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Review

Bioremediation of heavy metals using microalgae: Recent advances and mech-


anisms

Yoong Kit Leong, Jo-Shu Chang

PII: S0960-8524(20)30155-3
DOI: https://doi.org/10.1016/j.biortech.2020.122886
Reference: BITE 122886

To appear in: Bioresource Technology

Received Date: 20 December 2019


Revised Date: 20 January 2020
Accepted Date: 21 January 2020

Please cite this article as: Kit Leong, Y., Chang, J-S., Bioremediation of heavy metals using microalgae: Recent
advances and mechanisms, Bioresource Technology (2020), doi: https://doi.org/10.1016/j.biortech.2020.122886

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A revised manuscript (BITE-D-19-08978R1) submitted to Bioresource Technology

(All the changes made are marked with yellow highlight)

Bioremediation of heavy metals using microalgae: Recent advances and mechanisms

Yoong Kit Leonga, and Jo-Shu Changa,b,c*

aDepartment of Chemical and Materials Engineering, College of Engineering, Tunghai

University, Taichung, Taiwan


bDepartment of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan
cCenter for Nanotechnology, Tunghai University, Taichung, Taiwan

*Corresponding author: Prof. Jo-Shu Chang (changjs@mail.ncku.edu.tw)

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ABSTRACT

Five heavy metals namely, arsenic (As), cadmium (Cd), chromium (Cr), lead (Pb) and

mercury (Hg) are carcinogenic and show toxicity even at trace amounts, posing threats to

environmental ecology and human health. There is an emerging trend of employing

microalgae in phycoremediation of heavy metals, due to several benefits including abundant

availability, inexpensive, excellent metal removal efficiency and eco-friendly nature. This

review presents the recent advances and mechanisms involved in bioremediation and

biosorption of 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.

KEYWORDS: Microalgae, Bioremediation, Biosorption, Heavy Metals, Mechanism

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1. INTRODUCTION

“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 concentrations, 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 escalating the risk of cancers (Balaji et al., 2016a; Jaafari &

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

electrocoagulation) (Fu & Wang, 2011). However, these techniques have the disadvantages

of expensive operation and maintenance costs, as well as secondary pollution due to toxic

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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 outstanding

biological characteristics such as high photosynthetic efficiency and simple structure has

the ability to grow well under extreme environmental conditions such as presence of heavy

metals, high salinity, 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 and large surface area (Cameron et al.,

2018). Furthermore, both living cells and non-living biomass of microalgae can be

employed as biosorbents. 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 summary 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

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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 complexes 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 immobilization, gene regulation, exclusion

and chelation as well as antioxidants 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 separated insides vacuoles to help regulate the heavy

metal ions concentration in cytoplasm which subsequently mitigate their toxic effects

(Balaji et al., 2016b). In addition, heavy metals activate the biosynthesis of phytochelatins
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(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 ascorbate peroxidase, catalase, glutathione

reductase, peroxidase and superoxide 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). Cysteine indirectly or directly serves as the precursor for PCs, GSH,

metallothioneins 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 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).

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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 polysaccharides (cellulose and alginate), lipids

and organic proteins, provides 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 exchange of heavy metal ions with cell wall cation and binding of

heavy metal cations with negatively charged uronic acids of microalgae exopolysaccharides.

On the other hand, the process of heavy metal accumulation 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

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phytochelatins (Ibuot et al., 2017; Pradhan et al., 2019). Figure 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). Physical 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, infant morbidity, immune alteration, impairment to the central nervous system,

hepatic damage and hyperkerotosis (Li et al., 2019). Conversely, in plant and microalgae,

arsenic causes impairment to cell organelle and DNA, lipid peroxidation and protein

degradation (Upadhyay et al., 2016).

The toxicity and physico-chemical properties of arsenic is dependent 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

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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) reduction, 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 configuration as arsenate [As(V)] (Sun et al., 2015). Therefore, the high affinity

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 concentration

significantly reduced the arsenic uptake in microalgae such as Chlorella sp. (Bahar et al.,

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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 respectively, by

extracellular PO43-. However, it is reported that PO43- concentration has negligible impact

on the removal of arsenic by 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 Dunaliella 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

concentration 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

reported 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

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aeruginosa and Oscillatoria tenuis experienced up to 2.6-fold slower growth rate in As(V)-

containing medium compared to control. Among these microalgae, A. affinis and O. tenuis

removed As(V) by binding them to extracellular 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) concentration 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 essential 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 microalgae

species (Chlorella vulgaris, Chlamydomonas reinhardtii, Scenedesmus almeriensis and an

indigenous Chlorophyceae spp.), S almeriensis 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 bioaccumulation of arsenic, the total chlorophyll content

of Nannochloropsis sp. decreased, while carotenoids and lipid content increased.

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Nannochloropsis sp. treated with 100 µm As(III) has the highest lipid productivity 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 problems such as cancer, Parkinson’s and Alzheimer’s diseases, gastrointestinal

disorder, amyotrophic lateral sclerosis, hypertension, kidney damage, kidney stone,

peripheral neuropathy, osteoporosis and respiratory insufficiency (Zhang et al., 2019).

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Microalgae strains respond differently under cadmium stress. In addition to reducing

cell growth and chlorophyll content, Cd(II) induces 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 adsorption 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 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% respectively (Chia et al., 2013). Similarly, acid-

tolerant microalgae, Heterochlorella sp. MAS3 and Desmodesmus sp. MAS1 can

simultaneously remove 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 diluted 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 distinctive 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. vulgaris 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-

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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 improved 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

accumulation. A novel technique for Cd ions bio-immobilization utilizing Microcystis

aeruginosa and Selenastrum capricornutum cells as bio-templates by co-incubating with

selenium for bio-fabrication of fluorescent CdSe NPs has been performed. The reducing

biomolecules of the microalgae, 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+ concentration 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 -OH, -NH, P=O (phosphoryl group), P=S

(phosphorotioate group), C=O of amide and C-O 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

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Apsergillus niger fungi showed 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

functional groups such as carboxylic acid. Functional groups such as -OH, -NH, C=O of

amide and C-O of alcoholic groups contributes to cadmium biosorption (Dirbaz & Roosta,

2018). The residual lipid extracted biomass of Chlorella sp. CHA-01, Chlamydomonas sp.

TAI-03 and Coelastrum 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)

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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 digestive 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 anionic 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).

Several mechanisms have been proposed for the reduction and removal 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

16
adsorption 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 observed in non-living

microalgae biomass, which might due to the release of glutathione (Yen et al., 2017). Some

Cr(III) complexes are released 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 background 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 concentrations 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 tolerate high concentration of Cr(VI) up to 10 mg/L and 4 mg/L

17
respectively, 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, respectively, and the former has a Cr removal efficiency

of 71% (Dao et al., 2018). Four microalgae presented different tolerance when 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 content with increase in heavy metal

concentration, due to the accumulation 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)

18
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 5mg/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

respectively. 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 microalgae 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 immobilization damaged the

microalgae cell wall and resulted in reduced heavy metal removal efficiency (Ardila et al.,

2017). Among the different forms of dried S. quadricauda (powder, pellet and biochar),

microalgal biochar synthesized at 500°C showed 100% removal efficiency up to 10 mg/L

19
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 efficiency 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, carboxylic, 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 increasing temperature (Kayalvizhi et al., 2015; Nithya et al., 2019; Pradhan

20
et al., 2019). Sibi also reported that Cr(VI) removal of C. vulgaris increased with increasing

electrical conductivity up to 2.9 mS/cm, 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, reproduction 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, carboxyl, hydroxyl, phenols

and phosphate were identified for the biosorption 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
21
was observed 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 decreased 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 effective detection of lead

toxicity utilizing chlorophyll fluorescence 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
22
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 antibiotic 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

volatilization of mercury only happened under light illumination, while biological

volatilization was light-independent, but depended on metal concentration and cell density

(Devars et al., 2000). Accelerated volatilization was shown in microalgae pretreated with 5

mM dimethylfumarate (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

potential routes of Hg(II) removal are thiol chelation and bio-methylation to methylmercury

23
(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 together 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 mineral layer serves as a

protection layer for microalgae from heavy metals poisoning and the microalgae can grow

well in high concentration 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 deterred 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 wastewater with

the advantages of promoted removal yield as well as reduction in fouling and expensive cost

of membrane recovery. The dynamic membrane bioreactor demonstrated a higher Hg2+

24
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 content, 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 microalgae-

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

25
efficiency of heavy metal remediation and to reduce the operation costs. The future

prospects are thus discussed in detail below.

5. FUTURE PROSPECTS

Considering the multiple advantages provided by microalgae for heavy metal

bioremediation, the future prospects of large scale application 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 attributes: (i) able to accumulate high content of lipids and

other valuable co-products, (ii) high CO2 sequestration ability and low nutrient requirements,

(iii) resistant to predation by grazers and robustness toward existence of other

microorganism species, and (iv) ability to self-flocculate for cost-effective cell harvesting.

Therefore, genetic, metabolic 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 underlying

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 integration 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
26
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 inhibition 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 valuable 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.

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

accumulation, chelation and bio-methylation are involved in bioremediation and biosorption

of heavy metals. More efforts in the areas of genetic engineering, immobilization techniques,
27
pretreatment strategies and integration with other technologies are required to fully explore

the potential of microalgae in heavy metal phycoremediation and simultaneously value-

added products formation.

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.

28
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42
Figure 1. Mechanism of heavy metals removal by microalgae

43
Table 1. Performance of biosorption of arsenic (As) by different microalgae strains

Microalgae Initial metal Biomass conc. Max. sorption Removal


Temp (°C) Optimal pH Time (min) Reference
strains conc. (mg/L) (g/L) (mg/g) efficiency (%)

Maugeotia (Sarı et al.,


20 6 10 4 60 2.4 96
genuflexa 2011)
Mixture of green (Sulaymon et
(Chlorophyta) al., 2013a;
algae and blue- Sulaymon et
20 4 50 10 180 3.5 70
green al., 2013b)
(Cyanobacteria)
algae
(Doshi et al.,
Spirulina sp. 35 6 7276 12 240 365 60.2
2009)
Ulothrix (Tuzen et al.,
20 6 10 4 60 2.45 98
cylindricum 2009)
Chlamydomonas (Saavedra et
- 9.5 12 1 180 4.63 38.6
reinhardtii al., 2018)
Chlorella
- 5.5 12 1 180 3.89 32.4
vulgaris

Scenedesmus
- 9.5 12 1 180 5.0 41.7
almeriensis

44
Table 2. Performance of biosorption of cadmium (Cd) by different microalgae strains

Microalgae Temp (°C) Optimal pH Initial metal Biomass conc. Time (min) Max. sorption Removal Reference
strains conc. (mg/L) (g/L) (mg/g) efficiency (%)

Lipid-extracted 30 7.5 - 1 60 23.3 - (Zheng et al.,


Chlamydomonas 2016)
sp.
Lipid-extracted 30 7.5 - 1 60 25.5 -
Chlorella sp.
Lipid-extracted 30 7.5 - 1 60 32.8 -
Coelastrum sp.
Chlorella 28 6 - 4 20 303 - (Yang et al.,
minutissima 2015)
Immobilized - 6 10 1.3 - 15.51 92.5 (Shen et al.,
Chlorella spa 2018)
Parachlorella 35 7 100 1 - 96.2 - (Dirbaz &
sp. Roosta, 2018)
Scenedesmus-24 - 6 200 1.5 - 48.4 60.5 (Jena et al.,
2015)
aA complex of water-hyacinth leaf derived biochar pellets immobilized with Chlorella sp.

45
Table 3. Performance of biosorption of chromium (Cr(VI)) by different microalgae strains

Initial metal Biomass conc. Max. sorption Removal


Microalgae strain Temp (°C) Optimal pH Time (min) Reference
conc. (mg/L) (g/L) (mg/g) efficiency (%)

Spirulina 60 1 500 - 90 59.6 - (Nithya et al.,


platensis 2019)
Lipid-extracted 60 1 500 - 90 45.5 - (Nithya et al.,
Spirulina 2019)
platensis
Immobilized 25 2 250 1 - 49 19.6 (Kwak et al.,
Spirulina 2015)
platensisa
Methylated 25 - 18 - 25 2-4 120 - 240 16.7 > 80 (Finocchio et
Spirulina al., 2010)
platensis
Spirulina 25 1.5 250 1 600 148.64 59.5 (Gokhale et al.,
platensis 2008)
Pigment-extracted 25 1.5 250 1 600 174.64 69.9 (Gokhale et al.,
Spirulina 2008)
platensis
(Gokhale et al.,
Chlorella vulgaris 25 1.5 250 1 600 140 56 2008)

Chlorella vulgaris 25 2 147 1 240 63.2 43 (Sibi, 2016)

46
Scenedesmus 25 6 for Cr(III) 100 2 120 - 98.3 for (Shokri
quadricauda Cr(III) Khoubestani et
1 for Cr(VI) al., 2015)
47.6 for
Cr(VI)
Scenedesmus 22 2 10 2 240 - 100 (Daneshvar et
quadricauda al., 2019)
biochar
Rhizoclonium - 2 1000 1 45 67.3 6.7 (Kayalvizhi et
hookeri al., 2015)

Immobilized 30 2 100 60 % (w/v) 2160 57.33 99.7 (Singh et al.,


Chlorella 2012)
minutissimab

aSpirulina platensis beads prepared with 1 M LiCl/DMSO


bChlorella minutissima immobilized in calcium alginate beads

47
Table 4. Performance of biosorption of lead (Pb) by different microalgae strains

Microalgae Initial metal Biomass conc. Max. sorption Removal


Temp (°C) Optimal pH Time (min) Reference
strains conc. (mg/L) (g/L) (mg/g) efficiency (%)

Chaetoceros sp. 25 6 20 1.5 180 8 60 (Molazedah et


al., 2015)
Chlorella sp. 25 6 20 1.5 180 10.4 78

Immobilized 25 6 500 - 120 308.7 - (Bayramoğlu


Chlamydomonas et al., 2006)
reinhardtiia

Phormidium sp. 25 5 10 4 40 2.305 92.2 (Das et al.,


2016)

Rhizoclonium 40 4.5 - - - 81.7 - (Suganya et


hookeri al., 2016)

aChlamydomonas reinhardtii immobilized in calcium alginate beads

48
Table 5. Performance of biosorption of mercury (Hg) by different microalgae strains

Microalgae Initial metal Biomass conc. Max. sorption Removal


Temp (°C) Optimal pH Time (min) Reference
strains conc. (mg/L) (g/L) (mg/g) efficiency (%)

Immobilized 25 6 500 - 120 106.6 - (Bayramoğlu


Chlamydomonas et al., 2006)
reinhardtiia

Chlorella sp. DT 30 - 8 0.3 120 3.33 12.5 (Huang et al.,


2006)
Transgenic 30 - 8 0.3 120 7.33 27.5
Chlorella sp.b

Chlorella 20 5 48 2 120 17.49 72.9 (Solisio et al.,


vulgaris 2019)

Scenedesmus 25 5 20 0.125 180 - - (Huang et al.,


obtusus XJ-15 2019)

Spirogyra sp. 4 4 1 3 30 0.253 76 (Rezaee et al.,


2006)

aChlamydomonas reinhardtii immobilized in calcium alginate beads


bTransformed with the Bacillus megaterium strain MB1 merA gene

49
Table 6: Challenges and proposed strategies of heavy metal removal by microalgae

Challenges Proposed strategies


Raw heavy metal wastewater consists of bacteria, fungus  Pretreatment techniques (such as acidification, autoclaving, chlorination
and other microorganisms, which compete for nutrients by bleach, exposure to high level of ammonia, filtration, ozonation and
and possibly dominating because of their relatively higher UV-irradiation)
growth rate  Utilization of microalgae-bacteria, microalgae-fungus and others
consortium
Nutrient variability (such as excessive or deprivation in  Nutrient supplementation using waste nutrient rich materials
carbon, nitrogen and phosphorus content)  Dilute high strength wastewater with low nutrient wastewater
 Select the appropriate microalgae strain that can adapt to the specific
wastewater
 Screening and isolation of indigenous microalgae species from heavy
metal wastewater
Wastewater might contain huge amount of TSS and has  Pretreatment techniques (such as adding flocculants and sedimentation
high turbidity that reduce light penetration and hinder by gravity)
microalgae growth  Introduce turbulence such as in raceway ponds to increase light exposure
Microalgae biomass harvesting  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
Presence of heavy metal remaining in microalgal cell  Pretreatment techniques (such as recovery and regeneration of heavy
might interfere with downstream processing of valuable metals)
product  Integration and hybrid downstream purification strategies to remove
heavy metals

50
Yoong Kit Leong: Investigation, Writing- Original draft preparation; Jo-Shu Chang: Supervision, Conceptualization, Writing- Reviewing and Editing

Highlights

 Bioremediation of five toxic heavy metals by microalgae were reviewed in-depth.

 Feasibility and potential of value-added product accumulation were evaluated.

 Discussion of advanced techniques and integration with other technologies.

 Challenges and proposed strategies were summarized along with future prospects.

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the
work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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