2019)
Industrial wastewater 153.1 NH4-N 11 PO4-P 72 TOC Chlorella vulgaris 1.52 g/L (Yadav et al., 2019)
11

Page 43 of 49
12 Table 4. A list of environmental variables as reported in different studies in microalgae research.
Photoper Maximum
Light intensity Light CO2 Aeration
Microalgae iod Light source pH Temp. °C Salinity algal yield Reference
μmol m−2 s−1 wavelength % L min−1
(L:D) g L−1
5.0
Natural
Chlorella sorokiniana 1000–3000 - -
irradiance
and - 23.1–30.8 - 5 0.7 (Liu et al., 2020)
6.5
Eustigmatos vischeri 300 24:0 - - - 1 25 - - 8.08 (Xu et al., 2020)
Scenedesmus obliquus 100 12:12 - - 7.2 - 25 - - 0.897 (Qu et al., 2020)
400–750
Chlorella sp. and
Nannochloris oculata
100 - 440–500 LED - 1 25 - - - (Yuan et al., 2020)
500–550
Tisochrysis lutea 60 24:0 627 LED 8.7 0.6 25 40−50 1 1.5 (Fret et al., 2020)
Nannochloropsis oceanica 200 to 636 24:0 450–620 LED 7.8 15−30  1 - (Sá et al., 2020)
(Khichi et al.,
Botryococcus braunii 133 to 348 12:12 - LED 8 5 27 - - 2.52
2019)
(Molitor et al.,
Scenedesmus obliquus 280 24:0 - LED 6.8 0.04−34 27 - 0.1 >2
2019)
(Vasconcelos
Chlorella sorokiniana 175 14:10 - - 7 3 25 - 0.5 11.5 Fernandes et al.,
2015)
Cool-white
Chlamydomonas reinhardtii 110.3 24:0 -
fluorescent
7.2 0.04 25 - - 0.19 (Patel et al., 2015)
16:8 (Fagerstone et al.,
Nannochloropsis salina 90−120 - - 7.5 23 20 - -
2011)
(Blockx et al.,
Nannochloropsis oculata - - - - 8.5 - - 30 5 0.25
2018)
24:0 White
Pavlova lutheri 90–130
12:12
-
fluorescent
5-10 - 28 15−40 - - (Shah et al., 2014)
Cool-white 0.4−18. (Yoshimura et al.,
Botryococcus braunii 0–2000 14:10 - - 0.04−50 5−45 - 3.3
fluorescent 1 2013)
12:12
460
Chlorella sp. 300-900 14:10
660
LED 6.42 - 25 - - 0.532 (Yan et al., 2016)
16:8
Phaeodactylum tricornutum ~ 0.75
500 24:0 - - 8 5 20 - - (Choi et al., 2019)
13
Page 44 of 49
14 Table 5. A list of supplied microalgae and their suppliers for lab-scale research.
Microalgae Origin Supplier Country Reference
Scenedesmus quadricauda and a Tetraselmis Culture Collection of Algae and Protozoa
Culture collection Scotland (Daneshvar et al., 2019)
suecica (CCAP)
Institute of Hydrobiology, Chinese
Chlorella vulgaris FACHB-31 Culture collection China (Chang et al., 2016)
Academy of Sciences
Chlorella minutissima and Synechococcus Oceanographic Institute of the University of
Culture collection Brazil (Costa et al., 2018)
subsalsus São Paulo (USP)
Chlorella vulgaris, Scenedesmus obliquus,
The microalgae stock cultures of the
Psammothidium sp., and Monoraphidium Culture collection Turkey (Aghaalipour et al., 2020)
Biology Department of Gazi University
contortum
Chlorella sorokiniana SAG 211-8 k SAG Culture Collection of Algae Culture collection Germany (Holdmann et al., 2019)
Provasoli-Guillard National Center for
Nannochloropsis salina (1776) Culture collection United States (Fagerstone et al., 2011)
Culture of Marine Phytoplankton (NCMA)
UTEX Culture Collection of algae,
Chlorella vulgaris Culture collection United States (Almomani, 2020)
University of Texas at Austin
Haematococcus pluvialis National Institute for Environmental Studies Culture collection Japan (Hwang et al., 2019)
Acutodesmus dimorphus Industrial effluents Isolation India (Chokshi et al., 2015)
Eustigmatos vischeri JHsu-01 Subtropical lake Isolation China (Xu et al., 2020)
100 native microalgal strains Freshwater lakes and rivers Isolation Canada (Abdelaziz et al., 2014)
Chlorella sp. FC2 IITG Local freshwater pond Isolation India (Naira et al., 2019)
Secondary settler of the Carraixet
Scenedesmus sp. Isolation Spain (Viruela et al., 2016)
wastewater treatment plant
Scenedesmus obliquus Open pond at wastewater treatment plant Isolation South Africa (Ansari et al., 2019)

Page 45 of 49
16

Photoautotroph

Photoheterotroph

Light

Mixotroph
Organic
carbon

Heterotroph
17
18 Fig. 1. Light, inorganic carbon, and organic carbon requirement for the photoautotrophic, heterotrophic,
19 mixotrophic, and photoheterotrophic cultivation of microalgae.

20

21

Page 46 of 49
 Aeration tube Syringe for sampling
Gas venting

Sealing cap Sampling tube

Cultivation medium
Bioreactor
container
Gas bubbles

(A) (C)

(B)
22

23 Fig. 2. Different types of bioreactors used for microalgae research: (A) Schematic overview of common
24 handmade bioreactor, (B) High-tech photobioreactor with online monitoring system (Adapted from Naira et
25 al., 2019), (C) Microplate for a small volume of microalgae cultivation.

Page 47 of 49
 (A) (B)

(C) (D)

26
27 Fig. 3. Common lab-scale bioreactors for microalgae cultivation: (A) bottle bioreactors, (B) flat bioreactor,
28 (C) helical bioreactor, and (D) airlift bioreactor.
29

30 Highlights
31
32  This review discusses the important steps of upstream processing in microalgae research.

33  Critical aspects that influence microalgal cultivation and biomass production are discussed.

Page 48 of 49
34  Existing challenges and knowledge gaps are discussed with future recommendations.

35

36

Page 49 of 49