Resources, Conservation & Recycling 183 (2022) 106355

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Resources, Conservation & Recycling

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

Review

Overview on stress-induced strategies for enhanced microalgae lipid

production: Application, mechanisms and challenges
Xueting Song a, 1, Bing-Feng Liu a, 1, Fanying Kong b, Nan-Qi Ren a, Hong-Yu Ren a, *
a
State Key Laboratory of Urban Water Resource and Environment, School of Environment, Harbin Institute of Technology, Harbin 150090, China
b
School of Water Conservancy and Civil Engineering, Northeast Agricultural University, Harbin 150030, China

A R T I C L E I N F O A B S T R A C T

Keywords: Microalgae have been proposed as a potential renewable energy source for biofuel production. However, owing
Microalgae lipids to the low biomass concentration and lipid productivity, the sustainable and industrialized production of
Biofuels microalgae biofuels is still limited. New lipid improvement strategies have appeared at the technology forefront
Abiotic stress
to overcome the bottlenecks. This review comprehensively illustrates the stress-induced strategies of recent
Metals
studies and achievements in promoting the lipid accumulation of microalgae. Different stress-induced strategies
Phytohormone
Two-stage cultivation for improving lipid production such as the use of phytohormone, change of cultivation strategies, combined
chemical additives and abiotic stresses (nutrient stress, metal ions and wastewater, etc.) are addressed. After a
comprehensive analysis, it is shown that the use of phytohormone combined with abiotic stress under two-stage
culture condition is a promising stress-induced strategy to promote the microalgae lipid accumulation.
Furthermore, the possible mechanisms of using phytohormone and abiotic stress to enhance the microalgae lipid
biosynthesis are discussed. This review is dedicated to further accelerate the sustainable development and
practical application of microalgae-based biofuel production.

1. Introduction These sequential steps should be improved to achieve large-scale pro­

With the depletion of fossil fuels and deterioration of environment,
the pursuit for sustainable and renewable energy resources is becoming
urgent (Aziz et al., 2020; Kong et al., 2020; Singh et al., 2020). Micro­
algae have received a lot of attention from researchers as a sustainable
feedstock due to their potential to achieve high lipid content, fast cell
growth rate, high photosynthetic efficiency and environmental benefits
for biofuels and other high-value bio-products production (Chua et al.,
2022; Pang et al., 2019). In addition, microalgae biofuel could serve as
an eco-friendly and sustainable substitute to fossil fuels (Cheng et al.,
2022; Rosmahadi et al., 2021). However, commercial realization of
microalgae biofuel production has not yet been realized due to the low
biomass concentration, low lipid content, and the high cost in harvesting
and lipid extraction (Hanifzadeh et al., 2018). Meanwhile, the chal­
lenges associated with low biomass concentration is not only less
biomass, but also the greater need for dewatering (thus higher energy
usage, cost, etc.) (Cheng et al., 2021). The microalgae biodiesel pro­
duction usually involves a variety of sequential steps, including algal cell
cultivation, harvesting, lipid extraction and fatty acids methyl ester.
duction of microalgae biofuels (Vasistha et al., 2021). In addition,
cost-effective production of microalgae biofuels needs a sufficient
microalgae biomass productivity (Bellou et al., 2014). Therefore, im­
provements in biomass and lipid biosynthesis efficiencies in microalgae
cultivation phase is the prerequisite for higher productivity (Alvarez
et al., 2021).
The lipids are biosynthesized by different complex synthetic path­
ways within the algal cells, and lipid production is influenced by many
factors like temperature, pH, light intensity and color (Brindhadevi
et al., 2021; Vijay et al., 2021; Zienkiewicz et al., 2016). Microalgae
cultivation under these conditions treatments may increase the accu­
mulation of lipids, but it could impair the growth rate of algae, thereby
reducing the overall productivity of biomass and lipids, and increasing
the production cost of biofuels (Pancha et al., 2015a). In addition, pre­
sent papers have focused on the effect of induction strategies on lipid
accumulation of microalgae (Alishah Aratboni et al., 2019; Shi et al.,
2020; Zhu et al., 2016), and rare information is available on the novel
alternative strategies to improve the biomass and lipid productivity of
microalgae. Therefore, more alternative strategies should be explored to

* Corresponding author: 73 Huanghe Road, Harbin 150090, China. Tel./Fax: +86 451 86282110
E-mail addresses: rhy@hit.edu.cn, rhongyu@126.com (H.-Y. Ren).
1
Both authors contributed equally to this work.

https://doi.org/10.1016/j.resconrec.2022.106355
Received 31 December 2021; Received in revised form 6 April 2022; Accepted 10 April 2022
Available online 20 April 2022
0921-3449/© 2022 Elsevier B.V. All rights reserved.
X. Song et al.
eliminate the limitations of traditional cultivation methods and to
obtain the maximum possible productivities for biofuels, sustainability
and cost effectiveness (Ferreira et al., 2019).
Microalgae can survive in relatively extreme environments by
accumulating lipids in cells (Leong et al., 2022; Ren et al., 2019b). The
lipid accumulation of microalgae can be affected by some abiotic fac­
tors, and it has been found that the energy flow in the biosynthetic
pathways of bio-products can be transferred to neutral lipids (Paliwal
et al., 2017). Nutrient depletion or nutrient stress has been well inves­
tigated to stimulate the microalgae lipid production, like nitrogen
depletion, phosphate starvation, salinity stress, and metals (iron, mag­
nesium, etc.) stress (Giarikos et al., 2021; Khamoushi et al., 2020; Su
et al., 2021; Yaakob et al., 2021). Although these factors can increase the
accumulation of lipids, they may inhibit the growth of algal cells and
even reduce the concentration of biomass, which cannot significantly
improve the lipid productivity (Roleda et al., 2013; Sakarika & Kor­
naros, 2016). This phenomenon may be attributed to the reactive oxy­
gen species (ROS) (generated under abiotic stress/extreme
environments) or oxidative stress damaging the oxygen evolving in
complex and photosystem II reaction center, thereby suppressing the
electron transport, and the absorption, transfer, and utilization of light
energy (Ji et al., 2018). In addition, the reduced biomass concentration
of microalgae is also due to respiration and internal scavenging of
phospholipids etc. and rerouting of other cell resources. However,
microalgae biofuel synthesis requires an enormous amount of biomass
feedstock (Arun et al., 2020). This limitation can be overcome by the
addition of natural or synthetic chemical modulators to the stressed
microalgae. External supplementation of phytohormones or antioxi­
dants can promote the growth of microalgae cells and their tolerance to
the abiotic stress (Giridhar Babu et al., 2017; Touliabah & Almutairi,
2021). In addition, phytohormone can alleviate the microalgae cells
oxidative damage and medicate internal biochemical pathways under
adverse environment conditions, and can improve the lipid accumula­
tion (Giridhar Babu et al., 2017; Li et al., 2019; Zhao et al., 2021b).
This work shows a comprehensive overview on key factors and
cultivation strategies on biodiesel production of microalgae, including
an essential discussion of advantages and disadvantages of stress-
induced strategies. Detailed descriptions on effective cultivation mode
of lipid production as well as advanced cultivation strategies including
the use of two-stage cultivation are investigated. Moreover, combined
abiotic stress and chemical additives for cell growth and lipid accumu­
lation of microalgae are reviewed. Apart from that, the possible regu­
lation mechanism of increasing the lipid production by combining
phytohormones and abiotic stress is also discussed. Further, current
challenges and future perspectives for microalgae biofuel production are
evaluated. The key factors and cultivation strategies of microalgae to
improve lipid biosynthesis and productivities are discussed to provide
the basics for large-scale commercial production of microalgae-based
biofuels.
2. Effects of abiotic stress factors on the microalgae lipid
production
Under abiotic stress conditions, the yields of lipids and secondary
metabolites are higher in microalgae with higher lipid and high-value
bio-products accumulation (Singh et al., 2016b; Yu et al., 2018; Yu
et al., 2020). This is due to microalgae can accumulate more neutral
lipids and other metabolites under extreme environmental conditions to
protect algal cells against oxidative damage (Ding et al., 2018a; Qiao
et al., 2021; Ren et al., 2020). Enhancing lipid accumulation by alter­
ation of the culture medium conditions are widely used in microalgae
cultivation phase (Singh et al., 2016a). The effects of different abiotic
stress factors such as lighting, CO2 concentration, nitrogen, phosphorus,
salinity and metals have been investigated by several researches to
elevate the microalgae lipid accumulation (Qiao et al., 2021; Yu et al.,
2021; Zhao et al., 2020; Zhao et al., 2021a).
2
Resources, Conservation & Recycling 183 (2022) 106355
2.1. Lighting and CO2
Lighting (light intensity, light wavelength and time exposure to the
light, etc.) is one of the most essential abiotic stress factors for cell
growth and lipid production of microalgae. This may be due to the fact
that the photosynthetic process of microalgae is closely related to the
number of photons perceived by cells and the spectrum absorbed.
Moreover, under high light intensity condition, microalgae can produce
more reactive oxygen species, which directly affects the contents of
lipids, protein, and nucleic acids (Gaignard et al., 2021). Nzayisenga
et al. (2020) observed that the highest biomass (1.1 g L− 1) and fatty
acids content (6.2%) were obtained at 300 μmol photons m− 2 s− 1. The
wavelength and time exposure of light could also affect the biomass
production and fatty acid composition. It was shown that the highest
total MUFA (15.9%) and PUFA (27.4%) were acquired at 37.5 μmol
photons m− 2 s− 1 with 8 h:16 h photoperiod (Amini Khoeyi et al., 2011).
In Scenedesmus obliquus, the higher biomass concentration and lipid
content were obtained under blue and red LEDs compared with white
light (Abomohra et al., 2019).
In addition to lighting, CO2 is also an important factor for the
microalgae growth and lipid accumulation, which can control the effi­
ciency of photosynthesis and affect the carbon metabolism rate (Wang
et al., 2021). CO2 concentration within a certain range has been shown
to have positive effect on algal growth and lipid production. However,
excess concentration of CO2 can decrease the pH level of culture me­
dium, which leads to the decrease of intracellular related enzyme ac­
tivity and affects cell growth and lipid accumulation of microalgae
(Omar Faruque et al., 2020). It was reported that lipid accumulation
increased in different concentrations of CO2 in Chlorella vulgaris MSU
AGM 14 (Lakshmikandan et al., 2020). The highest lipid content
(18.6%) and lipid productivity (11.9 mg L− 1 d− 1) were obtained with
8% CO2 concentration. Moreover, 8% CO2 was found to be the optimum
concentration to stimulate cell growth and lipid production during the
outdoor cultivation of Chlorella pyrenoidosa. The maximum biomass
concentration and lipid content was 37.9% and 85.7% higher than that
of the control group, respectively (Wang et al., 2020).
2.2. Nutrients
Alterations of nutrients can be regarded as an effective strategy in
microalgae cultivation, which has positive effect on the lipid production
(Ren et al., 2018a; Vidotti et al., 2020). Nutrients like nitrogen (N),
phosphorus (P), salinity and metals are regarded as crucial for cellular
mechanism, photosynthesis, protein synthesis, and cell division of
microalgae (Hanifzadeh et al., 2018; Zhang et al., 2014). Under nutrient
deficiency or nutrient limitation condition, microalgae tend to produce
energy to protect cell resistance to oxidative stress in the form of neutral
lipids and other by-products (such as docosahexaenoic acid, eicosa­
pentaenoic acid, and carotenoidsand phycocyanin) (Dong et al., 2020).
2.2.1. Nitrogen source
Nitrogen depletion is one of the most effective methods to increase
the microalgae lipid content and productivity. Under nitrogen depletion
condition, cell division can be reduced (Ajjawi et al., 2017; Ren et al.,
2013). The reduction in cell division shifts the lipid biosynthesis path­
ways to accumulate more neutral lipids rather than membrane lipids
required by the formation of cell wall (Nayak et al., 2019; Poh et al.,
2020). Meanwhile, due to slower photosynthetic rate, nicotinamide
adenine dinucleotide (NADH) accumulation could induce the inhibition
of enzyme citrate synthase, which prevents acetyl-CoA from entering
into tricarboxylic acid cycle (Praveenkumar et al., 2012). High con­
centration of acetyl-CoA activates acetyl-CoA carboxylase that converts
acetyl-CoA into malonyl-CoA (Suastes-Rivas et al., 2020). This reaction
is the rate-limiting step of fatty acids synthesis, and induces the
enhancement of algal lipid production (Song et al., 2020). It was
observed that lipid content increased 28.8% and 12.5% in Chlorococcum
X. Song et al.
nivale and Scenedesmus destricola under nitrogen starvation, respectively
(Li et al., 2013). In another report, the increase in lipid content of up to
46.3% in Chaetoceros muelleri and 54.2% in Dunaliella salina was ob­
tained under nitrogen source and complete nutrient depletion condi­
tions, respectively (Gao et al., 2013). A previous study showed that
depletion of nitrogen source can stimulate the lipid production of
Schizochytrium sp. ABC101, and the maximum lipid yield reached 25.4 g
L− 1 (Ju et al., 2020). Similarly, a lipid content of 42.1% in the micro­
algae Chlorella PY-ZU1 was obtained when the algal cells were cultured
under nitrogen deprivation conditions (Chu et al., 2019). It has also been
reported that nitrogen starvation can reduce the thylakoid membrane
cellular level, activate acyl hydrolase and stimulate phospholipid hy­
drolysis, resulting in the increase of intracellular fatty acids acyl-CoA
content (Suastes-Rivas et al., 2020). Thus, nitrogen starvation is a
commonly used strategy to enhance the lipid accumulation in
microalgae.
2.2.2. Phosphorus source
Phosphorus is also an important macronutrient, as it is a crucial
ingredient for the formation of phospholipids, nucleotides, nucleic acids,
ATP and NADP that are involved in different metabolic processes
including energy generation/transformation, signaling pathways and
photosynthesis (Chu et al., 2013; Roopnarain et al., 2014). Under
phosphorus source limitation and starvation condition, Isochrysis gal­
bana U4 is known to accumulate lipids and carotenoids while reducing
chlorophyll (Roopnarain et al., 2014). In particular, the maximum lipid
content of 53% in Scenedesmus sp. was achieved under phosphorus
source limitation condition (0.1 mg L− 1) (Xin et al., 2010). Canavate
et al. (2017) reported that starvation of phosphorus source resulted in
lipid production and changes in microalgae fatty acid composition. In
their study, polar lipid remodeling accounted for 38.8% of total varia­
tion. This may be due to the fact that phosphorus limitation can support
the continuous algal growth and lipid synthesis in microalgae by redi­
recting the carbon distribution of photosynthesis to energy-rich mac­
romolecules (mainly lipids) under stress condition. Although this
nutrient starvation (such as nitrogen source and phosphorus source)
strategy can significantly improve the microalgae lipid accumulation,
the low biomass productivity is the major bottleneck to increase the lipid
yield.
2.2.3. Salinity
The role of salt in the medium is to promote the microalgae to grow
and uptake nutrients. Meanwhile, salinity stress can affect some algal
physiological and biochemical mechanisms. And it can also influence
the fatty acid metabolism, which results in the enhancement of micro­
algae lipid synthesis (Elloumi et al., 2020). An appropriate increase in
Na+ concentration (salinity stress) in the culture medium can contribute
to an improvement in microalgae lipid accumulation (BenMoussa-Dah­
men et al., 2016). It was also known that a high salinity level in medium
can induce oxidative stress to microalgae cells, thereby improving the
lipid accumulation (Pancha et al., 2015b). Teh et al. (2021) indicated
the addition of NaCl for promoted lipid accumulation of Chlorella vul­
garis. An increased salinity level from 5 to 30 ppt improved lipid content,
and the highest lipid content (63.5%) was obtained in 15 ppt salinity
condition. The effect of salinity level on lipid yield of Scenedesmus sp.
CCNM 1077 was investigated by Pancha et al. (2015b) and the lipid
production increased up to 74.6% for 3 days cultivation under 400 mM
NaCl. It has been well documented that increasing the salinity can in­
crease the palmitic acid and oleic acid compositions in microalgae
(Elloumi et al., 2020). In addition, it was reported during salinity stress,
microalgae tended to produce more neutral lipids. This is because
neutral lipids make cell membranes rigid, which helps maintain the
regulation of mineral ions in microalgae cells (Lu et al., 2012). The
carbohydrate content of 53.3 ± 2.8% and lipid content of 33.4 ± 2.3%
in green microalgae Acutodesmus dimorphus were achieved under 200
mM NaCl condition (Chokshi et al., 2017). Salt stress-induced lipid
3
Resources, Conservation & Recycling 183 (2022) 106355
production in microalgae may be due to increased level of oxidative
stress or ROS accumulation.
2.2.4. Metals
It is well known that some metals (Fe3+, Mg2+, Ca2+ and Cu2+) are
indispensable in the culture medium for algal cells to maintain cellular
metabolism and functions and can promote cell growth on a low con­
centration (Gallo et al., 2020). Metals such as iron and copper are
components of photosynthetic electron transport proteins. However,
some metals like cadmium, chromium, lead, mercury and arsenic could
cause negative effects (like impairing photosynthetic mechanism,
blocking cell division and inhibiting enzyme activity) in algal cells.
However, based on the analysis in different studies, it can be found that
appropriate concentrations of metals can stimulate the microalgae lipid
production, and the effectiveness of metals depends on their concen­
trations in culture medium (Kong et al., 2020; Sajjadi et al., 2018).
Iron is an important nutrient in all photosynthesis organisms and
plays a central role in nitrogen fixation, DNA synthesis, respiration, and
other metabolic processes (Concas et al., 2021; Zhang, 2014; Zhuo et al.,
2021). It has been reported that iron participated in fundamental
enzymatic reactions of photosynthesis and it can regulate the gene
expression and lipid accumulation in microalgae (Sajjadi et al., 2018).
Che et al. (2015) cultured Monoraphidium sp. in BG-11 medium with
different concentrations of FeC6H5O7•NH4OH solution, and the algal
lipid productivity was observed to increase with the elevation of Fe3+
level. The maximum lipid productivity of 10.7 mg L− 1 d− 1 was acquired
in culture with 50 μM FeC6H5O7•NH4OH concentration. The Fe3+
enhanced the cell growth and lipid synthesis of microalgae (Concas
et al., 2021). It was shown that when Fe3+ concentration was from 0 to
0.1 g L− 1, lipid content of Chlorella Vulgaris was improved from 9% to
13%. Moreover, the biomass yield of 3.47 g L− 1 and lipid content of
43.2% of Scenedesmus sp. was obtained at 1.2 mg L− 1 Fe3+, respectively
(Ren et al., 2014). The supplement of Fe3+ was reported for algal growth
and lipid synthesis of Chlorella sp. HQ (Liu et al., 2017). It was found that
Fe3+ can promote the algal growth and lipid accumulation, and the
highest algal biomass and lipid content reached 350 ± 31.5 mg L− 1 and
54.2 ± 11.9%, respectively, with the addition of 6.6 mg L− 1 Fe3+.
Therefore, an appropriate rise in iron addition to the medium could
increase the storage of excess carbon, resulting in an increased carbon
flow in the direction of lipid synthesis.
In addition, Mg2+ is also found to affect the cell growth and lipid
production in microalgae (Ren et al., 2014; Sirin et al., 2013). Mg2+
plays an important part in different metabolic processes and physio­
logical pathways of algae and is a major element in microalgae culture
medium. Mg2+ is a component of chlorophyll molecules, and Mg2+ level
in the culture medium directly affects the bio-formation of chlorophyll.
It has been reported that the increase of Mg2+ level could enhance the
acetyl-CoA carboxylase (ACCase) in vivo activity, and ACCase exerted
intense regulation of fatty acid synthesis in microalgae (Lv et al., 2010).
This is due to this enzyme can regulate the first step of microalgae lipid
biosynthesis and is necessary for chloroplast pyruvate dehydrogenase
complex (Volgusheva et al., 2015). Polat et al. (2020) reported biomass
productivity and lipid productivity of microalgae Auxenochlorella pro­
tothecoides treated by 20 mg L− 1 Mg2+ increased by almost 3.9- and
1.1-fold compared with the control. Algal biomass concentration was
30% higher compared with the control, and the maximum lipid content
of 59.8% was achieved under the group supplemented with 100 μM
Mg2+ (Huang et al., 2013). Hence, addition of proper concentration of
magnesium to the medium could promote the growth and lipid pro­
duction of microalgae.
In addition to iron and magnesium, appropriate concentrations of
calcium and copper can also be favorable for microalgae growth and
lipid synthesis. Calcium has been demonstrated to act a critical role in
signal transduction of environmental stimuli and the neutral lipid syn­
thesis can be mediated based on intracellular Ca2+ level by Ca2+ signals
in microalgae (Sajjadi et al., 2018). Moreover, it has been confirmed that
X. Song et al.
copper participates in signal transduction and induces the over­
expression of genes encoding antioxidant enzymes, which favors the
accumulation of metabolites (such as lipid and astaxanthin) (Guo et al.,
2021). Gim & Kim. (2018) reported that when Ca2+ concentration
increased to 10 mg L− 1, the maximum biomass production (6.2 g L− 1)
and lipid yield (25.1%) of Botryococcus braunii LB572 were achieved.
However, after continuing to increase the concentration of Ca2+, the
lipid production was seriously inhibited. The biomass concentration and
lipid content of microalgae increased 12.1% and 4.5% in the presence of
2 mM CaCl2 under strigolactone induction, respectively. Conversely, the
biomass concentration and lipid production were both markedly
decreased with the addition of calcium chelating agent of ethylene
glycol tetraacetic acid (Song et al., 2019). The biomass productivity and
lipid productivity of Chlorella minutissima UTEX2341 increased 12.4%
and 33.9%, respectively, with the addition of 0.4 mM copper in culture
medium (Yang et al., 2015). Aguilar-Ruiz et al. (2020) also found higher
lipid content of 77.1% was obtained under 4.6 mg L− 1 Cu2+ treatment of
microalgae Nannochloropsis oculate, twice as high as that achieved in the
control (33.1%). Based on these studies, it can be found that calcium and
copper are both vital metals for cell growth and lipid production of
microalgae.
Heavy metals in water could be accumulated in organisms and might
produce various toxic reactions at the cellular levels (Nguyen et al.,
2020; Rai et al., 2013). Although heavy metals usually exhibited nega­
tive influence during the microalgae cultivation, some studies also
suggested they had positive effect on the microalgae culture (Rai et al.,
2013; Salama et al., 2019). Previous researches showed the underlying
of heavy metals to enhance the lipid production and stimulate the
microalgae growth and metabolites accumulation (Yang et al., 2015). In
addition, it was reported that heavy metals can be effectively removed in
green microalgae culture process (Giarikos et al., 2021; Salama et al.,
2019). Sun et al. (2015) showed that arsenic could increase the lipid
production and arsenic adsorption of Nannochloropsis sp. The maximum
lipid content of 24.2% was examined with 10 mg L− 1 arsenic. In a study,
lipid content (52.8%) and productivity (96.8 mg L− l d− 1) increased by
160% and 140% in algal cells treated with 80 and 40 μM Cd2+,
respectively, compared to the control (0 μM Cd2+) (Zhao et al., 2019a).
The increase in lipid production might be due to the production of
Fig. 1. Schematic mechanism of metals translocation, sequestration, uptake in microalgae, and the possible mechanism of metal-mediated lipid accumulation. Men+,
Metal ion; AA, Amino acids; OA, Organic acids; Gly, Glycine; Cys, Cysteine; GSH, Glutathione; PC, Phytochelatin; PC-MeC, Phytochelatin metal ion complexes; LMW
PC-MeC, Low Molecular Weight Phytochelatin metal ion complexes; HMW PC-MeC, High Molecular Weight phytochelatin metal ion complexes; MTP - Metal­
lothionein protein; GCS, Glutamyl–cysteinyl synthase; GS, Glutathione synthase; PCS- phytochelatin synthase.
4
Resources, Conservation & Recycling 183 (2022) 106355
antioxidants, and anti-oxidizes could alleviate the oxidative stress
generated by cadmium stress. Yang et al. (2015) studied the influences
of Cd2+ and Cu2+ on triglyceride accretion of oleaginous microalgae
Chlorella minutissima UTEX2341. It was found under heterotrophic cul­
ture condition Cd2+ and Cu2+ were major impact factors for triglyceride
accumulation. The highest lipid productivity at 0.4 mM Cd2+ and 0.4
mM Cu2+ showed 249.4 and 154.2 mg L− 1 d− 1, respectively. Similarly,
Leon-Vaz et al. (2021) used heavy metal cadmium to increase the lipid
accumulation and obtained 560% increase of the plastid acyl-ACP
desaturase, which could take part in the polyunsaturated fatty acids
synthesis. In addition, cadmium also led to upregulation of starch
catabolism, photorespiration and anti-oxidant enzymes in Chlorella
sorokiniana. According to the Moreno-García et al. (2021), Cr (III)
increased the content of methyl oleate (C18:1) and led to a decrease in
methyl linolenate (C18:3) and methyl linolelaidate (C19:1), which
improved the oxidative stability of biodiesel. Afterwards, the adsorption
efficiency of 100 mg L− 1 Cr (III) by microalgae was studied, and it could
reach 99%. This probably occurred because microalgae cells resisted
oxidative stress by accumulation of lipids and metabolites under heavy
metal treatment condition. The complete overview of mechanism of
metals translocation, sequestration, uptake in microalgae, and the
possible mechanism of metal-mediated lipid accumulation are illus­
trated in Fig. 1. Heavy metals in the cytoplasm can affect photosynthetic
activity and stimulate ROS production of microalgae. To protect algal
cells affected by ROS, the cells activate different protection mechanisms,
among which the main ones are ROS scavenging enzymes and secondary
metabolites accumulation such as lipids and pigments (Guo et al., 2021;
Zhao et al., 2019a). Moreover, the bioremediation of heavy metals by
microalgae may be achieved by converting these metals into some
complexes (Salama et al., 2019).
Nutrients alteration is a preferred strategy to promote the algal lipid
accumulation because it is easy to use in lab and industrialization cul­
tures (Ananthi et al., 2021). Nevertheless, the major limitation of this
strategy is how to balance the biomass concentration and lipid pro­
ductivity. Some conventional abiotic stress factors for promoting
microalgae lipid production and their advantages and challenges are
outlined in Table 1. The abiotic stress factors like nitrogen starvation,
salinity stress and heavy metals treatment have shown significant roles
X. Song et al. Resources, Conservation & Recycling 183 (2022) 106355

Table 1 Table 2
Comparison of advantages and challenges of some stress-induced strategies for Comparison of the biomass concentration/productivity and lipid content/pro­
promoting microalgae lipid production. ductivity obtained from two-stage cultivation for different microalgae species.
Approaches Advantages Challenges Microalgae strain Biomass Lipid content/ Reference
concentration/ Lipid
Nutrient deficiency High lipid production Low biomass
Biomass productivity
productivity
productivity
High amount of saturated Low lipid productivity
1 1 1
fatty acids Chlorella sp. HS2 774.4 mg L− d− 289.6 mg L− (Nayak et al.,
Metals High lipid accumulation Low lipid productivity d− 1 2020)
− 1
Biosorption of heavy Chlorella 2.5 g L 37.5% (Kakarla et al.,
metals sorokiniana HS1 2018)
Wastewater High lipid accumulation Needs large-scale tests Tetraselmis sp. - 51.7% (Swain et al.,
Wastewater treatment 2020)
1 1
Two-stage cultivation High biomass production Needs large-scale tests Acutodesmus 143.0 mg L− d− 66.6 mg L− 1 (Renuka et al.,
in the first stage obliquus d− 1 2018a)
1
High lipid accumulation Monoraphidium sp. 389.1 ± 1.6 mg L− 120.8 ± 2.0 mg (Che et al.,
in the second stage FXY-10 d− 1 L− 1 d− 1 2016)
Combined multiple High lipid accumulation Needs to find cheap Monoraphidium 1.2 g L− 1 49.6% (Li et al., 2017)
stresses nutrient sources sp. QLY-1
1
Phytohormones High growth rate High cost of Nannochloropsis 1.0 g L− 40.0 % (Aleman-Nava
High lipid production phytohormones oculata et al., 2017)
1 1
Coupling abiotic stress High/maintain biomass Needs further research Chlorella 44.0 mg L− d− 20.2% (Amaral et al.,
and phytohormones production and optimization minutissima 2020)
− 1 1
High lipid productivity High cost of Chlorella 0.5 g L 28.8 mg L− (Xie et al., 2020)
phytohormones sorokiniana SDEC- d− 1
18
1 1
Nannochloropsis 490.0 mg L− d− 43.0% (Remmers et al.,
in lipid synthesis, and it is necessary to further study the physiological gaditana 2017)
1
and biological effects of these abiotic stresses on microalgae to better Chlorella vulgaris 18.2 g L− 35.3% (Wang et al.,
2016)
produce lipids. The performance comparison of microalgae being Neochloris 52.0 mg L − 1
d − 1
51.0% (Yoon et al.,
exposed to various abiotic stresses is shown in Table S1. oleoabundans 2015)

3. Effects of novel alternative strategies on the microalgae lipid

production
Although conventional approaches of producing microalgae lipids
under abiotic stress can promote lipid accumulation, these factors usu­
ally inhibit the growth of microalgae, thereby increasing the cost of
microalgae biofuel production. In order to overcome the bottlenecks of
conventional approaches, more efficient strategies are required,
focusing on increasing/maintaining the biomass concentration and
improving lipid accumulation. Some novel alternative strategies such as
two-stage cultivation of microalgae, combined multiple stresses, sup­
plementation of phytohormone and coupling abiotic stresses and
chemical additives have been used by several researches to improve the
microalgae lipid productivity for biofuel production (Shokravi et al.,
2020).
3.1. Two-stage cultivation
A two-stage cultivation strategy could be considered as an emerging
microalgae culture strategy because it can not only enhance the biomass
concentration, but also increase the microalgae lipid productivity (Aziz
et al., 2020; Luo et al., 2019; Nagappan et al., 2019). In the two-stage
culture, algal cells are firstly grown in the optimal growth conditions
to gain higher biomass concentration. In the second phase, algal cells are
treated with stimulating factors to trigger the production of lipids
(Nayak et al., 2020). Table 2 presents a summary of studies in which a
two-stage cultivation method is used to increase the biomass/lipid
accumulation. Table 2 describes the lipid content ranging from 20.2%
(%, dry weight) to 51.7% (%, dry weight) and biomass concentrations
from 0.5 to 18.2 g L− 1 under various algal strains and treatments. It was
observed two-stage cultivation can be used for simultaneous accumu­
lation of lipids, carotenoids, and antioxidant compounds in Chlorella
vulgaris. When Chlorella vulgaris was treated with 5 g L− 1 MgCl2 and
7700 lux light intensity in the second stage, the maximum lipid pro­
ductivity of 15.6 ± 0.1 mg L− 1 d− 1 was recorded (Ali et al., 2020).
Kakarla et al. (2018) investigated the effect of NaCl stress in a two-stage
microalgae culture. Algal cells were initially grown in freshwater BG-11
medium under mixotrophic conditions until late exponential stage, and
then were placed under high NaCl concentration to induce positive lipid
production. The maximum biomass yield of 2.5 g L− 1 and lipid content
of 37.5% were achieved in Chlorella sorokiniana HS1. Furthermore,
cultivation of microalgae was quite effective for simultaneous biomass
and lipid production by applying two-stage cultivation strategy (Gane­
san et al., 2020). In another report, the maximum lipid content of 51.7%
was obtained in two-stage cultivation of Tetraselmis sp. (Swain et al.,
2020). Microalgae biofuels production by the two-stage cultivation have
multifarious beneficial characteristics in comparison to the ones ac­
quired from single stage cultivation. For large-scale production of
microalgae biofuels, the use of photo-bioreactors in the first-stage
cultivation generates biomass with high algal cell density in a shorter
time and the use of open-pond in the second-stage cultivation increases
the lipid accumulation. This cultivation mode can simultaneously pro­
mote the biofuels and biomass accumulation. The results suggest that
two-stage cultivation strategy is also a promising research direction for
large-scale microalgae biofuel production in the future.
3.2. Combined multiple stresses
3.2.1. Multiple abiotic stresses
Recent studies have reported the potential to combine multiple
stresses factors to improve lipid productivity of microalgae. Some
combined multiple stresses, including the use of combing temperature
stress and nitrogen depletion, light and nutrient stress, high salinity and
nitrogen depletion are discussed. Table 3 shows the effect of combined
multiple stresses condition on the biomass concentration/productivity
and lipid content/productivity in various microalgae strains. And
different microalgae strains and cultivation conditions showed different
biomass concentrations and lipid contents under combined multiple
stresses conditions (Table 3). To achieve the maximum lipid yield, it is
necessary to understand the synergistic effects of these multiple stress
factors and the importance of each factor to lipid accumulation of
microalgae. Qiao et al. (2021) discussed the synergistic effect of
hydrogen peroxide and salinity stress on microalgae lipid accumulation.

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Table 3
The biomass concentration/productivity and lipid content/productivity of various microalgae under combined multiple stresses conditions.
Microalgaestrain Cultivation condition Biomass concentration/ Biomass Lipid content/Lipid Reference
productivity productivity
1 1
Monoraphidium sp. H2O2 (1.0 mM) 200.0 mg L− d− 53.3% (Qiao et al., 2021)
NaCl (171.1 mM)
Chlorella vulgaris MgCl2 (5.0 g L− 1) 2.4 g L − 1 − 1 − 1
15.6 mg L d (Ali et al., 2020)
2
Light intensity (140.0 μmol photons m−
s− 1)
Chlorella minutissima NaCl (43.7 g L− 1) 307.0 mg L− 1
d− 1
22.8% (Cao et al., 2014)
Fe3+(0.1 mM)
NaNO3 (0 mM)
1 1
Scenedesmus sp. NaCl (100 mM) 15.1 mg L− d− 38.5% (Anand et al., 2021)
NaNO3 (0 mM)
K2HPO4 (0.2 mM)
Monoraphidium sp. High temperature (30◦ C) 0.7 g L− 1 29.2 mg L− 1d− 1 (Wu et al., 2013)
Chlorella minutissima Wastewater 0.6 g L− 1 49.1 mg L− 1d− 1 (Arora et al., 2016)
Picochlorum sp. Nitrogen starvation 236.6 mg L− 1d− 1
138.9 mg L− 1d− 1 (Dahmen-Ben Moussa et al.,
Scenedesmus Phosphorus starvation 1.1 g L− 1 43.0% 2019)
rubescens Phosphorus starvation (Jo et al., 2020)
(NH4)2SO4 (1.0 g L− 1)
NaCl (400 mM)
2
Light intensity (1000 μmol photons m−
s− 1)

When 1 mM H2O2 and 10 g L− 1 NaCl treatment were combined, lipid
productivity of 107.3 mg L− 1 d− 1 was acquired, which was 14.3% and
43.3% higher compared with 10 g L− 1 NaCl treatment alone and control
(0 g L− 1 NaCl and 0 mM H2O2), respectively. Ali et al. (2020) stated that
there was a simultaneous increase in lipids, carotenoids, and antioxidant
compounds when Chlorella vulgaris was subjected to abiotic stress (light
and high salinity stress condition). Studies have shown that the
maximum lipid productivity of 15.6 ± 0.1 mg L− 1 d− 1 under 5 g L− 1
MgCl2 and 7700 lux light intensity condition. The addition of 43.7 g L− 1
NaCl and 0.1 mM FeCl3 6H2O resulted in the increase of lipid content of
Chlorella minutissima UTEX 2341 under nitrogen depletion condition,
and the lipid content reached 22.8% (Cao et al., 2014). Another report
also revealed the effect of multiple stresses on lipid production of Sce­
nedesmus sp. In their study, the highest lipid content of 385.4 ± 34.2 μg
mg− 1 was obtained under 100 mM NaCl, 0.2 mM K2HPO4 and nitrogen
starvation condition (Anand et al., 2021). In a study carried out by Wu
et al. (2013), increased accumulation of biomass concentration and
lipids in microalgae were observed. The highest lipid content of 34.7%
was obtained in high temperature and industrial wastewater condition.
Thus, the combination of multiple abiotic stresses could further enhance
the microalgae lipid synthesis compared with one stress alone by regu­
lating oxidative stress and osmotic balance.
maximum biomass concentration of 1.2 g L− 1 and lipid content of 68.7%
were achieved by using combined molasses wastewater and melatonin
treatment. Silambarasan et al. (2021) indicated that the microalgae
consortium (combined Chlorella sp. and Scenedesmus sp.) grew well in
75% diluted wastewater, and acquired the maximum biomass concen­
tration (1.8 g L− 1), chlorophyll content (27 μg mL− 1), protein level (175
μg mL− 1) and lipid content (34.8% dry cell weight) under 75% diluted
domestic wastewater. Similarly, supplementation of glycerol obtained
the maximum lipid content of 39.5% and lowest carbohydrate content of
8.2% for Chlorella sp. under heterotrophic condition by using municipal
wastewater as the culture medium. Higher biomass concentration of 650
mg L− 1 and lipid productivity of 29.2 mg L− 1 d− 1 were achieved by
coupling high temperature (30◦ C) with wastewater treatment (Nzayi­
senga et al., 2018). This confirms the response of algal cells to the
oxidative and intracellular lipid peroxidation environment under
wastewater treatment condition. Moreover, the potential of microalgae
to remove heavy metals, TN, TP, COD and antibiotics in actual waste­
water remains to be explored, and the simultaneous accumulation of
lipids, pigments (astaxanthin, chlorophyll and xanthophyll, etc.), pep­
tides or amino acids and antioxidants in the microalgae also needs to be
further clarified.

3.2.2. Multiple stresses induced by wastewater 3.3. Chemical additives

It has been known that wastewater is a mixture component con­
taining many nutrients, heavy metals and antibiotic residues, which
constitutes multi-stress environment (Ferreira et al., 2019; Pang et al.,
2019; Ren et al., 2022; Rosli et al., 2019; Usmani et al., 2020). Specif­
ically, diverse metabolites can be synthesized by microalgae using
wastewater, including lipids, antioxidants and pigments (Zhuang et al.,
2020). The use of wastewater to produce microalgae biomass provides
nutrients sources (such as carbon, nitrogen and phosphorus), energy and
organic matter in a cost-effective way, and at the same time offers an
environmentally friendly wastewater treatment method (M & K, 2021;
Ren et al., 2018b). In addition, algal cultivation can allow efficient
removal of heavy metals, total nitrogen (TN), total phosphorus (TP),
chemical oxygen demand (COD) and other organic materials from
wastewater, thus minimizing ecological risks (Grandclement et al.,
2017; Ji, 2022; Nguyen et al., 2019; Ren et al., 2019a). Furthermore,
wastewater is considered as an economical culture medium, which can
be used as nutrients source for microalgae growth and lipid production.
Dong et al. (2019) successfully adopted molasses wastewater to promote
the cell growth and lipid accumulation of Monoraphidium sp. The
In recent studies, some chemical additives were known to increase
cell density and lipid production in microalgae by regulating different
metabolic pathways such as respiration, signal transduction, antioxidant
system and oxidative evolution of PSII and ATP-synthase (Sun et al.,
2019).
3.3.1. Phytohormone
Addition of phytohormone has great potential as a sustainable and
eco-friendly approach to promote the microalgae lipid accumulation
(Arora & Mishra, 2020; Madani et al., 2020; Stirk & van Staden, 2020).
The role of phytohormone as effective “exogenous additives” for stim­
ulating microalgae biomass and lipid accumulation has been well
documented (Che et al., 2016; Li et al., 2017; Song et al., 2019). Earlier
studies have shown that specific role of phytohormone can participate in
regulating the microalgae growth and lipid production (Salama et al.,
2014; Stirk & van Staden, 2020; Yu et al., 2018). The effect of phyto­
hormone treatment (such as indole-3-acetic acid (IAA), kinetin, strigo­
lactone, and fulvic acid) on the biomass concentration and lipid
accumulation in microalgae is illustrated in Table 4. Yu et al. (2017)

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Table 4
Effect of phytohormone treatment on the cell growth and lipid production of microalgae. Results showed an increase (+) or a decrease (-) compared with the control.
IAA, Indole-3-acetic acid; GA3, Gibberellic acid; SL, Strigolactone; MeJA, Methyl jasmonic acid; FA, Fulvic acid; MT, Melatonin; EPA, Eicosapentaenoic acid; ME, Malic
enzyme; accD, Acetyl-CoA carboxylase beta subunit; GPAT, glycerol-3-phosphate acyltransferase; KAS I, β-ketoacyl ACP synthase I; C16:0, Palmitic Acid; C18:0, 12-
Hydroxy Stearic Acid.
Microalgaestrain Phytohormone Biomass concentration/ Lipid content/Lipid Other performances Reference
treatment Biomass productivity productivity
1 1 1
Scenedesmus obtusus IAA (25 μM) 0.8 g L− 17.8 ± 1.3 mg L− d− Carbohydrate productivity (13.9 ± (Vijay et al., 2021)
0.6 mg L− 1 d− 1)
Protein productivity
(19.0 ± 1.6 mg L− 1 d− 1)
1 1 1
Scenedesmus obtusus Kinetin (25 μM) 1.1 g L− 17.8 ± 1.5 mg L− d− Carbohydrate productivity (19.0 ± (Vijay et al., 2021)
1.6 mg L− 1 d− 1)
Protein productivity
(25.1 ± 0.4 mg L− 1 d− 1)
1
Monoraphidium sp. QLY-1 SL (1 μM) 1.0 g L− 48.8% Ca2+ level (+), NO level (+) (Song et al., 2019)
Monoraphidium sp. FXY-10 FA (25 mg L− 1) 2.3 g L− 1
54.3 % ME (+), accD (+), GPAT (+) (Che et al., 2016)
1
Monoraphidium sp. QLY-1 MT (1 μM) 1.2 g L− 49.6% Protein content (-) (Li et al., 2017)
Carbohydrate content (-)
1 1
Monodopsis subterranea GA3 (100 μM) 4.7 mg L− d− 9.7% EPA content (+) (Arora & Mishra,
CCALA 2020)
− 1 − 1
Monodopsis subterranea MeJA (100 μM) 3.9 mg L d 10.4% EPA content (+) (Arora & Mishra,
CCALA 2020)
1
Ankistrodesmus falcatus IAA (10 μM) 0.4 g L− (+) Chlorophyll-a (+) (Vijay et al., 2020)
CMSACR1001 Total chlorophyll (+)
Nannochloropsis oceanica IAA (40 μM) 579.5 × 106 cells mL− 1
60.9% EPA content (10.8%) (Udayan &
CASA CC201 Arumugam, 2017)
6 − 1
Chlorella vulgaris UMT-M1 GA3 (10 μM) 9.8 × 10 cells mL 40.0 % KAS I (+) (Jusoh et al., 2019)
C16:0 (+)
C18:0 (+)

reported that lipid content of Scenedesmus sp. and Chlorella sorokiniana Ren et al. (2014) reported that after treatment with EDTA and metal
were obviously affected by naphthylacetic acid (NAA) and ions, the highest biomass of 3.5 g L− 1 and lipid content of 47.4% was
indole-3-butyric acid (IBA) at very low concentrations (5 to 50 mg L− 1), observed in Scenedesmus sp. A previous study reported that supple­
and the maximum lipid content of Scenedesmus sp. and Chlorella sor­ mentation of 1 g L− 1 EDTA and 20 mg L− 1 fulvic acid (FA) influenced the
okiniana was 56.2% and 55.8%, respectively. Li et al. (2017) investi­ cell growth and lipid accumulation of Schizochytrium sp. Moreover,
gated the role of phytohormone melatonin on the lipid biosynthesis of higher biomass concentration of 130.7 g L− 1 and lipid productivity of
Monoraphidium sp. When Monoraphidium sp. was cultivated in medium 1.2 g L− 1 h− 1 were achieved, representing 36.4% and 3-fold increase
containing 1 μM melatonin, the highest lipid content of 49.6% was over the un-treated group, respectively (Sun et al., 2018a). In a study,
achieved. Moreover, Singh et al. (2020) have shown that in medium the lipid productivity of microalgae Chlorella sorokiniana was improved
composed of IAA, IBA and indole-3-propionic acid and cytokinins, lipid from 71.0 to 77.0 mg L− 1 d− 1 when EDTA (3 mg L− 1) was added to the
content of Desmodesmus sp. increased up to 40-62%. Phytohormone medium under metal stress condition (Singh et al., 2017). The addition
provides a potential strategy to enhance the lipid accumulation due to of different chemicals to culture medium can enhance the lipid synthesis
their intrinsic functions in microalgae cell growth and metabolites yield, (Table 5). Moreover, under different chemical treatments, biomass
which can increase the lipid productivity and improve the microalgae concentration and lipid content increased to different degrees (Table 5).
tolerance to adapt to more extreme environmental changes. In addition, Therefore, the addition of chemicals is a useful strategy at low doses, and
phytohormone can also be used as signal molecules to participate in
signaling pathways, and the signaling cascades could induce feedback Table 5
regulation of lipid and metabolites production in microalgae. Effects of some chemical additives on the microalgae lipid production.
Chemicals Microalgae Species Increase in lipid Reference
3.3.2. Other chemicals production
A few other chemical additives are also demonstrated to affect the
EDTA Scenedesmus sp. Lipid content (Ren et al.,
biomass concentration and lipid accumulation by indirect regulation of
increased 28.2% 2014)
lipid biosynthesis pathway in microalgae, such as regulating oxidative Brefeldin A Chlamydomonas Lipid content (Kim et al.,
stress or increasing cell permeability (Colling Klein et al., 2018; Sun reinhardtii increased 34% 2013)
et al., 2019). Additionally, brefeldin A is recorded to enhance the lipid Carbon dots Chlorella sp. Lipid content (Zhao et al.,
accumulation in Chlamydomonas reinhardtii. An increase of 34% in lipid increased 19.5% 2021c)
EDTA Schizochytrium sp. Lipid productivity (Sun et al.,
content was obtained under 75 μg mL− 1 brefeldin A treatment. This increased three-fold 2018a)
effect could be due to brefeldin A-induced endoplasmic reticulum stress, Azide Chlorella desiccata Lipid content (Rachutin
which rapidly triggered triglyceride accumulation in Chlamydomonas improved 60-70% Zalogin & Pick,
reinhardtii (Kim et al., 2013). Zhao et al. (2021c) investigated the role of 2014)
Tween-80 Thraustochytrium Lipid content (Taoka et al.,
carbon dots on lipid yield and protein content of Chlorella sp. The
aureum ATCC 34304 increased 1.2-fold 2011)
highest lipid content of 47.1% and protein content of 14.3 % were ob­ Malonate Schizochytrium sp. Lipid productivity (Zhao et al.,
tained at 1 mg L− 1 carbon dots treatment, which was 19.5% and 24.8% increased 1.8-fold 2018a)
higher than the control (0 mg L− 1), respectively. Ethylene diamine tet­ Ethanolamine Scenedesmus obliquus Lipid yield (Cheng et al.,
raacetic acid (EDTA) is known to increase the cell permeability through increased 22% 2012)
Sethoxydim Crypthecodinium cohnii Lipid yield (Diao et al.,
inducing the membrane pore and facilitating the uptake of nutrients into increased 50% 2019)
microalgae cells, which has been widely reported in increasing biomass Sesamol Crypthecodinium cohnii DHA productivity (Liu et al.,
concentration and lipid production of microalgae (Sun et al., 2018a). increased 20% 2015)

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X. Song et al. Resources, Conservation & Recycling 183 (2022) 106355

can be easily adopted in large-scale biofuel production.
3.4. Coupled abiotic stress and chemical additives
3.4.1. Coupled abiotic stress and phytohormone
Microalgae can adapt to some abiotic stresses by accumulating lipids
and other metabolites (Yu et al., 2018). However, the major drawback of
abiotic stress-induced lipid production is the substantial reduction in
biomass concentration. Phytohormone supplementation could sustain
the cell growth and eventually prevent the reduction of biomass con­
centration under abiotic stress condition (Song et al., 2020). This is
because phytohormone can alleviate the oxidative damage caused by
ROS and free radicals generated during abiotic stress in microalgae
(Zhao et al., 2019b). Some phytohormones might be involved in regu­
lating microalgae ROS homeostasis. Exogenous supplementation of five
diverse phytohormones, including IAA, GA, kinetin, 1-triacontanol and
ABA could exhibit stimulation effect on the microalgae cell growth.
Besides ABA, other phytohormones can improve the fatty acid methyl
esters accumulation under nitrogen limitation condition in Chlamydo­
monas reinhardtii (Park et al., 2013). Some phytohormones can be used
as signaling molecules to participate in the signaling cascade in algal
cells, thereby regulating the expression level of genes associated with
lipid synthesis and improving the microalgae lipid production (Song
et al., 2019; Zhao et al., 2021a). The highest biomass concentration of
0.9 g L− 1 and lipid content of 57.1% were achieved in Monoraphidium sp.
by coupling jasmonic acid with strigolactone under nitrogen deficiency
(Song et al., 2020). Under combined effect of IAA and nitrogen starva­
tion, the biosynthesis of pigments in Nannochloropsis oceanica was
enhanced, thereby promoting the algal cell growth and lipid accumu­
lation (Touliabah & Almutairi, 2021). Li et al. (2019) also found that
phytohormone FA could increase the microalgae biomass concentration
and lipid accumulation under NaCl treatment and achieved the highest
lipid content of 59.5% at combined 25 mg L− 1 FA and 20 g L− 1 NaCl
conditions. Some studies have confirmed that the coupled abiotic stress
and phytohormone can promote the lipid accumulation in microalgae
(Table 6). In general, coupling phytohormone and abiotic stresses as a
sustainable lipid production strategy has broad prospects. Further
investigation should be conducted to better understand the mechanisms
of phytohormone-medicated oxidative stress tolerance in enhancing the
lipid accumulation.
3.4.2. Coupled abiotic stress and antioxidants
Antioxidant plays a crucial role in the regulation of oxidative stress
in microalgae, protecting algal cells from oxidative damage. In addition,
antioxidant can promote the lipid production in microalgae under stress
conditions. As common antioxidants, butylated hydroxytoluene, glycine
betaine and butylated hydroxyanisole have been employed to enhance
the microalgae lipid production. A previous work (Zhao et al., 2018c)
found that 2 mg L− 1 antioxidant butylated hydroxytoluene can increase
the microalgae lipid content (46.0%) under high-light and nitrogen
depletion, thereby revealing its efficiency as a lipid promoter. Addition
of glycine betaine resulted in positive effect on the microalgae lipid
production. In their study, the highest lipid content of 48.5% and lipid
productivity of 121.2 mg L− 1 d− 1 were achieved under 5 mM glycine
betaine and 20 g L− 1 NaCl condition (Zhao et al., 2016). Ding et al.
(2019) found that butylated hydroxyanisole up-regulated and activated
the processes involved in cellular basal metabolism (such as TCA cycle,
glycolysis and amino acid metabolism) and signaling systems (such as
Ca2+ signal cascade and phosphatidylinositol signaling system), thereby
improving the lipid accumulation in H. pluvialis under nitrogen deple­
tion. However, the mechanisms of antioxidant and abiotic stress con­
ditions on the lipid accumulation of microalgae remain to be elucidated.
Application of these novel alternative strategies has great potential,
sustainability and scalability for the production of microalgae lipids
(Fig. S1). Furthermore, it is essential to choose different effective
nutrient sources or abiotic stresses, including phytohormones or chem­
ical additives to increase the cell growth and stress tolerance of algal
cells. The addition of exogenous phytohormone is one of the most effi­
cient strategies to increase the algal lipid production under abiotic
stress. Additionally, further research should be conducted to compre­
hensively understand the mechanism of phytohormone-mediated stress
tolerance to enhance the lipid production under abiotic stress condition
and facilitate its application in large-scale microalgae cultivation.
4. Possible mechanisms of phytohormone-mediated stress
tolerance to enhance the lipid production of microalgae
This review provides a possible mechanism for the stress tolerance of
phytohormone to promote the lipid accumulation and further under­
stand the role of phytohormone in abiotic stresses (Fig. 2). To date, there
are only a few studies on the functional roles of phytohormone and how

Table 6
The performance of various microalgae under combined phytohormones and abiotic stress conditions. KI, Kinrtin; TRIA, 1-triacontanol; JA, Jasmonic acid; NAA,
Naphthylacetic acid; DA-6, Diethylamine ethyl hexanoic acid; IBA, Indole-3-butyric acid; CK, Cytokinin; EBL, 24-epibrassinolide; FAMEs, Fatty acid methyl esters;
rETR, relative electron transport rate.
Microalgae Phytohormones Stress Performance References
− 1 − 1
Chlorella sorokiniana IAA Nitrogen The highest biomass productivity of 201.0 mg L d and lipid productivity of 69.0 (Giridhar Babu et al.,
limitation mg L− 1 d− 1 were obtained. 2017)
Chlorella sorokiniana DA-6 Nitrogen The highest biomass productivity of 236.0 mg L− 1 d− 1 and lipid productivity of 86.0 (Giridhar Babu et al.,
limitation mg L− 1 d− 1 were obtained. 2017)
Monoraphidium sp. FA Salinity stress The highest biomass of 1.2 g L− 1 and lipid content of 59.5% were obtained. (Li et al., 2019)
Scenedesmus SDEC-8 IBA Nitrogen The highest lipid productivity with 26.7 mg L− 1 d− 1 was obtained. (Yu et al., 2018)
Scenedesmus SDEC-8 NAA depletion The highest lipid productivities with 25.9 mg L− 1 d− 1 was obtained. (Yu et al., 2018)
Nitrogen
depletion
Monoraphidium sp. SL, JA Nitrogen The highest lipid content of 57.1% was obtained. (Song et al., 2020)
starvation
Chlamydomonas IAA, GA3, KI, Nitrogen Algal growth and FAMEs accumulation were both promoted. (Park et al., 2013)
reinhardtii TRIA limitation
Monoraphidium sp. MT Nitrogen The highest lipid content of 51.4% was obtained. (Zhao et al., 2018b)
Acutodesmus obliquus CK starvation Photosynthetic performance was improved and higher rETR values. (Renuka et al., 2017)
Acutodesmus obliquus Zeatin Nitrogen The highest biomass productivity of 176.8 mg L− 1 d− 1 was obtained. (Renuka et al., 2017)
Acutodesmus obliquus Kinetin limitation Lipid productivity increased 1.5-fold. (Renuka et al., 2017)
Scenedesmus IAA, EBL Nitrogen Fatty acids content increased 3.3-fold. (Kozlova et al., 2018)
quadricauda limitation
Nitrogen
limitation
Wastewater

8
X. Song et al.
Fig. 2. Possible mechanisms of phytohormone-mediated stress tolerance to enhance lipid production of microalgae. CaM, Calmodulin; MDA, Malonaldehyde; APX,
Ascorbate peroxidase; MAPK, Mitogen activated protein kinases; ACCase, acetyl-CoA carboxylase; ROS, reactive oxygen species; FAS, Fatty-acid synthase; GPAT,
Glycerol 3-phosphate acyltransferase; LPA, Lysophosphatidic acid; DAG, diacylglycerol; DGAT, Diacylglycerol acyltransferase; TAG, Triacylglycerol.
phytohormone regulates the algal lipid accumulation under abiotic
stress. Phytohormones play important roles in regulating the occurrence
and physiological functions in microalgae and each phytohormone has a
specific role (Che et al., 2016; Peng et al., 2021; Song et al., 2019; Zhao
et al., 2018b). Cytokinins (CK), salicylic acid (SA), auxins (IAA, IBA,
NAA, etc.) and gibberellins can promote the cell division, cell growth,
photosynthesis and cell elongation in microalgae and plants (Arora &
Mishra, 2020; Awad et al., 2020; Fu et al., 2021; Singh et al., 2020). IAA
can regulate carbon fixation related to certain metabolic pathways to
increase the biomass concentration and lipid production of green
microalgae Chlorella vulgaris (Trebouxiophyceae). Further analysis
revealed that IAA treatment can up-regulate the expression level of rbcL
(ribulose-1,5-bisphosphate carboxylase oxygenase large subunit) and
accD (acetyl-CoA carboxylase) (Piotrowska-Niczyporuk & Bajguz,
2013). In another study, the highest lipid productivity of 49.6% was
achieved by treating Monoraphidium sp. with melatonin, and it was
revealed that melatonin could regulate the carbon flow and photosyn­
thesis rate, thereby increasing lipid synthesis (Li et al., 2017). Conse­
quently, most plant hormones may have similar functions in regulating
cell growth, lipid production and metabolites accumulation of
microalgae.
In addition, ROS is a mediator in cellular responses and it is well
established that ROS will increase when algae are subjected to various
unfavorable environmental stresses (Zhu et al., 2019). Environmental
stress conditions can trigger ROS accumulation, and enhance the lipid
content and other metabolites production in microalgae (Li et al., 2021;
Zhao et al., 2019b). This phenomenon may be due to the ROS activation
of the antioxidant system to produce antioxidant enzymes (e.g., ascor­
bate peroxidase (APX), superoxide dismutase (SOD), peroxidase (POD)
and catalase (CAT)) and antioxidants (e.g., glutathione (GSH), carot­
enoids and polyunsaturated fatty acids (PUFAs)) under abiotic stress
conditions, which can defense against oxidative stress and keep the algal
cells in a state of redox balance (Rezayian et al., 2019; Sun et al., 2018b).
In particular, the SOD, CAT and APX activity of Dunaliella salina
significantly increased under nitrogen depleted conditions (Yilancioglu
et al., 2014). Under abiotic stress conditions, microalgae could reduce
the synthesis of proteins and nucleic acids, and carbon flow is directed
from protein synthesis to lipid synthesis to protect algal cells from
9
Resources, Conservation & Recycling 183 (2022) 106355
oxidative damage (Song et al., 2020; Yilancioglu et al., 2014). It is
suggested that abiotic stress induced by oxidative stress is an effective
means to increase the accumulation of neutral lipids in algae (Sun &
Zhang, 2021). However, the connection between ROS level and lipid
accumulation in microalgae species requires further research.
In a previous study, phytohormones were shown to correlate with
increased stress tolerance and improved cellular lipid accumulation
under abiotic stress conditions in microalgae. Microalgae have the
ability to convert carbon dioxide, nutrients and other substances into
lipids, proteins, carbohydrates and other metabolites though photo­
synthetic activity (Ruiz et al., 2016). However, microalgae can reduce
the gene expression of various proteins to form photosystem complexes I
and II under abiotic stress conditions, and change the photosynthesis
rate (Qiao et al., 2021). Moreover, phytohormone melatonin can
up-regulate the expression level of rbcL. Formation of ribulose 1,
5-bisphosphate carboxylase/oxygenase (RuBisCO) may result in alter­
ations in photosynthesis rate, which was consistent with the observed
increase in chlorophyll content under abiotic stress condition (Zhao
et al., 2018b). Hence, phytohormones may modify the photosystem to
increase the photosynthesis rate of microalgae under abiotic stress.
Interestingly, phytohormones act individually as well as influencing
other endogenous plant hormones (e.g., ethylene and jasmonic acid) and
could regulate signal transduction pathways (such as NO, Ca2+, mitogen
activated protein (MAPK) and cGMP) under abiotic stress conditions
(Song et al., 2020). This intricate network, known as “signaling
cascade”, can make specific changes in response to environmental cues
and stimulate defense mechanisms in various unfavorable environ­
mental stresses. Phytohormones can increase the intracellular calcium
and CaM gene transcription to improve the lipid synthesis under
photo-induction condition in microalgae. After treatment with CaCl2,
algal cells accumulate more lipids than untreated group, which might be
due to the phytohormone participates in signal transduction pathways,
and further regulates the expression levels of lipid biosynthesis related
genes (Song et al., 2019). Moreover, strigolactone could mediate jas­
monic acid-induced lipid and γ-aminobutyric acid synthesis in micro­
algae and increase the microalgae tolerance to protect algal cells from
oxidative damage by regulating ROS and γ-aminobutyric acid signaling
under nitrogen deficiency condition (Song et al., 2020). Furthermore,
X. Song et al.
under high light and nitrogen depletion condition, the NO-dependent
MAPK signaling cascade was activated in response to astaxanthin and
lipid synthesis induced by melatonin in H. pluvialis (Ding et al., 2018b).
The mechanisms of cell growth and lipid accumulation by micro­
algae under combined phytohormone and abiotic stress is explored. As
shown in Fig. 2, phytohormones may protect microalgae against
oxidative stress through different regulatory mechanisms under abiotic
stress condition. Phytohormones can mediate the level of ROS via pro­
duction of antioxidant enzymes (SOD, POD, CAT, etc.) and antioxidants
(GSH, astaxanthin) to adapt to the rapidly changing culture environ­
ment under abiotic stress condition (Zhao et al., 2018b). Moreover,
phytohormones can increase endogenous plant hormones level in
response to abiotic stress (Song et al., 2020). In addition, phytohor­
mones might promote/maintain the cell growth of microalgae by
regulating the photosynthesis rate, providing energy for Calvin cycle
under abiotic stress condition. Therefore, phytohormones could stimu­
late the lipid synthesis and stress tolerance by regulating signal cascades
and endogenous stress hormones contents to improve the genes
expression levels related to lipid biosynthesis in response to various
abiotic stresses.
5. Challenges and future perspective for microalgae biofuel
production
In order to achieve eco-friendly and renewable production of
microalgae lipids, an effective and sustainable strategy should be chosen
based on the biomass productivity and lipid productivity of microalgae.
However, most cultivation strategies are based on lab-scale studies and
it has not been verified whether they are applicable to all algal strains.
Table S2 summarizes the effects of different culture systems on biomass
and lipid production performances of microalgae. Moreover, industri­
alization and economic feasibility researches of these cultivation stra­
tegies are required to be continuously examined. In addition, the high
cost of large-scale production of microalgae biofuels remains a chal­
lenge. In the future, molecular tools need to be investigated to improve
the tolerance to abiotic stress to achieve higher biomass productivity in
extreme condition and improve the lipid yield of microalgae. In genetics,
metabolic engineering is used to transform algal species and overexpress
the lipid-producing genes or block competitive pathways to increase the
microalgae lipid production. In this respect, more strategies (such as
overexpression of functional genes, starch less mutant and altering fatty
acid chain length) should be adopted for the improvement production of
microalgae lipids. Flexible use of these cultivation strategies focusing
more on molecular approaches and analyzing the metabolic mechanisms
of lipid accumulation in microalgae are more conducive to the large-
scale production of microalgae biofuels.
6. Conclusions
Microalgae biomass is a feedstock with great potential for the pro­
duction of biofuels. This review discusses the stress-induced strategies
that affect the lipid accumulation of microalgae and provides novel in­
sights into the possible regulation mechanisms. By analyzing abiotic
factors and novel alternative strategies, it is shown that two-stage
cultivation is an effective and promising strategy to increase the
microalgae lipid production. On the basis of two-stage cultivation, the
use of phytohormones coupled with abiotic stresses can further promote
the microalgae lipid accumulation production. In addition, mechanisms
on phytohormone-mediated stress tolerance that lead to significant
improvement in microalgae lipid accumulation is examined. With the
most efficient culture strategy to improve the economics of lipid accu­
mulation, microalgae biofuel production can potentially be scaled up to
an industrial level.
10
Resources, Conservation & Recycling 183 (2022) 106355
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that cdocould have appeared to in­
fluence the work reported in this paper.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (No. 21906037), the Heilongjiang Postdoctoral Science Foun­
dation (No. LBH-TZ2012), the State Key Laboratory of Urban Water
Resource and Environment (Harbin Institute of Technology) (No.
2021TS18), the Fundamental Research Funds for the Central Univer­
sities, the Young Scientist Studio of Harbin Institute of Technology, and
the Heilongjiang Touyan Innovation Team Program.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.resconrec.2022.106355.
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