Bioresource Technology 303 (2020) 122886

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

Bioremediation of heavy metals using microalgae: Recent advances and T

mechanisms
⁎
Yoong Kit Leonga, Jo-Shu Changa,b,c,
a
Department of Chemical and Materials Engineering, College of Engineering, Tunghai University, Taichung, Taiwan
b
Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan
c
Center for Nanotechnology, Tunghai University, Taichung, Taiwan

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

Keywords: Five heavy metals namely, arsenic (As), cadmium (Cd), chromium (Cr), lead (Pb) and mercury (Hg) are carci-
Microalgae nogenic and show toxicity even at trace amounts, posing threats to environmental ecology and human health.
Bioremediation There is an emerging trend of employing microalgae in phycoremediation of heavy metals, due to several
Biosorption benefits including abundant availability, inexpensive, excellent metal removal efficiency and eco-friendly
Heavy metals
nature. This review presents the recent advances and mechanisms involved in bioremediation and biosorption of
Mechanism
these toxic heavy metals utilizing microalgae. Tolerance and response of different microalgae strains to heavy
metals and their bioaccumulation capability with value-added by-products formation as well as utilization of
non-living biomass as biosorbents are discussed. Furthermore, challenges and future prospects in bioremediation
of heavy metals by microalgae are also explored. This review aims to provide useful insights to help future
development of efficient and commercially viable technology for microalgae-based heavy metal bioremediation.

1. Introduction 2019). Hence, it is crucial to remediate these toxic heavy metals in

“Heavy metals” is a term generally referred to metals and metalloids
with density more than 5 g/cm3 which include arsenic (As), cadmium
(Cd), chromium (Cr), copper (Cu), iron (Fe), lead (Pb), mercury (Hg),
silver (Ag), zinc (Zn) and others (Duruibe et al., 2007). They are widely
involved in human activities, such as fossil fuel combustion, mining,
electroplating, dye and pigments manufacturing, fertilizers, and other
industrial activities, which are then released in large amount into the
environment daily via wastewater or other pathways (Zakhama et al.,
2011). Due to their non-biodegradable characteristics, heavy metals
tend to persist in nature, leading to bio-accumulation in food chain
which causes severe environmental and health issues (Yang et al.,
2015).
Following the regulations of US Environmental Protection Agency,
the maximum contamination limits (MCL) of As, Cd, Cr, Hg and Pb are
0.01, 0.005, 0.1, 0.002 and 0.015 mg/L respectively (USEPA, 2016).
These heavy metals and their compounds, even at very low con-
centrations, are highly toxic, carcinogenic, mutagenic and teratogenic.
Direct contact, inhalation and ingestion of these heavy metals poses
serious threats to human physical and mental health, causing mutations
and genetic damage, damaging central nervous system as well as es-
calating the risk of cancers (Balaji et al., 2016a; Jaafari & Yaghmaeian,
wastewater effluents before discharging them.
Conventional techniques employed for heavy metals removal from
wastewater include chemical precipitation, ion exchange, membrane
filtration, floatation, coagulation-flocculation and electrochemical
methods (such as electrodeposition, electrofloatation and electro-
coagulation) (Fu & Wang, 2011). However, these techniques have the
disadvantages of expensive operation and maintenance costs, as well as
secondary pollution due to toxic sludge formation. Furthermore, most
of these techniques are further expensive and/or ineffective at very low
concentrations of heavy metals, specifically below 100 mg/L (Birungi &
Chirwa, 2015; Jaafari & Yaghmaeian, 2019).
On account of their environmental-friendliness and cost-efficiency
at low heavy metals concentration, bioremediation of heavy metals
using different microorganisms including bacteria, microalgae, yeasts
and fungi have been widely applied as alternatives to conventional
methods. Among all microorganisms, microalgae which has out-
standing biological characteristics such as high photosynthetic effi-
ciency and simple structure has the ability to grow well under extreme
environmental conditions such as presence of heavy metals, high sali-
nity, nutrient stress and extreme temperature. Therefore, there is an
emerging trend of employing microalgae in phycoremediation of toxic
heavy metals due to its high binding affinity, abundance of binding sites

⁎
Corresponding author at: Department of Chemical and Materials Engineering, College of Engineering, Tunghai University, Taichung, Taiwan.
E-mail address: changjs@mail.ncku.edu.tw (J.-S. Chang).

https://doi.org/10.1016/j.biortech.2020.122886
Received 20 December 2019; Received in revised form 20 January 2020; Accepted 21 January 2020
Available online 23 January 2020
0960-8524/ © 2020 Elsevier Ltd. All rights reserved.
Y.K. Leong and J.-S. Chang
and large surface area (Cameron et al., 2018). Furthermore, both living
cells and non-living biomass of microalgae can be employed as bio-
sorbents. Other than outstanding removal capacity and being eco-
friendly, bioremediation of heavy metals using microalgae has the
benefits of robust and simple process, lack of toxicity constraint, rapid
growth rate compared to higher plants as well as formation of value-
added products such as biofuels and fertilizers (Abinandan et al., 2019;
Balaji et al., 2016b). In addition, oxidative stress induced by heavy
metals enhances the lipid content of microalgae (Upadhyay et al.,
2016). Furthermore, microalgae can also be employed for the recovery
of precious metal ions, such as thallium, silver and gold (Birungi &
Chirwa, 2015; Jaafari & Yaghmaeian, 2019).
In this review, we present the recent advances and in-depth sum-
mary of the development of bioremediation of five non-threshold toxic
heavy metals, which are arsenic (As), cadmium (Cd), chromium (Cr),
lead (Pb) and mercury (Hg) by microalgae. These heavy metals have
gained much attention and became a significant research focus due to
their severe deteriorating effects to the environmental ecology and
human health (Rahman & Singh, 2019; Suvarapu & Baek, 2017).
2. Mechanisms of heavy metal removal by microalgae
Microalgae consume heavy metals such as boron (B), cobalt (Co),
copper (Cu), iron (Fe), molybdenum (Mo), manganese (Mn), and zinc
(Zn) as trace elements for enzymatic process and cell metabolism,
though other heavy metals such as As, Cd, Cr, Pb and Hg are toxic to
microalgae. Due to hormesis phenomenon, low toxic heavy metal
concentrations can stimulate the growth and metabolism of microalgae
(Sun et al., 2015). Some cyanobacterial species, such as Anabaena,
Oscillatoria, Phormidium and Spirogyra can naturally grow in water
contaminated by heavy metals due to their tolerance towards heavy
metal stress (Balaji et al., 2016a). Other than heavy metals, microalgae
have reactive groups with active binding sites which can form com-
plexes with pollutants in wastewater. This leads to flocculation and
subsequently reduces the content of both total dissolved solids (TDS)
and total suspended solids (TSS) (Balaji et al., 2016a).
Microalgae species has multiple strategies of unique self-protection
mechanisms against toxicity of heavy metals, such as heavy metal im-
mobilization, gene regulation, exclusion and chelation as well as anti-
oxidants or reducing enzymes which reduce heavy metals via redox
reactions (Gómez-Jacinto et al., 2015). Microalgae can form cellular
protein-heavy metals complexes without changing its own activity
(Priatni et al., 2017). The organometallic complexes are further sepa-
rated insides vacuoles to help regulate the heavy metal ions con-
centration in cytoplasm which subsequently mitigate their toxic effects
(Balaji et al., 2016b). In addition, heavy metals activate the biosynth-
esis of phytochelatins (PCs) which are thiol-rich peptides and proteins
that minimize heavy metal stress by interacting with them (Gómez-
Jacinto et al., 2015).
To counteract with free radicals released by heavy metals during
adsorption, microalgae synthesize antioxidant enzymes such as ascor-
bate peroxidase, catalase, glutathione reductase, peroxidase and su-
peroxide dismutase (SOD) as well as non-enzymatic antioxidants such
as carotenoids, cysteine, ascorbic acid (ASC), glutathione (GSH) and
proline (Upadhyay et al., 2016). SOD acts as the first line of defence
against superoxide anion by breaking it down into oxygen molecules
and hydrogen peroxide. The hydrogen peroxide is further degraded by
catalase into water and oxygen molecules (Balaji et al., 2016b). Cy-
steine indirectly or directly serves as the precursor for PCs, GSH, me-
tallothioneins and other sulphur containing compounds, hence serving
as the indicator for the synthesis of different antioxidants.
GSH and ASC are important endogenous antioxidants synthesized
by microalgae and they play a key role in the reduction of reactive
oxygen species (ROS) and free radicals (Devars et al., 2000). Other than
maintaining the equilibrium of ROS production and elimination, ASC
protects microalgal cells by regulating metal-containing enzyme
2
Bioresource Technology 303 (2020) 122886
activity and ascorbic acid-glutathione (ASC-GSH) pathway as well as
sustaining the dissipation of surplus excitation energy and scavenging
ROS. Furthermore, high level of ASC is secreted by microalgae as a
hydrophilic redox buffer that is responsible for the protection of cytosol
and other cellular components against oxidative threats. On the other
hands, high level of GSH protect the microalgae by providing tolerance,
scavenging free radicals, facilitating PCs and ASC synthesis as well as
restoration of substrate for other antioxidants (Gómez-Jacinto et al.,
2015; Upadhyay et al., 2016).
Removal of heavy metals by microalgae is attained via a two-stage
mechanism. The first stage is the rapid extracellular passive adsorption
(biosorption), while the second stage is the slow intracellular positive
diffusion and accumulation (bioaccumulation). In addition to cell
polymeric substances such as peptides and exopolysaccharides with
uronic groups, cell wall of microalgae are mainly composed of poly-
saccharides (cellulose and alginate), lipids and organic proteins, pro-
vides many functional groups (such as amino, carboxyl, hydroxyl,
imidazole, phosphate, sulfonate, thiol and others) capable of binding
heavy metals (Priatni et al., 2017). Furthermore, they consisted of huge
amount of monomeric alcohols, laminaran, deprotonated sulphate and
carboxyl groups which attract both anionic and cationic species of
different heavy metals. Microalgae biomass also has different functional
groups, such as amide, carbonyl, carboxylic acid, ether and hydroxyls
which contribute to the biosorption (Pradhan et al., 2019). Fourier-
transform infrared (FTIR) analysis and nuclear magnetic resonance
(NMR) spectroscopy can be employed to determine the functional
groups in microalgae that are responsible for formation of complexes
with heavy metals as well as examine the interaction between them.
The knowledge of biosorbents chemical structure provides important
information for prediction of their affinities for heavy metal ions.
Adsorption of heavy metal on the surface of microalgae is a rapid
process and can happen via different paths, which are formation of
covalent bond between ionized cell wall with heavy metals, ionic ex-
change of heavy metal ions with cell wall cation and binding of heavy
metal cations with negatively charged uronic acids of microalgae exo-
polysaccharides. On the other hand, the process of heavy metal accu-
mulation inside the cell is much slower. The heavy metals are actively
transported across the cell membrane and into the cytoplasm followed
by diffusion and subsequent binding with internal binding sites of
proteins and peptides such as GSH, metal transporter, oxidative stress
reducing agents and phytochelatins (Ibuot et al., 2017; Pradhan et al.,
2019). Fig. 1 illustrates the mechanisms of heavy metals removal by
microalgae.
3. Bioremediation and biosorption of heavy metals
3.1. Arsenic (As)
Classified by the United States Environmental Protection Agency
(USEPA) as class A and category 1 carcinogen, arsenic (As) is one of the
major toxic heavy metals that cause contamination of potable water in
many countries, such as Argentina, Bangladesh, Chile, China, Finland,
India, South East Asia and USA (Arora et al., 2018; Upadhyay et al.,
2016; Wang et al., 2014). Widespread pollution of arsenic is caused by
anthropogenic activities, such as combustion of fossil fuels, mining,
medical use, fertilizers and pesticides, smelting, electrolytic process,
sewage sludge as well as pigments, semiconductor, glass and alloy
manufacturing processes (Arora et al., 2017; Singh et al., 2016). Phy-
sical exposure to As even at low concentrations (0.1 µg/mL) cause lung,
skin, kidney and bladder cancers, while higher concentration causes
arsenicosis, arsenical dermatitis, cardiovascular disease, diabetes, in-
fant morbidity, immune alteration, impairment to the central nervous
system, hepatic damage and hyperkerotosis (Li et al., 2019). Con-
versely, in plant and microalgae, arsenic causes impairment to cell or-
ganelle and DNA, lipid peroxidation and protein degradation
(Upadhyay et al., 2016).
Y.K. Leong and J.-S. Chang
Fig. 1. Mechanism of heavy metals removal by microalgae.
The toxicity and physico-chemical properties of arsenic is depen-
dent on it oxidation state and chemical form. Arsenic has four oxidation
states, which are arsenic (As0), arsine [As(-III)], arsenite [As(III)] and
arsenate [As(V)]. The most commonly occurring oxidation states of As
are trivalent [As (III)] and pentavalent [As(V)] forms, of which the
former is more toxic (Zhu et al., 2018). In aqueous solution, As (III)
exists predominantly as H3AsO3 with other forms such as H4AsO3+,
H2AsO3−, HAsO32− and AsO33−, while As(V) exists as H3AsO4,
H2AsO4−, HAsO42− and AsO43− (Arora et al., 2017). The inorganic
form of arsenic is much more toxic than its organic form. Thus, it is
beneficial to transform arsenic into its organic form, which includes
monomethylarsonate (MMA), dimethylarsinate (DMA), arsenocholine
(ArsC) and arsenobetaine (ArsB) (Wongrod et al., 2019). Microalgae
reduces the toxicity of inorganic arsenic by As (III) oxidation and
complex formation with phytochelatins and glutathione, As(V) reduc-
tion, biotransformation into arsenolipids/arsenosugars or methylated
arsenic species, extracellular adsorption and excretion from the cell
(Papry et al., 2019). Oxidation of As(III) mainly happens outside the
cells by interacting with functional groups such as –OH, –NH, –CH of
aldehyde and aliphatic as well as –CN of amino groups (Arora et al.,
2018; Wang et al., 2014). On the other hand, cellular metabolism of
arsenic starts with rapid reduction of As(V) to As (III) followed by a
slower step-by-step methylation first to MMAs, and then to DMAs.
Microalgae have been shown to excrete both methylated arsenic species
and/or reduced As(III) (Baker & Wallschläger, 2016).
Essential for protein synthesis, regulation of genetic materials and
modification of protein via phosphorylation, phosphate (PO43−) plays
an important role in speciation, bioaccumulation and detoxification of
arsenic in microalgae due to the similarity in outer electron config-
uration as arsenate [As(V)] (Sun et al., 2015). Therefore, the high af-
finity phosphate transporters of microalgae (such as PIT and PST) can
participate in the uptake of arsenic. PO43− limitation induces the
synthesis of arsenic transporter and thus, accelerates arsenic uptake
(Wang et al., 2014). On the other hand, increased phosphate con-
centration significantly reduced the arsenic uptake in microalgae such
as Chlorella sp. (Bahar et al., 2016), C. vulgaris (Baker & Wallschläger,
2016; Li et al., 2019), C. salina (Karadjova et al., 2008), Dunaliella salina
(Wang et al., 2017; Wang et al., 2016), Chlamydomonas reinhardtii and
Scenedesmus obliquus (Wang et al., 2013). This is because both As(III)
and As(V) experienced non-competitive and competitive inhibition re-
spectively, by extracellular PO43−. However, it is reported that PO43−
concentration has negligible impact on the removal of arsenic by
3
Bioresource Technology 303 (2020) 122886
fungal-algal pellets and high removal capacity was achieved, though
arsenite methylation did not occur (Li et al., 2019). Wang and co-
workers reported that PO43− and As(V) has a synergistic interaction
and significantly promoted the initial growth of axenic cultures of Du-
naliella salina, though the growth experienced inhibition after 9 days of
cultivation. The presence of symbiotic bacteria and PO43− limitation
are shown to promote the reduction of As(V) and induce the excretion
of As (III) and DMA by D. salina (Wang et al., 2016).
Different freshwater and marine microalgae have varying tolerance
to arsenic, thus experience different growth rate and bioaccumulation
capacity in the presence of arsenic. Examining the bioaccumulation of
arsenate by marine microalgae Phaeoductylum tricornutum, Morelli and
colleagues observed a gradual decrease in growth rate when the con-
centration of As(V) was above 0.1 µm and the microalgae experienced
maximum inhibition of 35% at 1.0 µm arsenic (Morelli et al., 2005).
Comparing between two freshwater microalgae, Levy and co-workers
found that Chlorella sp. has more tolerance to arsenic compared to
Monoraphidium arcuatum (Levy et al., 2005). Jiang and co-workers re-
ported high tolerance of Chlorella vulgaris to As(V) up to 200 mg/L with
constant arsenic removal of 70%. Extracellular heavy metal adsorption
serves a critical role in As(V) removal by microalgae at concentrations
below 100 mg/L (Jiang et al., 2011). Huang and colleagues observed
cyanobacterial species such as Anabaena affinis, Microcystis aeruginosa
and Oscillatoria tenuis experienced up to 2.6-fold slower growth rate in
As(V)-containing medium compared to control. Among these micro-
algae, A. affinis and O. tenuis removed As(V) by binding them to ex-
tracellular cell wall active sites, while intracellular bioaccumulation in
the cytoplasm for most arsenic removal occurred in M. aeruginosa
(Huang et al., 2014). Upadhyay and colleagues has reported that
Nannochloropsis sp. can grow well in As (III) concentration up to 100 µm
and have a significant decrease in biomass content at As (III) con-
centration further increases to 500 µm. Rapid decrease in biomass at
high concentration of arsenic might be caused by the blocking of -SH
group, leading to disruption of metabolism by disintegration of essen-
tial cellular proteins (Upadhyay et al., 2016). Singh and colleagues also
reported high accumulation of arsenic up to 760 µg/g in microalgae
species such as Hydrodictiyon reticulatom, Diatoms, Pithophora sp.,
Phormidium sp. and Oscillatoria sp. found in As-contaminated area,
showing potential in bioremediation of As (Singh et al., 2016). Arora
and co-workers found that Chlorella minutissima and Scenedesmus sp.
IITRIND2 can tolerate up to 500 mg/L arsenic with removal up to
161 µg/g (Arora et al., 2017). Comparing between four green
Y.K. Leong and J.-S. Chang
microalgae species (Chlorella vulgaris, Chlamydomonas reinhardtii, Sce-
nedesmus almeriensis and an indigenous Chlorophyceae spp.), S almer-
iensis demonstrated highest removal of As up to 41.7% at pH 9.5
(Saavedra et al., 2018).
In addition, microalgae respond differently under oxidative stress
induced by arsenic. Responding to arsenic stress, decrease in protein
and chlorophyll content was observed in both C. minutissima and
Scenedesmus sp. In addition, cell size reduction was recorded in C.
minutissima, while Scenedesmus sp. has increased lipid content at the
cost of carbohydrate reserve (Arora et al., 2017). Upon bioaccumula-
tion of arsenic, the total chlorophyll content of Nannochloropsis sp.
decreased, while carotenoids and lipid content increased. Nanno-
chloropsis sp. treated with 100 µm As(III) has the highest lipid pro-
ductivity of 20.27 mg/L/day and produce biofuel meeting EN 14214
standards (Upadhyay et al., 2016). Sun and co-workers observed an
increase in the external cell surface area of Nannochloropsis sp. due to
formation of measles-like granules and wrinkles caused by arsenic
toxicity. The presence of arsenic increased the accumulation of short-
chain and monounsaturated fatty acids which is desirable for high
quality biofuel production (Sun et al., 2015). Scenedesmus sp. IITRIND2
cultured in As-containing synthetic soft water produced microalgal lipid
with linoleic acid, oleic acid, palmitic acid and stearic acid as major
constituents. The biodiesel produced from this lipid not only met the
requirement of biodiesel standards (EN14214 and ASTM D6751-52),
but also has higher oxidative stability, leading to a longer shelf life
(Arora et al., 2017). Table 1 summarizes the biosorption of arsenic (As)
by different microalgae strains.
3.2. Cadmium (Cd)
Characterized as a highly toxic invasive heavy metal, cadmium is
released into the environment through anthropogenic activities such as
waste incineration, manufacture of metals and alloys, ceramics, colour
pigment, fertilizers and pesticides, nickel-cadmium batteries, plastic,
and also electroplating, mining, smelting and welding process
(Abinandan et al., 2019). Due to its carcinogenic and teratogenic
nature, cadmium induces severe damage to reproductive organs,
kidney, liver and lungs even at trace amount, leading to health pro-
blems such as cancer, Parkinson’s and Alzheimer’s diseases, gastro-
intestinal disorder, amyotrophic lateral sclerosis, hypertension, kidney
damage, kidney stone, peripheral neuropathy, osteoporosis and re-
spiratory insufficiency (Zhang et al., 2019).
Microalgae strains respond differently under cadmium stress. In
addition to reducing cell growth and chlorophyll content, Cd(II) in-
duces the production of phytochelatins, superoxide dismutase, catalase
and peroxidase as defence mechanism in microalgae such as
Chlamydomonas moewusii and Monoraphidium sp. (Mera et al., 2016;
Zhao et al., 2019). On the other hand, Chlorella minutissima UTEX2341
demonstrated strong adaptability to cadmium with maximum adsorp-
tion capacity of 35.65 mg/g. Uptake of 0.6 mM Cd(II) have shown to
enhance both microalgal lipid content and productivity up to 42.1%
and 2.17 fold compared to control. The maximum lipid productivity of
Table 1
Performance of biosorption of arsenic (As) by different microalgae strains.
Microalgae strains Temp (°C) Optimal pH Initial metal
conc. (mg/L)
Maugeotia genuflexa 20 6 10
Mixture of green (Chlorophyta) algae and 20 4 50
blue-green (Cyanobacteria) algae
Spirulina sp. 35 6 7276
Ulothrix cylindricum 20 6 10
Chlamydomonas reinhardtii – 9.5 12
Chlorella vulgaris – 5.5 12
Scenedesmus almeriensis – 9.5 12
Bioresource Technology 303 (2020) 122886
249.36 mg/L day was also higher than many reported in literature
(Yang et al., 2015). Monoraphidium sp. QLY-1 treated with 80 µm Cd(II)
showed enhanced lipid content (52.78%) of 1.59 fold compared to
control (Zhao et al., 2019). A 0.02 µm of free Cd(II) improved growth
rate and total lipids of Chlorella vulgaris by 13.4% and 51.5% respec-
tively (Chia et al., 2013). Similarly, acid-tolerant microalgae, Hetero-
chlorella sp. MAS3 and Desmodesmus sp. MAS1 can simultaneously re-
move 2 mg/L Cd(II) with > 58% efficiency and accumulated lipids rich
in aliphatic long-chain hydrocarbons and triglycerides at pH 3.5
(Abinandan et al., 2019).
With the advantage of cost-effective biomass harvesting from di-
luted culture, self-flocculating microalgae, such as Chlorella vulgaris
JSC-7 (Alam et al., 2015) and Scenedesmus obliquus AS-6-1 (Zhang et al.,
2016), have been utilized for the removal of Cd(II). Due to the dis-
tinctive cell wall components, enhanced synthesis of phytohormones
and higher antioxidation activity, C. vulgaris JSC-7 was more tolerant to
Cd2+ with an improved heavy metal removal capacity of 21.5 mg/g
compared to 6.5 mg/g of non-flocculating control species, (i.e., C. vul-
garis CNW11) (Alam et al., 2015). Other than the main contribution
from phosphate and carboxyl groups, functional groups such as amide,
C-N, hydroxyl and S-O groups also responsible for adsorption of Cd(II)
in S. obliquus AS-6-1. Successive adsorption/desorption cycle studies
showed that S. obliquus AS-6-1 maintained its flocculating properties as
cadmium chloride did not impair the flocculating properties and might
even help induce chemical flocculation (Zhang et al., 2016).
Different strategies such as genetic engineering, bio-immobilization,
microalgae immobilization and bio-pellets have been studied to im-
proved cadmium biosorption. Gene manipulation has been performed
to overexpress metal tolerance protein in Chlamydomonas reinhardtii,
which showed a 2.29 and 3.06 fold increased in Cd2+ tolerance and
uptake, respectively, when compared with wild type C. reinhardtii
(Ibuot et al., 2017). However, the natural strains adapted to wastewater
growth such as Parachlorella hussii, Parachlorella kessleri and Chlorella
luteoviridis still demonstrated better heavy metal tolerance and accu-
mulation. A novel technique for Cd ions bio-immobilization utilizing
Microcystis aeruginosa and Selenastrum capricornutum cells as bio-tem-
plates by co-incubating with selenium for bio-fabrication of fluorescent
CdSe NPs has been performed. The reducing biomolecules of the mi-
croalgae, such as glutathione, NADPH and NADPH dependent reductase
were speculated to play a crucial role in the formation of CdSe NPs
(Zhang et al., 2019). Immobilized Chlorella sp. in alginate bead has
demonstrated biosorption of Cd(II) up to 60% at low Cd2+ concentra-
tion of 20 ppm (Valdez et al., 2018). In addition, bioremediation of
cadmium by Chlorella sp. – water hyacinth biochar complex achieved
214.7 mg/g higher than both Chlorella sp. and biochar alone due to
enhancement of the surface potential. Functional groups such as eOH,
eNH, P]O (phosphoryl group), P]S (phosphorotioate group), C]O of
amide and CeO of alcoholic groups contributes to cadmium biosorption
(Shen et al., 2017). Similarly, Chlorella sp. immobilized with water-
hyacinth leaf biochar pellets achieved 92.5% Cd(II) removal efficiency
and remained viable at 10 mg/L Cd(II) (Shen et al., 2018). Bio-pellets
composed of C. vulgaris microalgae and Apsergillus niger fungi showed
Biomass conc. Time Max. sorption Removal Reference
(g/L) (min) (mg/g) efficiency (%)
4 60 2.4 96 (Sarı et al., 2011)
10 180 3.5 70 (Sulaymon et al., 2013a;
Sulaymon et al., 2013b)
12 240 365 60.2 (Doshi et al., 2009)
4 60 2.45 98 (Tuzen et al., 2009)
1 180 4.63 38.6 (Saavedra et al., 2018)
1 180 3.89 32.4
1 180 5.0 41.7
4
Y.K. Leong and J.-S. Chang
better performance for the removal of Cd(II) at low level of 1 µg/L up to
56% compared to 40% of microalgae alone, with the advantages of
lower final pH values and simpler harvesting (Bodin et al., 2017).
On the other hand, utilization of non-living microalgae cells for the
removal of cadmium via passive surface binding have been studied as
well. Dirbaz and Roosta have reported Parachlorella sp. as a better
biosorbent for Cd(II) compared to Nannochloropsis sp., Scenedesmus sp.
and Spirulina sp. due to the presence of a large amount of acidic func-
tional groups such as carboxylic acid. Functional groups such as eOH,
eNH, C]O of amide and CeO of alcoholic groups contributes to cad-
mium biosorption (Dirbaz & Roosta, 2018). The residual lipid extracted
biomass of Chlorella sp. CHA-01, Chlamydomonas sp. TAI-03 and Coe-
lastrum sp. PTE-15 with broken cell wall have been investigated for the
biosorption of Cd2+ and suggested that the intracellular cell wall
components also contribute in heavy metal binding (Zheng et al.,
2016). Freeze-dried biomass of Chlorella minutissima UTEX2341 was a
superior biosorbent compared to growing algae (Yang et al., 2015).
Nostoc commune and Chlamydomonas angulosa microalgae-cellulose
composite aerogel beads have been utilized for Cd2+ removal with at
least 63.1% adsorption efficiency up to 50 ppm Cd2+ at pH 6 (Hwang
et al., 2018). Most of the literature reported that the adsorption of Cd2+
followed Langmuir isotherm, suggesting that the predominant sorption
step is a single surface reaction at constant adsorption energy (Dirbaz &
Roosta, 2018; Jena et al., 2015; Shen et al., 2017; Zhang et al., 2016;
Zheng et al., 2016). Table 2 summarizes the biosorption of cadmium
(Cd) by different microalgae strains.
3.3. Chromium (Cr)
Chromium compounds have industrial applications in dyes, paint,
ink and pigment manufacturing, leather tanning, metal electroplating,
production of steel and alloys as well as wood preservation (Gupta &
Rastogi, 2008). Chromium has several oxidation states ranging from
+2 to +6, with the two most common and stable states are trivalent Cr
(III) and hexavalent Cr(VI) (Shokri Khoubestani et al., 2015). Cr(VI) is
much more toxic than Cr(III) as the former can penetrate cell membrane
without difficulty and disturb its integrity, while the cell membrane is
almost impermeable to the latter. Cr(VI) shows toxicity even at very
low concentration in parts per billion (ppb) due to its carcinogenic and
mutagenic properties (Pradhan et al., 2019). Furthermore, Cr(VI) is
highly water-soluble and have strong oxidizing properties to damage
genetic materials and modify the DNA transcription process (Gokhale
et al., 2008; Shen et al., 2019). Ingestion of Cr(VI) can cause lung, skin
and stomach cancer as well as chronic bronchitis, epigastric pain, liver
damage, kidney problem, tissue neurosis, ulcers or irritation in diges-
tive system, internal haemorrhage, emphysema and DNA impairment
by interference with DNA polymerase (Daneshvar et al., 2019;
Kayalvizhi et al., 2015). Cr(III) and Cr(VI) occurs as cationic and an-
ionic species in the solution respectively, while H2CrO4 is predominant
at pH < 1, HCrO4− dominates between pH 1 and 6, CrO42− prevails at
pH > 7 (Gagrai et al., 2013).
Table 2
Performance of biosorption of cadmium (Cd) by different microalgae strains.
Microalgae strains Temp (°C) Optimal pH Initial metal conc.
(mg/L)
Lipid-extracted Chlamydomonas 30 7.5 – 1
sp.
Lipid-extracted Chlorella sp. 30 7.5 – 1
Lipid-extracted Coelastrum sp. 30 7.5 – 1
Chlorella minutissima 28 6 –
Immobilized Chlorella spa – 6 10
Parachlorella sp. 35 7 100
Scenedesmus-24 – 6 200
a
A complex of water-hyacinth leaf derived biochar pellets immobilized with Chlorella sp.
Bioresource Technology 303 (2020) 122886
Several mechanisms have been proposed for the reduction and re-
moval of Cr(VI) by microalgae, depending on the binding properties of
the functional groups and the nature of operating conditions.
Biosorption of Cr(VI) on the extracellular polymeric substances has
been reported as the major mechanism contributing to the chromium
bioremediation by Phaeodactylum tricornutum and Navicula pelliculosa
(Hedayatkhah et al., 2018). Organelles, granules and cytosolic heat-
stable peptides and proteins accumulated the majority of Cr(VI) inside
microalgal cells (Aharchaou et al., 2017). Other than extracellular ad-
sorption and intracellular accumulation of Cr(VI), enzymatic chromium
reductase is also responsible for removal of the heavy metal ions (Lee
et al., 2017; Yen et al., 2017). Furthermore, Cr(VI) also interacts with
the electron donor of reducing agents (such as hydroxyl group and
secondary alcohol groups) on the biomass surface favourably at acidic
pH and is reduced to Cr(III), which subsequently binds to the negatively
charged functional groups (such as sulfonate group and carboxyl
group). The reduction of Cr(VI) anionic species to Cr(III) is as following
(Gagrai et al., 2013):
HCrO4 + 6H+ + 3e− ⇌ Cr3+ + 4H2O E0 = 1.33 V vs. NHE
HCrO4− + 7H+ + 3e− ⇌ Cr3+ + 4H2O E0 = 1.35 V vs. NHE
CrO42− + 8H+ + 3e− ⇌ Cr3+ + 4H2O E0 = 1.48 V vs. NHE
The reduction of hexavalent to trivalent chromium was even ob-
served in non-living microalgae biomass, which might due to the re-
lease of glutathione (Yen et al., 2017). Some Cr(III) complexes are re-
leased into the medium due to complex formation with adjacent
negatively charged functional groups, ion exchange with competitor
ions (such as Ca2+, Mg2+ and Na+) as well as electrostatic repulsion
between Cr(III) complex and positively charged functional groups
(Pagnanelli et al., 2013; Shen et al., 2019). HPO42− and H2PO4 ions
significantly inhibited the adsorption of Cr(VI), while common back-
ground anions have a minor detrimental effect on Cr(VI) reduction in
the order of SO42− < Cl− < NO3− (Gagrai et al., 2013; Singh et al.,
2012).
Many species of microalgae can tolerate and accumulate high con-
centrations of Cr(VI). Chlorella sorokiniana can tolerate up to 100 ppm
Cr(VI) for three days and achieved removal efficiency up to 99.7% after
24 h contact time (Husien et al., 2019). Navicula subminuscula can tol-
erate high concentration of Cr(VI) up to 10 mg/L and 4 mg/L respec-
tively, in laboratory conditions and natural chromium-containing
water. The microalgae can almost completely remove the heavy metal
up to 98% in culture containing 20 mg/L Cr(VI) (Cherifi et al., 2016).
Phaeodactylum tricornutum and Navicula pelliculosa can tolerate Cr(VI)
up to 1 mg/L and simultaneously accumulated lipids at optimized
conditions of 55 µmol/m2s light intensity and 3 mM sodium nitrate
concentration (Hedayatkhah et al., 2018). Pseudanabeane mucicola and
Pediastrum duplex can tolerate Cr(VI) up to 1.936 and 0.224 g/L, re-
spectively, and the former has a Cr removal efficiency of 71% (Dao
et al., 2018). Four microalgae presented different tolerance when
Biomass conc. Time Max. sorption Removal efficiency Reference
(g/L) (min) (mg/g) (%)
60 23.3 – (Zheng et al., 2016)
60 25.5 –
60 32.8 –
4 20 303 – (Yang et al., 2015)
1.3 – 15.51 92.5 (Shen et al., 2018)
1 – 96.2 – (Dirbaz & Roosta,
2018)
1.5 – 48.4 60.5 (Jena et al., 2015)
5
Y.K. Leong and J.-S. Chang Bioresource Technology 303 (2020) 122886

cultivated with Cr(VI) in the order of Lyngbya sp. > Chlorella sp. >
Scenedesmus dimorphus > Oscillatoria sp. (Nath et al., 2017). Also,
Scenedesmus acutus and Chlorella vulgaris can tolerate Cr(VI) up to 15
and 45 mg/L, respectively (Travieso et al., 1999).
For Cr(VI) removal using living Anabaena, Oscillatoria, Phormidium
and Spirogyra sp., the functional groups responsible for metal ion
binding on microalgal cell wall were carboxylate, ester and hydroxyl
groups (Balaji et al., 2016b). The growth rate of microalgae decreased
with increasing chromium concentration due to delay of exponential
growth and reduction of cell division caused by heavy metal stress.
Similarly, microalgae also experience a reduction in total protein con-
tent with increase in heavy metal concentration, due to the accumu-
lation of NADH and H+ caused by decreasing activity of the electron
transport system. The microalgae Oscillatoria sp. is a better candidate
for phycoremediation of chromium, with better biomass growth and
biosorption efficiency as well as higher antioxidant activity (Balaji
et al., 2016a; Balaji et al., 2016b). Jacome-Pilco and colleagues have
studied the removal of Cr(VI) in continuous cultivation system using
Scenedesmus incrassatulus grown in a split-cylinder internal-loop airlift
photobioreactor. The culture reached steady state after 14 days; uptake
and removal efficiency of Cr(VI) achieved were 1.7 mg/g and 43.5%,
respectively (Jácome-Pilco et al., 2009). Shen and co-workers have
studied Cr(VI) removal potential of three heterotrophic microalgae
strains namely, Botryococcus sp. NJD-1, Scenedesmus sp. NJD-6 and
Chlorella sp. NJD-9 and the strain NJD-1 showed higher tolerance to Cr
(VI) compared to the other two strains. Cultivating at 3% v/v of sodium
acetate and 5 mg/L Cr(VI), the microalgae achieved removal efficiency
of 94.2%, 66.9%, 99.2% and 98.2% for Cr(VI), NO3, PO4 and TOC re-
spectively. The functional groups such as –OH, –NH, aliphatic C–H,
amino, carboxyl and secondary alcohol groups participated as electron
donors in the reduction of Cr(VI) to Cr(III) up to 87.2% (Shen et al.,
2019).
On the other hand, literature has reported the utilization of non-
living biomass of microalgae as biosorbent for the removal of Cr(III)
and Cr(VI) which summarized in Table 3. Pre-treatment techniques
such as methylation (Finocchio et al., 2010) and treatment with NaOH
and SDS (Nath et al., 2017) were performed to improve Cr(VI) removal
by microalgal biomass. Methylated S. platensis biomass demonstrated
satisfactory removal efficiency of > 80% for Cr(VI) up to 25 mg/L
with 2–4 g/L biomass (Finocchio et al., 2010). Pre-treated consortia of
Scenedesmus dimorphus, Chlorella sp., Oscillatoria sp., and Lyngbya sp.
with 0.1 N NaOH and 0.01% SDS both showed better Cr(VI) removal
performance compared to untreated non-living consortia and live
consortia cells (Nath et al., 2017). In addition, immobilization of mi-
croalgae biomass on PVA (Ardila et al., 2017) and calcium alginate
beads (Kwak et al., 2015; Singh et al., 2012) were performed for the
purpose of easier harvesting. However, the harsh process of im-
mobilization damaged the microalgae cell wall and resulted in reduced
heavy metal removal efficiency (Ardila et al., 2017). Among the dif-
ferent forms of dried S. quadricauda (powder, pellet and biochar), mi-
croalgal biochar synthesized at 500 °C showed 100% removal efficiency
up to 10 mg/L Cr(VI) (Daneshvar et al., 2019). For cost-effective Cr(VI)
bioremediation, lipid-extracted spent biomass (Nithya et al., 2019) and
β-carotene pigment-extracted biomass of Spirulina plantesis (Gokhale
et al., 2008) were utilized for Cr(VI) removal. The pigment extracted
biomass of S. plantesis demonstrated higher maximum biosorption ef-
ficiency of 86.2% compared to 73.6% of fresh biomass, which might
due to increased surface area of biomass caused by cell membrane
rupture during pigment extraction process (Gokhale et al., 2008).
Ion exchange mechanism is the major contributor for chromium
adsorption by microalgae where heavy metal ions carrying positive or
negative charge adsorbed on the oppositely charged biomass surface or
indirect reduction followed by subsequent adsorption (Kwak et al.,
2015). Cr(VI) removal can also be explained by an adsorption-coupled
reduction mechanism where Cr(VI) is removed by direct reduction of
the biomass electron donor (Park et al., 2007). The functional groups
such as aldehydes, alkyl chains, amide, amine, alcohols/phenols, car-
boxylic, ester, organic halide compounds, phosphate, sulfoxide and
aliphatic organic chains of cellulose were identified as functional
groups for the biosorption of chromium. The microalgae biosorbents
generally has maximum Cr(III) removal efficiency at pH 6, due to the
increase of species such as CrOH2+ and CrOH2+ which have stronger
binding affinity to negative functional groups. On the contrary, the
maximum removal of Cr(VI) was achieved at pH 1–2, due to formation
of anionic species of Cr(VI) such as hydrogen chromate (HCrO4−),
chromate (CrO42−), dichromate (Cr2O72−) and tetraoxyhydrochromate
(HCrO4−) which can be adsorbed on the protonated binding sites.
Furthermore, the biomass surface consisted of high levels of hydronium
ions at acidic conditions which promoted interaction between Cr(VI)
and microalgae binding sites (Kayalvizhi et al., 2015). The adsorption
efficiency of chromium by microalgal biosorbents increased with in-
creasing temperature (Kayalvizhi et al., 2015; Nithya et al., 2019;
Pradhan et al., 2019). Sibi also reported that Cr(VI) removal of C. vul-
garis increased with increasing electrical conductivity up to 2.9 mS/cm,

Table 3
Performance of biosorption of chromium (Cr(VI)) by different microalgae strains.
Microalgae strain Temp (°C) Optimal pH Initial metal Biomass conc. Time (min) Max. sorption Removal ReferenceS
conc. (mg/L) (g/L) (mg/g) efficiency (%)

Spirulina platensis 60 1 500 – 90 59.6 – (Nithya et al., 2019)(Nithya

Lipid-extracted Spirulina 60 1 500 – 90 45.5 – et al., 2019)
platensis
Immobilized Spirulina 25 2 250 1 – 49 19.6 (Kwak et al., 2015)
platensisa
Methylated Spirulina platensis 25 – 18–25 2–4 120–240 16.7 > 80 (Finocchio et al., 2010)
Spirulina platensis 25 1.5 250 1 600 148.64 59.5 (Gokhale et al., 2008)
Pigment-extracted Spirulina 25 1.5 250 1 600 174.64 69.9 (Gokhale et al., 2008)
platensis (Gokhale et al., 2008)
Chlorella vulgaris 25 1.5 250 1 600 140 56
Chlorella vulgaris 25 2 147 1 240 63.2 43 (Sibi, 2016)
Scenedesmus quadricauda 25 6 for Cr(III) 100 2 120 – 98.3 for Cr(III) (Shokri Khoubestani et al.,
1 for Cr(VI) 47.6 for Cr(VI) 2015)
Scenedesmus quadricauda 22 2 10 2 240 – 100 (Daneshvar et al., 2019)
biochar
Rhizoclonium hookeri – 2 1000 1 45 67.3 6.7 (Kayalvizhi et al., 2015)
Immobilized Chlorella 30 2 100 60% (w/v) 2160 57.33 99.7 (Singh et al., 2012)
minutissimab

a
Spirulina platensis beads prepared with 1 M LiCl/DMSO.
b
Chlorella minutissima immobilized in calcium alginate beads.

6
Y.K. Leong and J.-S. Chang
but decreased sharply at the electrical conductivity of 3.8 mS/cm due to
competition between anionic species of Cr(VI) with chloride ion for
binding sites and inhibition of cell membrane permeability by NaCl
(Sibi, 2016).
3.4. Lead (Pb)
Lead has various applications in paint, battery making, cosmetics,
weaponries and building materials. Being one of the most toxic heavy
metal, lead seriously endangers the safety of human health and aquatic
organisms. Other than causing serious physical and mental health
problems in children, Pb2+ can cause anaemia, brain disease, damage
to the central nervous system, dementia, kidney malfunction, re-
production abnormality and even death (Das et al., 2016).
Literature has reported the utilization of Spirulina platensis
(Malakootian et al., 2016), Chlamydomonas reinhardtii (Bayramoğlu
et al., 2006), Phormidium sp. (Das et al., 2016), Rhizoclonium hookeri
(Suganya et al., 2016) as well as Chaetoceros and Chlorella sp.
(Molazedah et al., 2015) as the biosorbent for the removal of lead. Das
and co-workers have further studied semi-packed bed adsorption of
Pb2+ and found that bed height and flow rate were the significant
parameters in the semi-batch biosorption process (Das et al., 2016). The
functional groups such as acyl-amino, amide, amine, carbonyl, car-
boxyl, hydroxyl, phenols and phosphate were identified for the bio-
sorption of Pb2+. The adsorption of lead ions decreased significantly at
low pH due to electrostatic repulsion caused by high positive charge
density on the binding sites and competition with H+ for occupancy of
sorption sites. The biosorbent generally has the maximum efficiency of
Pb2+ removal at pH 5–6, due to the increase of species such as Pb2+
and Pb(OH)+. The formation of Pb(OH)2 which precipitated was ob-
served at pH > 6 (Akhtar et al., 2004). Ion exchange mechanism can
be employed to explain the adsorption of cationic species of Pb2+ on
Chlorella sp. surface. The organometallic complex formation model is as
follows (Molazedah et al., 2015):
2(-R-OH) + Pb2+ → 2(RO)Pb + 2H+
-R-OH + Pb(OH)+ → (-RO)PbOH + H+
The adsorption enthalpy value and enthalpy change showed that the
adsorption of Pb2+ by microalgae biomass is spontaneous, endothermic
and chemical in nature. Most studies agreed that the biosorption of
Pb2+ by microalgae followed a pseudo-second order kinetic model (Das
et al., 2016; Malakootian et al., 2016; Molazedah et al., 2015; Suganya
et al., 2016). This suggested that the chemical sorption which involves
ion exchange and electron sharing between Pb2+ and the biomass
might be the rate-limiting step.
Upon the exposure to lead, Scenedesmus incrassatulus showed a de-
creased peripheral cell type “incrassatulus” and increased morphology
type “obliquus”. Therefore, peripheral morphology of S. incrassatulus
can be employed as an effective bio-indicator for the detection of lead
pollution (Batsalova et al., 2017). Similarly, marine microalgae Nitzchia
closterium has become a potential candidate for rapid, simple and ef-
fective detection of lead toxicity utilizing chlorophyll fluorescence
Table 4
Performance of biosorption of lead (Pb) by different microalgae strains.
Microalgae strains Temp (°C) Optimal pH Initial metal
conc. (mg/L)
Chaetoceros sp. 25 6 20
Chlorella sp. 25 6 20
Immobilized Chlamydomonas 25 6 500
reinhardtiia
Phormidium sp. 25 5 10
Rhizoclonium hookeri 40 4.5 – –
a
Chlamydomonas reinhardtii immobilized in calcium alginate beads.
Bioresource Technology 303 (2020) 122886
technology (Gan et al., 2019). Table 4 summarizes the biosorption of
lead (Pb) by different microalgae strains.
3.5. Mercury (Hg)
Mercury has been primarily released by industrial activities such as
mining, smelting, waste incineration and coal combustion in gaseous
and aqueous form (Peng et al., 2017). Mercury and its compounds are
considered as one of the most toxic and hazardous heavy metals in the
environment as mercury can be converted into the potent neurotoxin
methyl-mercury (MeHg). Mercury toxicity is mainly associated with its
capability to penetrate the blood-brain barrier, interference with the
uptake of essential metals, modification of cell redox status, and the
disruption of proteins and metal thiolate bonds (Huang et al., 2006;
Rezaee et al., 2006). They can cause adverse health issues such as an-
tibiotic resistance, mental retardation, reproductive disturbance and
others. Bioaccumulation of mercury is similar to hydrophobic organic
compounds, rather than inorganic metal ions, as “biomagnification” of
mercury happens at every level of aquatic food chain (Mason et al.,
1996).
Microalgae have the ability to bio-transform acid reducible Hg2+
into elemental Hg0 and metacinnabar (β-HgS) to varying degrees (Kelly
et al., 2007). After enzymatic reduction of Hg2+ to Hg0 catalysed by
mercuric reductase, the volatile Hg0 is removed by both biological and
non-biological volatilization and most of the remaining unreduced
Hg2+ is converted into β-HgS. Majority of Hg0 volatilization happens
rapidly, within 20 min to few hours, as shown in microalgae such as
Selenastrum minutum, Chlorella fusca var. fusca, diatom Navicula pellicosa
and thermophilic alga Galdiera sulphuraria. Non-biological volatiliza-
tion of mercury only happened under light illumination, while biolo-
gical volatilization was light-independent, but depended on metal
concentration and cell density (Devars et al., 2000). Accelerated vola-
tilization was shown in microalgae pretreated with 5 mM dimethylfu-
marate (Kelly et al., 2007). Genetic engineering has been performed to
express mercuric reductase (MerA) from Bacillus megaterium B1 in
Chlorella sp. DT. The transgenic strains have improved Hg(II) removal
ability, up to two fold compared to control and experienced a reduced
level of oxidative stress (Huang et al., 2006). In addition, other po-
tential routes of Hg(II) removal are thiol chelation and bio-methylation
to methylmercury (MeHg). However, it was reported that unlike other
heavy metals, accumulation of mercury poorly induces phytochelatin
synthesis (Devars et al., 2000). Growth photoperiods have reported to
influence the composition of binding ligands and its complexation with
Hg. Hg-binding ligands are more homologous and aromatic in nature at
longer light exposure period, while formation of smaller, more aliphatic
Hg-ligand complexes rich in thiols and sulphur was observed at darker
growth conditions (Mangal et al., 2019).
A novel pretreatment technique, polyelectrolyte self-assembly to-
gether with biomimetic mineralization was used to pretreat Chlorella
vulgaris for Hg2+ removal. The mineralized microalgae cells were
wrapped in a coating of polyelectrolytes and a transparent amorphous
calcium phosphate mineral layer doped with sulphur atoms. The mi-
neral layer serves as a protection layer for microalgae from heavy
Biomass conc. Time Max. sorption Removal Reference
(g/L) (min) (mg/g) efficiency (%)
1.5 180 8 60 (Molazedah et al.,
1.5 180 10.4 78 2015)
– 120 308.7 – (Bayramoğlu et al.,
2006)
4 40 2.305 92.2 (Das et al., 2016)
– 81.7 – (Suganya et al., 2016)
7
Y.K. Leong and J.-S. Chang
metals poisoning and the microalgae can grow well in high con-
centration of mercury up to 100 µg/L with no obvious reduction in lipid
yield. Raw C. vulgaris has the optimal pH of 5.5 for the removal of Hg2+
up to 62.85%, as Hg2+ compete with H+ for binding sites at lower pH,
while complex formation of (Hg)OH2 at higher pH significantly de-
terred the binding of Hg2+ with organic functional groups. On the other
hand, the biomimetic mineralized C. vulgaris has enhanced adsorption
efficiency with increasing pH up to 94.7% at pH 7, due to higher degree
of crystallinity and improved loading of S atoms on the mineralized
layer (Peng et al., 2017).
Integration of membrane filtration and activated sludge utilizing
membrane bioreactor systems for wastewater treatment has gained a lot
of attention due to its high efficiency. Microalgae dynamic membrane
formed using C. vulgaris powder was utilized in dynamic membrane
bioreactor for the bioremediation of Hg2+ from synthetic dental was-
tewater with the advantages of promoted removal yield as well as re-
duction in fouling and expensive cost of membrane recovery. The dy-
namic membrane bioreactor demonstrated a higher Hg2+ removal
efficiency compared to control membrane bioreactor at 300–800 ppb
concentrations (Fard & Mehrnia, 2017).
The development of a rapid and sensitive biological monitoring
technology for heavy metal pollutants is of utmost importance for the
protection of aquatic environment. Other than significant increase in
SOD gene expression levels and activity as well as chlorophyll a con-
tent, the frustule of Halamphora veneta deformed and increased in size
when exposed to mercury. Therefore, SOD gene expression levels and
activity, changes in frustule morphology and chlorophyll a content of
Halamphora veneta are promising candidates for mercury toxicological
assessment in aquatic habitats (Mu et al., 2017). Table 5 summarizes
the biosorption of mercury (Hg) by different microalgae strains.
4. Challenges
Although microalgae offer a number of attractive benefits for heavy
metal bioremediation, it still faces some challenges for widespread
applications, such as contamination by other microorganisms, nutrient
variability, high content of TSS and high turbidity, microalgae biomass
harvesting, difficulty in downstream processing and others. Table 6
summarizes the issues that could challenge the feasibility of using mi-
croalgae-based biosorbents for heavy metal removal as well as the
proposed strategies that could solve the problems raise when applying
this technology. From the information provided in Table 6, heavy metal
removal by microalgae seems to be a promising and efficient alternative
to conventional methods when an appropriate pretreatment step is
employed or when it is integrated with other technologies. Still, more
powerful microalgal strains should be isolated and better treatment
processes should be developed to further improve the efficiency of
heavy metal remediation and to reduce the operation costs. The future
prospects are thus discussed in detail below.
Table 5
Performance of biosorption of mercury (Hg) by different microalgae strains.
Microalgae strains Temp (°C) Optimal pH Initial metal
conc. (mg/L)
Immobilized Chlamydomonas 25 6 500
reinhardtiia
Chlorella sp. DT 30 – 8
Transgenic Chlorella sp.b 30 – 8
Chlorella vulgaris 20 5 48
Scenedesmus obtusus XJ-15 25 5 20
Spirogyra sp. 4 4 1 3
a
Chlamydomonas reinhardtii immobilized in calcium alginate beads.
b
Transformed with the Bacillus megaterium strain MB1 merA gene.
Bioresource Technology 303 (2020) 122886
5. Future prospects
Considering the multiple advantages provided by microalgae for
heavy metal bioremediation, the future prospects of large scale appli-
cation look promising by taking into accounts few considerations.
Firstly, screening and choosing the suitable microalgae strains is an
important step in bioremediation of heavy metals wastewater. Other
than having high tolerance to heavy metal pollutants with fast and
stable growth, it is beneficial if the microalgae has the following at-
tributes: (i) able to accumulate high content of lipids and other valuable
co-products, (ii) high CO2 sequestration ability and low nutrient re-
quirements, (iii) resistant to predation by grazers and robustness to-
ward existence of other microorganism species, and (iv) ability to self-
flocculate for cost-effective cell harvesting. Therefore, genetic, meta-
bolic and molecular engineering to increase adaptive ability, specificity
and robustness of microalgae strains is a potential research area.
Similarly, continuous research for better understanding of the un-
derlying mechanisms as well as development of more comprehensive
equilibrium and kinetic model for heavy metals biosorption and
bioaccumulation by microalgae is also crucial. Recently, techniques
such as whole-cell immobilization, pelletisation and microalgal biofilm
for heavy metals removal and recovery have gained much attention
because of their potential in industrial applications. Also, strategies
such as surface and chemical modifications techniques as well as in-
tegration with other heavy metal removal techniques for microalgae
biomass can help to improve heavy metals removal efficiency.
Microalgae biomass harvesting has always been a challenging task as
microalgae are minute in size and their negatively charged surface keep
them in stable dispersed state. Innovation of conventional harvesting
technologies and development of new techniques are essential to reduce
the overall operating cost.
On the other hands, heavy metals have both enhancement and in-
hibition effect on microalgae growth as well as accumulation of lipid
and other valuable co-products. Although usually having inhibition
effect on microalgae growth, some heavy metals such as Al, As, Cd Co,
Pb, elements from the lanthanide group, metallic nanoparticles and
others at certain concentration demonstrated a positive influence on the
growth of specific microalgae strains as reported in a recent review
(Miazek et al., 2015). Similarly, certain heavy metals also played a
positive role in the accumulation of lipids (such as As, Cd, Cu, Ni),
exopolymers (such as Ag, Cd, Co, Cu), pigments (such as As, Cd, Cu, Fe,
Ni and Te), phytochelatin (such as As, Cd, Cu, Pb) and phytohormones
(such as Cd, Cu, Pb) due to the inducing effect by metal stress. In
particular, heavy metal stress can be exploited to modify microalgal
fatty acids composition in order to produce biodiesel with desirable
properties and quality (Miazek et al., 2015). However, the presence of
heavy metals might interfere with downstream processing of these va-
luable products. Therefore, more research work should be done on
upstream cultivation and downstream purification process to achieve
satisfactory simultaneous heavy metal bioremediation and value-added
product formation.
Biomass conc. Time Max. sorption Removal Reference
(g/L) (min) (mg/g) efficiency (%)
– 120 106.6 – (Bayramoğlu et al.,
2006)
0.3 120 3.33 12.5 (Huang et al., 2006)
0.3 120 7.33 27.5
2 120 17.49 72.9 (Solisio et al., 2019)
0.125 180 – – (Huang et al., 2019)
30 0.253 76 (Rezaee et al., 2006)
8
Y.K. Leong and J.-S. Chang
Table 6
Challenges and proposed strategies of heavy metal removal by microalgae.
Challenges
Raw heavy metal wastewater consists of bacteria, fungus and other microorganisms, which
compete for nutrients and possibly dominating because of their relatively higher
growth rate
Nutrient variability (such as excessive or deprivation in carbon, nitrogen and phosphorus
content)
Wastewater might contain huge amount of TSS and has high turbidity that reduce light
penetration and hinder microalgae growth
Microalgae biomass harvesting
Presence of heavy metal remaining in microalgal cell might interfere with downstream
processing of valuable product
6. Conclusions
Microalgae strains demonstrated varied tolerance and response as
well as bioaccumulation capability towards heavy metals. Different
functional groups as well as proteins and peptides are responsible for
metal-binding. Mechanisms such as extracellular adsorption, reduction,
volatilization, complex formation, ion exchange, intracellular accumu-
lation, chelation and bio-methylation are involved in bioremediation
and biosorption of heavy metals. More efforts in the areas of genetic
engineering, immobilization techniques, pretreatment strategies and
integration with other technologies are required to fully explore the
potential of microalgae in heavy metal phycoremediation and si-
multaneously value-added products formation.
CRediT authorship contribution statement
Yoong Kit Leong: Investigation, Writing - original draft. Jo-Shu
Chang: Supervision, Conceptualization, Writing - review & editing.
Declaration of competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
The authors gratefully acknowledge financial support received from
Taiwan’s Ministry of Science and Technology under grant number
MOST 109-3116-F-006 -016 -CC1; 108-2621-M-006-020, 108-2218-E-
029-002-MY3, and 107-2221-E-006-112-MY3.
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Proposed strategies
• Pretreatment techniques (such as acidification, autoclaving, chlorination by
bleach, exposure to high level of ammonia, filtration, ozonation and UV-
irradiation)
• Utilization of microalgae-bacteria, microalgae-fungus and others consortium
• Nutrient supplementation using waste nutrient rich materials
• Dilute high strength wastewater with low nutrient wastewater
• Select the appropriate microalgae strain that can adapt to the specific wastewater
• Screening
wastewater
and isolation of indigenous microalgae species from heavy metal
• gravity)
Pretreatment techniques (such as adding flocculants and sedimentation by
• Introduce turbulence such as in raceway ponds to increase light exposure
• Combination of different harvesting techniques and integration with other
technologies (such as membrane technology and electrokinetics technologies)
• Exploring the potential of novel natural organic flocculants, coagulants, magnetic
and nanoparticles separator
• Strategies such as immobilization, microalgal biofilm, pelletisation, co-cultivation
with flocculating microorganisms
• Pretreatment techniques (such as recovery and regeneration of heavy metals)
• Integration and hybrid downstream purification strategies to remove heavy metals
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