Ecotoxicology and Environmental Safety 113 (2015) 329–352

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

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

Review

Microalgae – A promising tool for heavy metal remediation

K. Suresh Kumar a, Hans-Uwe Dahms b,c, Eun-Ji Won d, Jae-Seong Lee d, Kyung-Hoon Shin a,n
a
Department of Marine Sciences and Convergent Technology, College of Science and Technology, Hanyang University, Ansan 426-791, South Korea
b
Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 80424, Taiwan, ROC
c
Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, ROC
d
Department of Biological Sciences, College of Natural Sciences, Sungkyunkwan University, Suwon 440-746, South Korea

art ic l e i nf o a b s t r a c t

Article history: Biotechnology of microalgae has gained popularity due to the growing need for novel environmental
Received 25 June 2014 technologies and the development of innovative mass-production. Inexpensive growth requirements
Received in revised form (solar light and CO2), and, the advantage of being utilized simultaneously for multiple technologies (e.g.
3 December 2014
carbon mitigation, biofuel production, and bioremediation) make microalgae suitable candidates for
Accepted 8 December 2014
several ecofriendly technologies. Microalgae have developed an extensive spectrum of mechanisms
(extracellular and intracellular) to cope with heavy metal toxicity. Their wide-spread occurrence along
Keywords: with their ability to grow and concentrate heavy metals, ascertains their suitability in practical appli-
Microalgae cations of waste-water bioremediation. Heavy metal uptake by microalgae is affirmed to be superior to
Heavy metals
the prevalent physicochemical processes employed in the removal of toxic heavy metals. In order to
Bioremediation
evaluate their potential and to fill in the loopholes, it is essential to carry out a critical assessment of the
Mechanism
existing microalgal technologies, and realize the need for development of commercially viable tech-
nologies involving strategic multidisciplinary approaches. This review summarizes several areas of heavy
metal remediation from a microalgal perspective and provides an overview of various practical avenues
of this technology. It particularly details heavy metals and microalgae which have been extensively
studied, and provides a schematic representation of the mechanisms of heavy metal remediation in
microalgae.
& 2014 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
2. Hazardous effects of heavy metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
3. Conventional vs. novel approaches in heavy metal remediation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
4. Microalgae and their potential in metal remediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332
5. Metal removal mechanisms adapted by microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336
5.1. Live microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
5.1.1. The role of the algal cell wall in heavy metal binding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
5.1.2. The plasma membrane and heavy metal flux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
5.1.3. The ion exchange concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
5.1.4. Physical adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
5.1.5. Complexation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
5.1.6. Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
5.1.7. Metallothioneins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
5.1.8. Sequestration and compartmentalization in the vacuole. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
5.1.9. Polyphosphate bodies in algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
5.1.10. Sequestration to the chloroplast and mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
5.1.11. Other mechanisms of heavy metal remediation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
5.2. Biosorption by biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340

n
Corresponding author. Fax: þ 82 31 416 6173.
E-mail address: shinkh@hanyang.ac.kr (K.-H. Shin).

http://dx.doi.org/10.1016/j.ecoenv.2014.12.019
0147-6513/& 2014 Elsevier Inc. All rights reserved.
330 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352

5.2.1. Significance of functional groups in heavy metal uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

6. Factors affecting heavy metal remediation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
6.1. Biotic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
6.1.1. Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
6.1.2. Tolerance capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
6.1.3. Biomass concentration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
6.1.4. Size and volume of microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
6.2. Abiotic factors influencing metal removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
6.2.1. pH. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
6.2.2. Ionic strength. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
6.2.3. Salinity and hardness. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
6.2.4. Temperature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
6.2.5. Metal speciation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
6.2.6. Effect of combined metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
7. Potential microalgal species for metal remediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
8. Strategic approaches in heavy metal remediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
8.1. Immobilized microalgae and metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
8.2. Transgenic microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
8.3. Metal desorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
8.4. Recycling of microalgal biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
9. Algal biosorbent technologies currently in use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
10. Conclusion and future avenues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348

1. Introduction
Heavy metals (HMs) occur as natural constituents of the earth
crust and soil. Although there is no clear definition of a HM, in
most cases density is the defining factor; conventionally, HMs are
defined as elements with metallic properties having an atomic
number 420 (Jing et al., 2007; Srivastava, 2007). They refer to a
group of metals and metalloids with atomic density greater than
4 g cm  3, or five times or more, greater than water; approximately
53 chemical elements fall into the category of HM (Duruibe et al.,
2007; Herrera-Estrella and Guevara-Garcia, 2009). In general, the
term HM refers to any metallic chemical element that has a rela-
tively high density and is toxic or poisonous at low concentrations
(http://www.lenntech.com/processes/heavy/heavy-metals/heavy-
metals.htm#ixzz2PfTfZ3eN). However, in an ecological sense, any
metal or metalloid that causes environmental pollution, or that
cannot be biologically degraded (and is therefore bioaccumulated),
could be considered as a HM (Herrera-Estrella and Guevara-Garcia,
2009). Some of these metals are micronutrients necessary for
plant growth (e.g. Zn, Cu, Mn, Ni, and Co), while others have un-
known biological function and are toxic (e.g. Cd, Pb, and Hg) (Gaur
and Adholeya, 2004). Herrera-Estrella and Guevara-Garcia (2009)
provide an ecological perspective stating that “a heavy metal” is a
metal or metalloid element that causes environmental pollution,
which does not have any vital function and is toxic at low con-
centrations [such as lead (Pb) and mercury (Hg)], and it has a vital
function but is harmful to organisms at high concentrations [such as
copper (Cu) and molybdenum (Mo)]. Wang and Chen (2009) cate-
gorized HMs of concern into three categories: toxic metals [such as
Hg, Cr, Pb, Zn, Cu, Ni, Cd, As, Co, Sn, etc.], precious metals [such as Pd,
Pt, Ag, Au, Ru etc.] and radionuclides [such as U, Th, Ra, Am, etc.].
Population growth, urbanization, industrialization, and una-
bated agrarian practices, have caused global concern worldwide.
Increasing urbanization and industrialization have made HMs
reach alarming toxic levels in the environment; thereby suggest-
ing HM enrichment in many ecosystems to be strongly related to
human activities (Lasat, 2000; Herrera-Estrella and Guevara-Gar-
cia, 2009). Various reports elucidate the variegated traditional uses
of HMs (Järup, 2003; Torres et al., 2008), their use in several in-
dustrial (Wang and Chen, 2009) and agricultural practices (Brower
et al., 1997), as well as, their indiscriminate disposal in the en-
vironment (Duffus, 2002). The most common HM contaminants
include Cd, Cr, Cu, Hg, Pb, and Zn. Especially, water pollution by
HM ions is one of the worldwide environmental problems (Hong
et al., 2011). Activities leading to enrichment of HMs in the aquatic
environment include: natural geological weathering, as well as
human industrial processing, such as mining, smelting and me-
tallurgical industry, chemical industry, textile printing and dyeing
zincification, plastics, paint and tire manufacturing, power plants,
gasoline and fossil fuel combustion, battery, petroleum refining,
cement and ceramic production, steel production, agrochemical
and animal feed industries, electroplates, paper and pulp, alloy
preparation, viscose rayon yarn and fiber production (Förstner and
Wittmann, 1983; Olade, 1987; Guo, 1994; Periasamy and Nama-
sivayam, 1996; Cheng, 2003; Freitas et al., 2008; http://pacifi-
cenvironment.org/downloads/GCA%20IT%20Campaign%20Report%
20Phase%20One.pdf).
Several metallic species mobilized and released into the en-
vironment by various technological activities by humans, persist
indefinitely or undergo transformations, circulating and eventually
accumulating throughout the food chain, thus posing a serious
threat to the environment (Volesky and Holan, 1995). They thereby
have a large environmental, public health, and economic impact
(Brower et al., 1997; White et al., 1995). In brief, unlike many other
pollutants, removal of HMs from the environment is truly chal-
lenging as they cannot be chemically or biologically degraded, and,
are ultimately indestructible. HMs discharged into water bodies
through wastes have an incorrigible impact on the aquatic system,
and destroy the self-purification ability of an aquatic body (Khan
et al., 2008).
2. Hazardous effects of heavy metals
The center for Hazardous Substance Research (US) clearly ca-
tegorized HMs as individual metals and metal compounds that can
impact human health (http://www.engg.ksu.edu/chsr/files/chsr/
outreach-resources/15HumanHealthEffectsofHeavyMetals.pdf).
HMs are stable elements (i.e. they cannot be metabolized by the
body), which could be bio-accumulated (passed up the food chain
 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352
to humans). Though the adverse health effects of HMs are known
since a long time, exposure to HMs continues and is even in-
creasing in some regions (Järup, 2003). HMs are key environ-
mental pollutants in regions with high anthropogenic pressure;
and their presence, even in traces, can cause serious problems to
several organisms and humans (Islam et al., 2007). Arbitrary dis-
posal of industrial and domestic wastes (containing heavy metals)
into aquatic ecosystems, threatens the inhabiting aquatic organ-
isms (De Filippis and Pallaghy, 1994). Organisms actively respond
to protect themselves from metal poisoning at contaminated sites.
Due to their mobility in aquatic ecosystems, and, their toxicity to
higher life forms, HMs in surface and groundwater supplies have
been prioritized as the major inorganic contaminants in the en-
vironment (Khan et al., 2008). Even if they are present in dilute
undetectable quantities, their recalcitrance and consequent per-
sistence in water bodies implies that, through natural processes
such as biomagnification, concentrations may become elevated to
such an extent that they start exhibiting toxic characteristics
(http://www.ces.iisc.ernet.in/biodiversity/pubs/ces_tr/TR112_Ahalya/
CESTechnical%20report%20-Metals210607.pdf). In this context,
Khan et al. (2008) stated that HMs beyond the permissible limits
in aquatic environment causes direct toxicity to humans and other
life forms. Water comprises one of the main portals of entry of
these obnoxious elements into the human body; therefore, even
the EPA (2009) has defined maximum contaminant level for cer-
tain metals (i.e. Hg, Pb, Cr, Cu, Cd, Zn and Ni concentrations in
drinking water should not exceed 0.002, 0.015, 0.1, 1.3, 0.005, 5,
and 0.04 mg l  1, respectively).
Traditional technologies for the removal of HMs (such as ion
exchange or lime precipitation) are often ineffective and/or very
expensive, and are generally used for the reduction of HM ions to
very low concentrations (Wilde and Benemann, 1993). Therefore,
it has become imperative to look for new-fangled technologies for
HMs remediation/removal to meet these requirements (Sheng
et al., 2004). It is essential for a remediation technology to be ef-
fective, economic/affordable, and consistent; moreover, it should
effectively reduce HM concentrations to environmentally accep-
table levels, and be applicable to field conditions such as effluents
and aquatic bodies.
3. Conventional vs. novel approaches in heavy metal
remediation
Conventional methods for HM removal comprise chemical
precipitation (hydroxide precipitation, carbonate precipitation and
sulfide precipitation), chemical oxidation or reduction, lime coa-
gulation, ion exchange (using resins, starch xanthate, etc.), reverse
osmosis, solvent extraction, evaporation recovery, cementation,
adsorption (involving use of activated carbon), electrodeposi-
tion, reverse osmosis and electro-dialysis (Rich and Cherry, 1987;
Ahalya et al., 2003; Gray, 1999; Ahluwalia and Goyal, 2007).
However, these conventional approaches are often ineffective or
expensive, especially when the metals in solution are in the range
of 1–100 mg l  1 (Nourbakhsh et al., 1994). Other technologies
either employ inorganic adsorbents [such as natural minerals,
ores, clay and waste materials from various industries like me-
tallurgical solid wastes like bauxite red muds, slag, ash, water
treatment (alum) sludge and seawater-neutralized red mud], or
involve organic adsorbents [such as waste materials of organic
origin derived from plants or animals] (Rai et al., 1998; Khan et al.,
2008; Zvinowanda et al., 2009; Zhou and Haynes, 2010, 2011).
However, most conventional techniques involved in HM re-
mediation provide incomplete metal removal (Volesky, 1990), re-
quire large amounts of reagents and energy (Ahalya et al., 2003),
have limited tolerance to pH change (Ahluwalia and Goyal, 2007),
331
have moderate or no metal selectivity (Antunes et al., 2003), need
very high or low working levels of metals (a method that is ef-
fective at lower concentrations of HM would not be good for high
concentrations) (Antunes et al., 2003), produce toxic sludge or
other waste products (Ahalya et al., 2003; Ahluwalia and Goyal,
2007), and suffer from high investment and regeneration costs
(Oboh et al., 2009). Therefore, the scientific community is under
tremendous pressure to develop new, innovative, cost-effective,
efficient and sustainable methods for the removal of toxic sub-
stances from aquatic bodies as well as wastewaters. There is a
growing impulse for the production of cheaper adsorbents to re-
place costly wastewater treatment methods; thereby non-con-
ventional HM remediation technologies have been gaining im-
mense popularity. Researchers have intensified their efforts in
developing suitable technologies for (i) prevention of HM pollu-
tion, (ii) decreasing HMs concentrations to a lower level (for e.g. by
decreasing the efflux of HMs into the receiving bodies such as
rivers, sewer and lake, etc.), and, (iii) removal of HMs from con-
taminated media.
Bioremediation technologies have a promising potential to
contribute in achieving this goal in an eco-friendly manner. In
general, biological remediation technologies could either involve:
i) Biosorption that comprises a metabolically passive process
(where the amount of contaminants that a sorbent can remove is
dependent on kinetic equilibrium and the composition of the
cellular surface of a sorbent); here the pollutant could be adsorbed
onto the cellular structure, and ii) bioaccumulation which is an
active metabolic process driven by energy from a living organism
and requires respiration where the absorbing contaminants which
are transferred onto and within the cellular surface (also see
Section 4). These biological technologies comprise low-cost, high-
efficiency techniques for HM removal from dilute solutions, and
may also involve regeneration; in addition, they could provide
metal recovery. The accumulation and concentration of pollutants
from aqueous solutions by the use of biological materials [such as
plant biomass, animal polymers (e.g. chitosan, tannins), or mi-
crobial biomass], facilitating the recovery and/or environmentally
acceptable disposal of the pollutant is termed as “bioremoval”
(Wilde and Benemann, 1993). However, the removal capacity can
be affected by factors such as: i) characteristics of the metal ion
(atomic weight, and valency), ii) environmental conditions (pH,
temperature, ionic strength, contact time, biomass concentration),
and iii) the nature of the biosorbent, which may determine dif-
ferences in selectivity and affinity to metal ions; here the type and
species of organisms, conditions of growth, physiological state and
cell age, may all affect the HM binding mechanism (Wang and
Chen, 2006; Chen and Wang, 2008; Perpetuo et al., 2011).
Since the past few decades, bacteria (or bacterial exopoly-
saccharides), land plants (or their products), aquatic plants, algae,
fungi and peat moss, have received substantial attention for their
capacity to eliminate HMs (Sandau et al., 1996a; Iyer at al., 2004,
2005; Kumar et al., 2007). In this context, the efficacy of live or-
ganism vs. dead biomass has been extensively debated. For ex-
ample, Rayson and Williams (2011) studied the plant, Datura in-
noxia, and reported that non-living biomaterials exhibit high ca-
pacity, rapid binding, and selectivity towards HMs. They corre-
spondingly state that functional groups of the lipids, carbohy-
drates, and proteins found in the cell walls of the biomaterial are
responsible for uptake (biosorption) of metal ions. On the other
hand, Siegel et al. (1990) mention that fungal systems offer con-
siderable versatility with respect to metals as they efficiently take
up and bind cations (Fe, Ni, Cu, Zn, Ag, Cd, La, Pb, Th, and U) as well
as anions (Cr and Mo). They suggest that even though filamentous
fungi possess a high potential of accumulating HMs from aqueous
solutions, the direct application of living fungal cells as biosor-
bents for HMs is unfavorable. Further, derivatives of fungal
332 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352
biomass (purified cell wall fractions, whole or heat killed myce-
lium) are known to possess a moderate degree of regenerability,
and superior HM remediation ability compared to commercial ion-
exchange resins, carbons and metal oxides (Siegel et al., 1990).
Among various technologies available for biological removal of
HMs, the most widely studied microbial know-how comprises the
use of sulfate-reducing bacteria (SRB) that remove HMs via the
production of metal-sulfide precipitates at a large-scale (Perales-
Vela et al., 2006). However, its main drawbacks include: long re-
sidence time (weeks), the need for a continuous supply of organic
substrates and large steel bioreactors (White et al., 1997; Perales-
Vela et al., 2006). Even though both living and dead cells, as well
as, products derived from or produced by microorganisms can be
effective metal accumulators, the lack of awareness regarding
metal–microbe interactions remain unexploited (Gadd, 1990), and
at times in indicipherable. Ozdemir et al. (2004) mention that
certain types of microbial biomass can retain relatively high
quantities of metals by means of passive processes known as
biosorption, which is dependent on the affinity between the me-
tallic species or its ionic forms and the binding sites on the mo-
lecular structure of the cellular membrane, cell-wall and capsule.
On another stance, algae as renewable natural biomass exhibit
different affinities toward different metals, and, are therefore, very
important candidates, employed as biosorbent materials (Mallick,
2002; Doshi et al., 2006). Macro- and microalgae exhibit con-
stitutive mechanisms for the removal of free metal ions from
waters, thereby detoxifying and remediating the water in ques-
tion. For these reasons, phycoremediation (the use of macro- and
micro-algae for the removal or biotransformation of pollutants)
has gained popularity. Metal-uptake capacities of certain marine
and river algae (studied for adsorption and elution of Au, Ag, and
Co) are reported to be much higher than activated carbon, natural
zeolite, and synthetic ion-exchange resin (Doshi et al., 2006).
Discussing potential removal of Cd (II) using marine brown algae,
Khan et al. (2008) report biomass of Sargassum had superior metal
binding capacity as compared to organic and inorganic sorbents.
Kumar et al. (2007, 2008) reported HM remediation using live
Kappaphycus alvarezii as well as its biomass.
Perales-Vela et al. (2006) discuss the removal of Zn, Cu and Mn
with the help of various algae-based technologies such as High
Rate Algal Ponds (HRAP), and Algal Turf Scrubber (ATS) employing
suspended biomass of common green algae (Chlorella, Scene-
desmus, and Cladophora), cyanobacteria (Spirulina, Oscillatoria,, and
Anabaena), or a consortia of both. Khan et al. (2008) reported
Chlorella vulgaris to have Cd (II) removal efficiency while Sphag-
num moss peat could remove Cu (II) and Ni (II). In addition, Pérez-
Rama et al. (2002) report cadmium removal by living marine mi-
croalga Tetraselmis suecica. According to Becker (1983, 1994),
planktonic algae (with a high potential to absorb HMs) could be
used for the removal of residual metals from wastewaters in an
economic method, resulting in high quality reusable effluent water
and valuable biomass (which in turn could have diverse applica-
tions like the production of biogas, fertilizer, fodder, etc.).
4. Microalgae and their potential in metal remediation
Microalgae are microscopic photosynthetic organisms found in
both marine and freshwater environments, and possess a photo-
synthetic mechanism that is fairly similar to land plants. In terms of
biomass, they form the world's largest group of primary producers,
responsible for at least 32% of global photosynthesis (Priyadarshani
et al., 2011). Microalgae are aquatic organisms possessing molecular
mechanisms that allow them to discriminate, non-essential HMs
from essential ones, for their growth (Perales-Vela et al., 2006). Re-
searchers have globally emphasized on the advantages of using
microalgae in metal biosorption. The benefits include: rapid metal
uptake capability, time and energy saving, eco-friendly, user-friendly,
year round occurrence, ease of handling, recyclable/ reusable, low-
cost, faster growth rate (as compared to higher plants), high effi-
ciency, large surface to volume ratio, ability to bind upto 10% of their
biomass, with high selectivity (which enhances their performance),
no toxic waste generation, no synthesis required, useful in both batch
and continuous systems, and, applicability to waters containing high
metal concentrations or relatively low contaminant levels (Monteiro
et al., 2012). Monteiro et al. (2012) particularly justify the worthiness
of microalgae (living and non-living biomass) by disclosing that they
aid in noteworthy removal of metals even at low contaminant levels.
Apart from possessing greater HM remediation efficacy, microalgae
enable easy recovery of HMs involving a few simple desorption
chemicals. Live algal biomass needs minimum nutrients and en-
vironmental conditions, while dead biomass does not require specific
nutrients or oxygen; moreover, they could remove HMs from multi-
metal solutions too (Figueira et al., 1999; Rajamani et al., 2007). In
addition, they are suitable for aerobic and anaerobic systems, and no
immobilization is required as compared to microbes. Rajamani et al.
(2007) elaborate how microalgae can efficiently sequester HMs, and
mention about the transgenic approaches used to enhance the HM
binding efficiency of microalgae including the use of fluorescent HM
biosensors developed using transgenic Chlamydomonas.
Microalgal affinities for polyvalent metals help establish their
potential application in cleansing wastewater containing dissolved
metallic ions (de-Bashan and Bashan, 2010); particularly, Chlorella
and Scenedesmus are microalgae of choice for metal removal.
However, Brinza et al. (2007) elaborate the role of marine micro-
and macro-algal species as biosorbents for metal uptake (K, Mg,
Ca, Fe, Sr, Co, Cu, Mn, Ni, V, Zn, As, Cd, Mo, Pb, Se and Al). They
mention the particular use of the following microalgae: Chlamy-
domonas reinhardtii, Chlorella salina, Chlorella sorokiniana, C. vul-
garis, Chlorella miniata, Chlorococcum spp., Cyclotella cryptica,
Lyngbya taylorii, Phaeodactylum tricornutum, Porphyridium pur-
pureum, Scenedesmus abundans, Scenedesmus quadricauda, Scene-
desmus subspicatus, Spirogyra spp., Spirulina platensis, Stichococcus
bacillaris and Stigeoclonium tenue (Brinza et al., 2007). Perales-Vela
et al. (2006) also mentioned Cd, U, and Cu removal capabilities of
Chlorella, Scenedesmus and P. tricornutum. Perales-Vela et al. (2006)
reported that microalgae, related eukaryotic photosynthetic or-
ganisms, preferentially produce peptides capable of binding HMs.
These peptide molecules bind the HMs forming organometallic
complexes, which are further positioned inside vacuoles to facil-
itate appropriate controls of the cytoplasmic concentration of HM
ions; this prevents or neutralizes the potential toxic effect of the
HMs (Cobbett and Goldsbrough, 2002).
Table 1 describes the HM remediation potential of various micro-
algae. According to this table, reports comprising cadmium removal
generally encompass studies on Cd2þ , and the pH range studied var-
ied from 4 to 8 (Table 1). Moreover, the microalgae C. reinhardtii,
S. platensis, as well as, various Scenedesmus spp., Tetraselmis spp. and
Chlorella spp. efficiently contribute to Cd removal. However, live cells
of Planothidium lanceolatum are reported to take up significant quan-
tities of Cd2þ (275.51 mg g  1; Sbihi et al., 2012). Investigations on
cobalt remediation comprise pH ranging from 4 to 7.5; here, Spirogyra
spp. (12.82 mg g  1) and Oscillatoria angustissima (15.32 mg g  1) are
promising. As witnessed in Table 1, amongst the three forms chro-
mium (i.e. Cr3 þ , Cr þ O72 and Cr6þ ) studied, more emphasis has been
laid on its hexavalent form (Cr6 þ ), wherein Chlorella and Spirulina spp.
as good contenders, and, a wide pH range (2–8.2) has been in-
vestigated. Among the several studies on chromium remediation using
live, dead, heat-treated and acid-treated forms of microalgae, re-
markable chromium uptake (333 mg g  1) by live Spirulina spp. has
been reported by Doshi et al. (2007). On the other hand, most studies
on copper removal mainly focus on Cu2þ (Table 1), where, the most
 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352 333

Table 1
Heavy metal removal efficiency of some microalgae.

Metal Speciation Organism pH Type of biomass Metal uptake (mg/g) Reference Country

Cd Cd2 þ AER Chlorella 3–7 Non-living 7.74 Sandau et al., 1996a Germany
AER Porphyridium 3–7 Non-living 7.55 Sandau et al., 1996a Germany
AER Spirulina 3–7 Non-living 7.28 Sandau et al., 1996a Germany
Aulosira fertilissima 5 Non-living 14.57 Singh et al., 2007 India
Calothrix parietina TISTR 8093 7 Non-living 79 Inthorna et al., 2002 Thailand
Chaetoceros calcitrans 8 Live 1055.27 Sjahrul and Arifin, 2012 Indonesia
Chlamydomonas reinhardtii 5.5 Cell wall 5.75 Macfie and Welbourn, 2000 Canada
Chlamydomonas reinhardtii 5.5 Without cell wall 3 Macfie and Welbourn, 2000 Canada
Chlamydomonas reinhardtii 6 Ca-alginate 28.9 Bayramoğlu et al., 2006 Turkey
Chlamydomonas reinhardtii 6 Immobilized 79.7 Bayramoğlu et al., 2006 Turkey
Chlamydomonas reinhardtii 6 Non-living 42.6 Tüzün et al., 2005 Turkey
Chlamydomonas reinhardtii 2.3 Munoz and Guieysse, 2006 Sweden
Chlorella homosphaera 8.4 Munoz and Guieysse, 2006 Sweden
Chlorella pyrenoidosa 6.8–7.0 2.8 Munoz and Guieysse, 2006 Sweden
Chlorella sorokiniana 5 Non-living 33.5 Akhtar et al., 2003 Pakistan
Chlorella sorokiniana 5 Immobilized 192 Akhtar et al., 2003 Pakistan
Chlorella sp. HA-1 21.6 Chen et al., 2012 Taiwan
Chlorella vulgaris 4 86.6 Aksu and Dönmez, 2006 Turkey
Chlorella vulgaris 4 Non-living 62.3 Aksu, 2001 Portugal
Chlorella vulgaris 4 Non-living 33.72 Klimmek et al., 2001 Germany
Chlorella vulgaris 6 Non-living 12.45 Sandau et al., 1996a Germany
Chlorella vulgaris 3–7 Non-living 8.41 Sandau et al., 1996a Germany
Chlorella vulgaris 6.8–7.0 2.6 Munoz and Guieysse, 2006 Sweden
Chlorella vulgaris BCC 15 7 Non-living 76 Inthorna et al., 2002 Thailand
Chlorella vulgaris CCAP211/11B 7 Non-living 62 Inthorna et al., 2002 Thailand
Cyclotella cryptica 6 Non-living 22.24 Schmitt et al., 2001 Germany
Desmodesmus pleiomorphus Non-living 58.6 Monteiro et al., 2011c Portugal
Desmodesmus pleiomorphus (ACOI 561) 4 Live 85.3 Monteiro et al., 2010 Portugal
Desmodesmus pleiomorphus (L) 4 Live 61.2 Monteiro et al., 2010 Portugal
Hydrodictyon reticulatum 5 Non-living 7.2 Singh et al., 2007 India
Isochrysis galbana Live 0.02 Sbihi et al., 2012 Morocco
Phaeodactylum tricornutum 6 Non-living 1.24 Schmitt et al., 2001 Germany
Phormidium spp. 5 Non-living 9.6 Wang et al., 1998 USA
Pithophora odeogonia 5 Non-living 13.07 Singh et al., 2007 India
Planothidium lanceolatum 7 Live 275.51 Sbihi et al., 2012 Morocco
Porphyridium cruentum 3–7 Non-living 8.84 Sandau et al., 1996a Germany
Porphyridium purpureum 6 Non-living 0.42 Schmitt et al., 2001 Germany
Pseudochlorococcum typicum 7 Live 5.48 Shanab et al., 2012 Egypt
Scenedesmus abundans 7.8–8 Live 574 Monteiro et al., 2009 Portugal
Scenedesmus abundans 0.64 Chen et al., 2012 Taiwan
Scenedesmus acutus IFRPD 1020 7 Non-living 110 Inthorna et al., 2002 Thailand
Scenedesmus obliquus 11.4 Monteiro et al., 2009 Portugal
Scenedesmus obliquus Non-living 60.8 Monteiro et al., 2011c Portugal
Scenedesmus obliquusCNW-N 6 Non-living 24.4–108.5 Chen et al., 2012 Taiwan
Scenedesmus subspicatus 6 Non-living 7.29 Schmitt et al., 2001 Germany
Spirogyra hyalina Non-living 18.18 Kumar and Oommen, 2012 India
Spirogyra insignis 6 Non-living 22.9 Romera et al., 2007 Spain
Spirogyra neglecta 5 Non-living 27.95 Singh et al., 2007 India
Spirulina platensis 6 Non-living 12.08 Sandau et al., 1996a Germany
Spirulina platensis 3–7 Non-living 8.06 Sandau et al., 1996a Germany
Spirulina platensis 7–8 Non-living 357 Solisio et al., 2008 Italy
Spirulina platensis Live 44.56 Murugesan et al., 2008 India
Spirulina platensis Immobilized 47.89 Murugesan et al., 2008 India
Spirulina platensis TISTR 8217 6 Alginate immobilized 70.92 Rangsayatorn et al., 2004 Thailand
Spirulina platensis TISTR 8217 7 Non-living 98.04 Rangsayatorn et al., 2002 Thailand
Spirulina platensis TISTR 8217 4–7 Silica-immobilized 36.63 Rangsayatorn et al., 2004 Thailand
Spirulina spp. 7.5 Non-living 0.46 Chojnacka et al., 2004 Poland
Spirulina spp. 0.463 Chen et al., 2012 Taiwan
Synechocystis sp. Live 199.83 Tiantian et al., 2011 China
Tetraselmis chuii 8 Live 13.46 Sjahrul and Arifin, 2012 Indonesia
Tetraselmis chuii Live 292.6 da Costa and de Franca, 1998 Brasil
Tetraselmis chuii Non-living 210.54 da Costa and de Franca, 1998 Brasil
Tolypothrix tenuis TISRT 8063 7 Non-living 90 Inthorna et al., 2002 Thailand
Co Co Chlamydomonas reinhardtii 5.5 Cell wall 0.89 Macfie and Welbourn, 2000 Canada
Chlamydomonas reinhardtii 5.5 Without cell wall 1.3 Macfie and Welbourn, 2000 Canada
Oscillatoria angustissima 4 Non-living 15.32 Mehta and Gaur, 2005 India
Spirogyra hyalina Non-living 12.82 Kumar and Oommen, 2012 India
Spirulina spp. 7.5 Non-living 0.01 Chojnacka et al., 2004 Poland
Cr Cr3 þ Chlorella miniata 3 Non-living 14.17 Han et al., 2006 Hong Kong
Chlorella miniata 4 Non-living 28.72 Han et al., 2006 Hong Kong
Chlorella miniata 4.5 Non-living 41.12 Han et al., 2006 Hong Kong
Chlorella spp. Non-living 98 Doshi et al., 2008 India
Chlorella spp. 9.62 Akhtar et al., 2008 Pakistan
Spirulina Live 304 Doshi et al., 2007 India
Spirulina spp. Non-living 167 Doshi et al., 2007 India
334 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352

Table 1 (continued )

Metal Speciation Organism pH Type of biomass Metal uptake (mg/g) Reference Country

Spirulina sp.(HD-104) Live 306 Doshi et al., 2008 India

Cr6 þ Chlamydomonas angulosa 8.2 Non-living 5.32 Dwivedi et al., 2010 India
Chlamydomonas reinhardtii 2 Non-living native 18.2 Arıc et al., 2005 Turkey
Chlamydomonas reinhardtii 2 Heat-treated 25.6 Arıc et al., 2005 Turkey
Chlamydomonas reinhardtii 2 Acid-treated 21.2 Arıc et al., 2005 Turkey
Chlorella vulgaris 2 Non-living 23 Dönmez et al., 1999 Turkey
Chlorella spp. 9.62 Akhtar et al., 2008 Pakistan
Chlorella vulgaris 4 Non-living 23.6 Aksu and Kutsal, 1990 Turkey
Dunaliella sp. 1 2 Non-living 58.3 Dönmez and Aksu, 2002 Turkey
Dunaliella sp. 2 2 Non-living 45.5 Dönmez and Aksu, 2002 Turkey
Nostoc muscorum 3 Non-living 22.92 Gupta and Rastogi, 2008 India
Oscillatoria nigra 8.2 Non-living 1.86 Dwivedi et al., 2010 India
Oscillatoria tenuis 8.2 Non-living 7.35 Dwivedi et al., 2010 India
Phormidium bohneri 8.2 Non-living 8.55 Dwivedi et al., 2010 India
Scenedesmus obliquus 2 Non-living 15.6 Dönmez et al., 1999 Turkey
Spirulina Live 333 Doshi et al., 2007 India
Spirulina spp. Non-living 143 Doshi et al., 2007 India
Synechocystis spp. 2 Non-living 19.2 Dönmez et al., 1999 Turkey
Ulothrix tenuissima 8.2 Non-living 4.56 Dwivedi et al., 2010 India
2
Cr2O7 Chlorella spp. Non-living 104 Doshi et al. 2008 India
Spirulina sp.(HD-104) Live 226 Doshi et al., 2008 India
Cu Cu2 þ Anabaena cylindrica 4.0–5.0 Live 12.62 Tien et al., 2005 Taiwan
Anabaena spiroides 4.0–5.0 Live 8.73 Tien et al., 2005 Taiwan
Asterionella formosa 4.0–5.0 Live 1.1 Tien et al., 2005 Taiwan
Asterionella formosa 4.0–5.0 Non-living 0.53 Tien et al., 2005 Taiwan
Aulacoseira varians 4.0–5.0 Live 2.29 Tien et al., 2005 Taiwan
Aulacoseira varians 4.0–5.0 Non-living 3.03 Tien et al., 2005 Taiwan
Aulosira fertilissima 5 Non-living 21.77 Singh et al., 2007 India
Ceratium hirundinella 4.0–5.0 Live 2.3 Tien et al., 2005 Taiwan
Ceratium hirundinella 4.0–5.0 Non-living 5.75 Tien et al., 2005 Taiwan
Chlamydomonas reinhardtii 5.5 Cells with Cell wall 6.42 Macfie and Welbourn, 2000 Canada
Chlamydomonas reinhardtii 5.5 Cells without cell wall 7.54 Macfie and Welbourn, 2000 Canada
Chlorella fusca 6 Live 3.2 Dönmez et al., 1999 Turkey
Chlorella miniata 6 Non-living 23.26 Lau et al., 1999 Hong Kong
Chlorella pyrenoidosa 7 Live 2.4 Yan and Pan, 2002 China
Chlorella spp. 7 Immobilized 33.4 Maznah et al., 2012 Malaysia
Chlorella spp. Live 220 Doshi et al., 2006 India
Chlorella spp. Non-living 108 Doshi et al., 2008 India
Chlorella vulgaris 2 Non-living 16.14 Romera et al., 2006 Spain
Chlorella vulgaris 4 Non-living 37.6 Aksu and Kutsal, 1990 Turkey
Chlorella vulgaris 4 Non-living 34.89 Romera et al., 2006 Spain
Chlorella vulgaris 4.5 Non-living 40 Dönmez et al., 1999 Turkey
Chlorella vulgaris 4.5 Non-living 48.17 Romera et al., 2006 Spain
Chlorella vulgaris 4.5 Free 76.71 Mehta and Gaur, 2001 India
Chlorella vulgaris 4.5 Immobilized 63.08 Mehta and Gaur, 2001 India
Chlorella vulgaris 5 Non-living 1.8 Dönmez et al., 1999 Turkey
Chlorella vulgaris 6 Non-living 10.9 Sandau et al., 1996a Germany
Chlorella vulgaris 6 7.5 Dönmez et al., 1999 Turkey
Chlorella vulgaris 6 Non-living 18.72 Lau et al., 1999 Hong Kong
Chlorella vulgaris 4.0–5.0 Live 3.63 Tien et al., 2005 Taiwan
Chlorella vulgaris 4.0–5.0 Non-living 4.26 Tien et al., 2005 Taiwan
Closterium lunula 7 Live 0.5 Yan and Pan, 2002 China
Cyclotella cryptica 6 Non-living 26.28 Schmitt et al., 2001 Germany
Eudorina elegans 4.0–5.0 Live 3.96 Tien et al., 2005 Taiwan
Eudorina elegans 4.0–5.0 Non-living 2.13 Tien et al., 2005 Taiwan
Hydrodictyon reticulatum 5 Non-living 08.72 Singh et al., 2007 India
Isochrysis galbana Live 0.11 Sbihi et al., 2012 Morocco
Microcystis aeruginosa 4.0–5.0 Live 8.21 Tien et al., 2005 Taiwan
Microcystis aeruginosa 4.0–5.0 Non-living 2.47 Tien et al., 2005 Taiwan
Microcystis spp. 9.2 Non-living 0.003 Singh et al., 1998 India
Phaeodactylum tricornutum 6 Non-living 1.67 Schmitt et al., 2001 Germany
Phormidium spp. 5 Non-living 10.1 Wang et al., 1998 USA
Pithophora odeogonia 5 Non-living 23.08 Singh et al., 2007 India
Planothidium lanceolatum 7 Live 134.32 Sbihi et al., 2012 Morocco
Porphyridium purpureum 6 Non-living 0.27 Schmitt et al., 2001 Germany
Scenedesmus obliquus 4.5 Non-living 20 Dönmez et al., 1999 Turkey
Scenedesmus obliquus 7 Live 1.8 Yan and Pan, 2002 China
Scenedesmus subspicatus 6 Non-living 13.28 Schmitt et al., 2001 Germany
Scenedesmus quadricauda 4 Non-living 2.8 Dönmez et al., 1999 Turkey
Spirogyra insignis 4 Non-living 19.3 Romera et al., 2007 Spain
Spirogyra neglecta 5 Non-living 40.83 Singh et al. 2007 India
Spirulina Live 389 Doshi et al. 2007 India
Spirulina platensis 6 Non-living 10.33 Sandau et al., 1996a Germany
Spirulina platensis 6 10 Dönmez et al., 1999 Turkey
Spirulina platensis 9 Live 0.85 Nalimova et al., 2005 Russia
Spirulina spp. Non-living 100 Doshi et al., 2007 India
 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352 335

Table 1 (continued )

Metal Speciation Organism pH Type of biomass Metal uptake (mg/g) Reference Country

Spirulina spp. 0.271 Chojnacka et al., 2004 Poland

Spirulina sp.(HD-104) Live 576 Doshi et al., 2008 India
Synechocystis spp. 4.5 Non-living 23.4 Dönmez et al., 1999 Turkey
Fe Fe3 þ Chlorella vulgaris 2 Non-living 24.52 Romera et al., 2006 Spain
Microcystis sp. 9.2 Non-living 0.03 Singh et al., 1998 India
Hg Hg2 þ Calothrix parietina TISTR 8093 7 Non-living 19 Inthorna et al., 2002 Thailand
Chlamydomonas reinhardtii 6 Ca-alginate immobilized 35.9 Bayramoğlu et al., 2006 Turkey
Chlamydomonas reinhardtii 6 Ca-alginate immobilized 106.6 Bayramoğlu et al., 2006 Turkey
Chlamydomonas reinhardtii 6 Non-living 72.2 Tüzün et al., 2005 Turkey
Chlorella vulgaris BCC 15 7 Non-living 18 Inthorna et al., 2002 Thailand
Chlorella vulgaris CCAP211/11B 7 Non-living 16 Inthorna et al., 2002 Thailand
Cyclotella cryptica 4 Non-living 11.92 Schmitt et al., 2001 Germany
Phaeodactylum tricornutum 4 Non-living 0.51 Schmitt et al., 2001 Germany
Porphyridium purpureum 4 Non-living 0.51 Schmitt et al., 2001 Germany
Pseudochlorococcum typicum 7 Live 15.13 Shanab et al., 2012 Egypt
Scenedesmus acutus IFRPD 1020 7 Non-living 20 Inthorna et al., 2002 Thailand
Scenedesmus subspicatus 4 Non-living 9.2 Schmitt et al. 2001 Germany
Spirogyra hyalina Non-living 35.71 Kumar and Oommen 2012 India
Spirulina spp. 7.5 Non-living 1.34 Chojnacka et al. 2004 Poland
Tolypothrix tenuis TISRT 8063 7 Non-living 27 Inthorna et al., 2002 Thailand
2þ
Ni Ni Arthrospira (Spirulina) platensis 5.0–5.5 Non-living 20.78 Ferreira et al., 2011 Brazil
Aulosira fertilissima 5 Non-living 4.16 Singh et al., 2007 India
Chlamydomonas reinhardtii 5.5 Cell wall 0.4 Macfie and Welbourn, 2000 Canada
Chlamydomonas reinhardtii 5.5 Without cell wall 0.63 Macfie and Welbourn, 2000 Canada
Chlorella miniata 6 Non-living 20.37 Lau et al., 1999 Hong Kong
Chlorella miniata 7.4 Live 1.37 Wong et al., 2000 Hong Kong
Chlorella spp. Live 122 Doshi et al., 2006 India
Chlorella spp. Non-living 183 Doshi et al., 2008 India
Chlorella vulgaris 4.5 58.4 Aksu and Dönmez, 2006 Turkey
Chlorella vulgaris 4.5 Free 59.29 Mehta and Gaur, 2001 India
Chlorella vulgaris 4.5 Immobilized 111.41 Mehta and Gaur, 2001 India
Chlorella vulgaris 4.7 Non-living 24.06 Klimmek et al., 2001 Germany
Chlorella vulgaris 5 Live 15.4 Al-Rub et al., 2004 U.A.E
Chlorella vulgaris 5 Non-living 15.6 Al-Rub et al., 2004 U.A.E
Chlorella vulgaris 5 Immobilized 28.6 Al-Rub et al., 2004 U.A.E
Chlorella vulgaris 5 Non-living 42.3 Dönmez et al., 1999 Turkey
Chlorella vulgaris 6 Non-living 12.06 Lau et al., 1999 Hong Kong
Chlorella vulgaris 7.4 Live 0.64 Wong et al., 2000 Hong Kong
Chlorella vulgaris 5.0–5.5 Non-living 29.29 Ferreira et al., 2011 Brazil
Hydrodictyon reticulatum 5 Non-living 13.86 Singh et al., 2007 India
Phormidium spp. 5 Non-living 5.7 Wang et al., 1998 USA
Pithophora odeogonia 5 Non-living 11.81 Singh et al., 2007 India
Scenedesmus obliquus 5 Non-living 18.7 Dönmez et al., 1999 Turkey
Spirogyra insignis 6 Non-living 17.5 Romera et al., 2007 Spain
Spirogyra neglecta 5 Non-living 26.3 Singh et al., 2007 India
Spirulina Live 1378 Doshi et al., 2007 India
Spirulina spp. 7.5 Non-living 0.19 Chojnacka et al., 2004 Poland
Spirulina spp. Non-living 515 Doshi et al., 2007 India
Spirulinasp.(HD-104) Live 1108 Doshi et al., 2008 India
Synechocystis spp. 5 Non-living 15.8 Dönmez et al., 1999 Turkey
Pb Pb2 þ Anabaena flosaquae 70 Arunakumara et al., 2008 China
Arthrospira (Spirulina) platensis 5–5.5 Non-living 102.56 Ferreira et al., 2011 Brazil
Aulosira fertilissima 5 Non-living 31.12 Singh et al., 2007 India
Calothrix parietina TISTR 8093 7 Non-living 45 Inthorna et al., 2002 Thailand
Chlamydomonas reinhardtii 5 Non-living 96.3 Tüzün et al., 2005 Turkey
Chlamydomonas reinhardtii 6 Ca-alginate immobilized 230.5 Bayramoğlu et al., 2006 Turkey
Chlamydomonas reinhardtii 6 Ca-alginate immobilized 380.7 Bayramoğlu et al., 2006 Turkey
Chlorella vulgaris 4 Non-living 97.38 Klimmek et al., 2001 Germany
Chlorella vulgaris 4 Non-living 90 Aksu and Kutsal, 1990 Turkey
Chlorella vulgaris 6 Non-living 17.13 Sandau et al., 1996a Germany
Chlorella vulgaris 5–5.5 Non-living 131.36 Ferreira et al., 2011 Brazil
Chlorella vulgaris BCC 15 7 Non-living 127 Inthorna et al., 2002 Thailand
Chlorella vulgaris CCAP211/11B 7 Non-living 39 Inthorna et al., 2002 Thailand
Cyclotella cryptica 6 Non-living 36.68 Schmitt et al., 2001 Germany
Hydrodictyon reticulatum 5 Non-living 24 Singh et al., 2007 India
Microcystis novacekii 5 Non-living 80 Ribeiro et al., 2010 Brazil
Oscillatoria laete-virens 5 Live 21.6 Miranda et al., 2012 India
Phaeodactylum tricornutum 6 Non-living 1.49 Schmitt et al., 2001 Germany
Phormidium spp. 5 Non-living 13.6 Wang et al., 1998 USA
Pithophora odeogonia 5 Non-living 71.13 Singh et al., 2007 India
Porphyridium purpureum 6 Non-living 0.32 Schmitt et al., 2001 Germany
Pseudochlorococcum typicum 7 Live 4.49 Shanab et al., 2012 Egypt
Scenedesmus acutus IFRPD 1020 7 Non-living 90 Inthorna et al., 2002 Thailand
Scenedesmus subspicatus 6 Non-living 38.71 Schmitt et al., 2001 Germany
Spirogyra hyalina Non-living 31.25 Kumar and Oommen, 2012 India
Spirogyra insignis 5 Non-living 51.5 Romera et al., 2007 Spain
336 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352

Table 1 (continued )

Metal Speciation Organism pH Type of biomass Metal uptake (mg/g) Reference Country

Spirogyra neglecta 5 Non-living 90.19 Singh et al., 2007 India

Spirogyra spp. 5 Non-living 140.84 Gupta and Rastogi, 2008 India
Spirulina (Arthrospira) platensis 7 Live 188 Arunakumara et al., 2008 China
Spirulina maxima 5.5 Intact biomass  32 Gong et al., 2005 China
Spirulina maxima 5.5 Pretreated biomass  42 Gong et al., 2005 China
Spirulina platensis 6 Non-living 16.97 Sandau et al., 1996a Germany
Stigeoclonium tenue 6.8 Non-living 0.86 Pawlik-Skowrońska, 2003b Poland
Stigeoclonium tenue 8.2 Non-living 0.38 Pawlik-Skowrońska, 2003b
Synechocystis spp. Live 155.63 Tiantian et al., 2011 China
Tolypothrix tenuis TISRT 8063 7 Non-living 31 Inthorna et al., 2002 Thailand
Zn Zn2 þ Arthrospira (Spirulina) platensis 5.0–5.5 Non-living 33.21 Ferreira et al., 2011 Brazil
Aulosira fertilissima 5 Non-living 19.15 Singh et al., 2007 India
Chlorella homosphaera 15.6 Munoz and Guieysse, 2006 Spain
Chlorella spp. 7 Immobilized 28.5 Maznah et al., 2012 Malaysia
Chlorella vulgaris 4 Non-living 24.19 Klimmek et al., 2001 Germany
Chlorella vulgaris 4 Non-living 24.5 Aksu and Kutsal, 1990 Turkey
Chlorella vulgaris 6 Non-living 6.42 Sandau et al., 1996a Germany
Chlorella vulgaris 5.0–5.5 Non-living 43.41 Ferreira et al., 2011 Brazil
Cyclotella cryptica 6 Non-living 242.9 Schmitt et al., 2001 Germany
Desmodesmus pleiomorphus 5 Non-living 360.2 Monteiro et al., 2009 Portugal
Euglena gracilis 7.5 Munoz and Guieysse, 2006 Sweden
Hydrodictyon reticulatum 5 Non-living 3.7 Singh et al., 2007 India
Isochrysis galbana Live 0.3 Sbihi et al., 2012 Morocco
Phaeodactylum tricornutum 6 Non-living 14.52 Schmitt et al., 2001 Germany
Phormidium spp. 5 Non-living 9.4 Wang et al., 1998 USA
Pithophora odeogonia 5 Non-living 8.98 Singh et al., 2007 India
Planothidium lanceolatum 7 Live 118.66 Sbihi et al., 2012 Morocco
Porphyridium purpureum 6 Non-living 2.01 Schmitt et al., 2001 Germany
Scenedesmus obliquus Non-living 22.3 Monteiro et al., 2011c Portugal
Scenedesmus obliquus Non-living 6.67 Omar, 2002 Egypt
Scenedesmus quadricauda Non-living 5.03
Scenedesmus subspicatus 6 Live 72.06 Schmitt et al., 2001 Germany
Spirogyra insignis 6 Non-living 21.1 Romera et al., 2007 Spain
Spirogyra neglecta 5 Non-living 31.51 Singh et al., 2007 India
Spirulina platensis 6 Non-living 7.36 Sandau et al., 1996b Germany
Spirulina spp. 7.5 Non-living 0.17 Chojnacka et al., 2004 Poland
Stigeoclonium tenue 6.8 Non-living 0.88 Pawlik-Skowrońska, 2003b Poland
Stigeoclonium tenue 8.2 Non-living 0.77 Pawlik-Skowrońska, 2003b

used organism was C. vulgaris in various forms (non-living, living, free,
and immobilized), and the range of pH tested includes pH 4–7.
However, proficient copper removal was achieved by live Spirulina
spp. (389 mg g  1; Doshi et al. 2007). Besides, the trivalent forms of
ferrous metal have been studied with various microalgae. Even more,
in case of Hg2 þ , C. vulgaris and C. reinhardtii proved their potentiality,
while the pH range studied was 4–7.5. Immobilized C. reinhardtii
showed remarkable removal capacities (106.6 mg g  1; Bayramoğlu
et al., 2006). The metals nickel and zinc have been studied in their
divalent form with pH ranges of 4.5–7.5 and 5–7.5, respectively. Po-
tential organisms that remediate nickel and zinc, include live Spirulina
spp. (1378 mg g  1; Doshi et al. 2008), and P. lanceolatum
(118.66 mg g  1; Sbihi et al., 2012), respectively. In contrast, Spirulina
and Chlorella spp. have potential Pb2þ removal abilities. In addition,
immobilized cells of C. reinhardtii are also reported to have significant
lead removal (380.7 mg g  1; Bayramoğlu et al., 2006).
As shown in Table 1, most studies on HMs and microalgae focus
on Cu, followed by Cd, Ni, Pb, Zn, Hg and Cr. Based on the tabu-
lation, it could be stated that live as well as immobilized micro-
algae take up higher quantities of metal rather as compared to
dead biomass; however, most reports project the ease of using dry
or dead biomass. Moreover, most researchers report higher metal
removal at pH approximating 5 (Table 1).
5. Metal removal mechanisms adapted by microalgae
As mentioned earlier, the metal accumulative bioprocesses
generally fall into one of the two categories based on the extent of
metabolic dependence (Gadd, 1990). In particular, the mechanisms
by which microorganisms remove metals from solutions include:
(i) extracellular accumulation/precipitation which can be fa-
cilitated by the use of viable microorganisms; (ii) cell-surface
sorption or complexation which occurs with living as well as dead
microorganisms; and (iii) intracellular accumulation that requires
microbial activity (Cossich et al., 2002). Notably, although both
living and dead cells are capable of metal accumulation, the me-
chanisms involved differ. Generally, HM ions are entrapped in the
cellular structure, and, subsequently biosorbed onto the binding
sites present in the cellular structure. This method of uptake is
independent of the biological metabolic cycle and is known as
“biosorption” or “passive uptake” (Malik, 2004). Additionally, HMs
could also pass into the cell, across the cell membrane through the
cell metabolic cycle; this mode of metal uptake is referred to as
“active uptake”. The metal uptake by both active and passive
modes can be termed “bioaccumulation”.
Likewise, the phenomenon of remediation by microalgae could
also be broadly categorized into two categories: (i) bioaccumulation
by living cells, and, (ii) biosorption by non-living, non-growing bio-
mass or biomass products. This first process (comprising bioaccu-
mulative uptake) forms the principle for waste detoxification pro-
cesses (for e.g. biological fluidized beds employing continually
growing biofilms). Here, a continually self-replenishing system can be
left to run continuously for extended periods if the problem of metal
toxicity to the growing cell is overcome by the use of metal-resistant
organisms. On the other hand, few reports suggest that the dead
(heat-killed, dried, acid and/or otherwise chemically treated) cells can
accumulate HM ions to a similar or greater extent than growing or
 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352
resting cells. This resulting adsorption is quite rapid, occurs to a high
extent, and is frequently selective (Aksu, 1998).
While numerous evaluations have been carried out comparing
selective removal of metals by particular microalgae, few reports
particularly compare uptake by live microalgal cells and their
biomass. For example, Matsunaga et al. (1999) screened 191
marine microalgae for bioremediation of polluted seawater and
found the marine green alga Chlorella spp. NKG16014 had the
highest Cd-removal capacity (48.7%). In this species, 67% of the
removed Cd was accumulated intracellularly and 25% of the re-
moved Cd was adsorbed on algal cell surfaces. They claimed a
faster removal of Cd by dried cells rather than by living cells.
Nevertheless, Pérez-Rama et al. (2002) reported that the growing
cultures of marine microalgae indicate that intracellular Cd levels
are often higher than the biosorbed ones. A cumulative account of
metal remediation by live, dead, immobilized and chemically
modified microalgae has been provided in Table 1.
5.1. Live microalgae
Algae possess the ability to take up toxic HMs from the
environment; resulting in higher concentrations than those in the
surrounding water (Megharaj et al., 2003; Priyadarshani et al., 2011).
Microalgae, in particular, are known to exhibit a number of HM
uptake processes involving different metabolisms (Ajayan et al.,
2011). Biosorption of HMs is a complex phenomenon, however,
Monteiro et al. (2012) emphasize that accumulation of HMs by
microalgae typically comprises a two-stage process: (i) an initial ra-
pid (passive) removal of metals by the cell, occurring at the cell
surface, and (ii) a much slower one that occurs inside the cell.
The first process (i.e. the passive removal), is non-metabolic,
rapid, and essentially reversible, occurring in both living and non-
living cells; here, HM ions are adsorbed to functional groups
present on the cell surface by electrostatic interactions. However,
these groups differ in their affinity for metal and specificity
binding. This process includes physical adsorption, ion exchange,
chemisorption, coordination, complexation, chelation, micro-
precipitation, entrapment in the structural polysaccharide net-
work, and diffusion through the cell wall and membrane (Mon-
teiro et al., 2012). Generally, when dealing with dispersed cells,
such metal ion adsorption is fast, reversible and not a limiting
factor in bioremoval kinetics (Al-Qunaibit, 2004).
Nevertheless, the second phase (occurring within the cell) is a
metabolism-dependent process, involving transport of metal ions
across the cell membrane barrier and a subsequent accumulation
inside the cell, with posterior binding to intracellular compounds
and/or organelle containment. This latter process of metal uptake
is restricted to living cells only, and, is slow and usually irrever-
sible. Moreover, as most of the HMs are hydrophilic, their trans-
port through the partially lipophilic biological membrane sur-
rounding the cell is mediated by specific proteins (Monteiro et al.,
2011a). Al-Qunaibit (2004) suggests this second phase could be
due to several mechanisms such as covalent bonding, surface
precipitation, redox reactions, crystallization on the cell surface, or
most often, diffusion into the cell interior. According to Monteiro
et al. (2012), when the extracellular concentration of metal ions is
considerably higher than its intracellular concentration, binding
groups on the surface may enable transport of those cations across
the cell membrane into the cytoplasm, where they will eventually
become compartmentalized in distinct subcellular organelles. The
authors describe two mechanisms: i) whereby metal ions compete
for binding to multivalent ion carriers, or, alternatively enter the
cell by active transport after binding to low-molecular-weight
thiols (e.g., cysteine); and ii) that metal ions bound to chelating
proteins (e.g., metallothioneins) may enter the cell by endocytosis.
In an attempt to detail algal cell/metal interactions, Monteiro
337
et al. (2012) stated that the algal resistance mechanism comprises
of the following stages: binding of metal ions at the cell surfaces;
precipitation of insoluble metal complexes thereon; complexation
of metal ions with excreted metabolites that may extracellularly
mask a toxic metal; development of energy-driven efflux pumps
that keep toxic element levels low in the interior of the cell;
change of the oxidation state, so a toxic form of a metal may be
enzymatically (and intracellularly) converted to a less toxic one;
vaporization and elimination via converting a toxic metal into a
volatile chemical species; binding of metal ions to proteins or
polysaccharides in the cytoplasm that may constrain metal toxi-
city; and enzymatic methylation that prevents a toxic element
from reacting with ASH groups inside the cell. However, Mehta
and Gaur (2005) reviewed the most common toxic metal re-
sistance mechanisms in microalgae and claimed that, complexa-
tion and microprecipitation are the most efficient mechanisms,
even though ion exchange dominates.
Reports on utilization of live microalgae for metal remediation
suggest various specific pH values for efficient remediation, but
the suggested pH values generally range from 4 to 9; in addition,
live microalgae are prevalently studied for copper remediation
(Table 1).
5.1.1. The role of the algal cell wall in heavy metal binding
At the interface between the physical environment and cyto-
plasm, is the cell wall, which acts as the first defense line against
toxic HM poisoning; it has been known that the relatively high
metal-binding capacities of microalgae could be attributed to the
intrinsic composition of their cell walls, which contain negatively
charged functional groups. The microalgal cell wall, in particular, is
the first barrier to metal cation uptake, and thereby interaction
with the cell walls or with membranes have been proposed as the
initial process of exposure to HM. The proteins, carbohydrates and
lipids present in these exterior surfaces (i.e. the cell walls and
membranes) could react with metallic species (Crist et al., 1981;
Dönmez et al., 1999; Monteiro et al., 2012). Crist et al. (1981) ex-
clusively elaborate that the cell walls of microalgae constitute
proteins, which in turn are made up of amino acids, that provide
groups (for e.g. –NH2) facilitating metal binding; additionally, the
polysaccharides of the cell wall also provide amino and carboxyl
groups, as well as sulfate. The authors further state that the amino
and carboxyl groups, the imidazole of histidine, and the nitrogen
and oxygen of the peptide bond, facilitate characteristic co-
ordination bonding with metallic ions like Cu2 þ . However, such
bond formation could be accompanied by the displacement of
protons dependent in part on the extent of protonation, as de-
termined by the pH. Metallic ions could also be electrostatically
bonded to unprotonated carboxyl oxygen and sulfate. Similarly,
Monteiro et al. (2012) stated that the microalgal cell wall consisted
mainly of polysaccharides, proteins, and lipids, which offer several
functional groups (e.g., carboxyl, –COOH; hydroxyl, –OH; phos-
phate, –PO3; amino, –NH2; and sulfhydryl, –SH) that confer a net
overall negative charge to the cell surface, and concomitantly a
high binding affinity for metal cations via counterion interactions.
Chojnacka et al. (2005) confirmed this by stating that algal cell
wall components such as peptidoglycan, teichuronic acid, teichoic
acid, polysaccharides and proteins, are polyelectrolytes that carry
charged groups, such as carboxyl, phosphate, hydroxyl or amine.
The presence of anionic and cationic sites gives the algal cell wall
amphoteric properties. The functional groups play vital roles in HM
uptake by live as well as non-living microalgae. Moreover, these
groups are either protonated or deprotonated depending on pH.
5.1.2. The plasma membrane and heavy metal flux
Membrane transport of the metal ions into the cytoplasm of the
cell is a vital process. Particularly, the plasma membrane metal
338 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352
transporters are pivotal in the interaction of algae with their en-
vironment. These membrane transporters represent the first line
of defense during cellular perturbations occurring during HM ex-
posure. Analysis of the algal metal transporter repertoires could
provide insight into a fundamental aspect of algal biology. Ac-
cording to Blaby-Haas and Merchant (2012), Group A transporters
are responsible for moving metal ions into the cytoplasm; they
include include members from the NRAMP (Natural Resistance-
Associated Macrophage Proteins), ZIP (Zrt-, Irt-like Proteins), FTR
(Fe TRansporter) and CTR (Cu TRansporter) families. The assim-
ilative transporters found in the plasma membrane also fall within
this group; however, they particularly increase the intracellular
concentration of metal, when the equilibrium between chelating
sites and metal ions is perturbed due to deficiency. Group A
transporters are also found in the vacuole membrane and have the
same role as assimilative transporters, except that, the source of
metal is an intracellular storage compartment vs. the external
environment. Group B transporters decrease the cytoplasmic
concentration of metal. Within this group are distributive trans-
porters, which provide metal for organelle-localized metal-de-
pendent proteins. When present in the membranes of the secre-
tory pathway, Group B transporters when present in the mem-
branes of the secretory pathway mediate the exocytosis of excess
metal. This group includes members from the CDF (Cation Diffu-
sion Facilitator), P1B-type ATPases, FPN (FerroPortiN) and Ccc1 (Ca
(II)-sensitive Cross-Complementer 1)/ VIT1 (Vacuolar Iron Trans-
porter 1) families (Blaby-Haas and Merchant, 2012).
The molecular mechanisms for metal ion transport have been
recently detailed in Chlamydomonas using functional genomics,
biochemical studies and bioinformatic approaches (Rajamani et al.,
2007), thereby, eleven gene families encoding metal ion trans-
porters were uncovered. The few studies on metal transporters in
Chlamydomonas that are available until now indicate that DMT1
mediates Mn, Fe, Cd and Cu uptake, but not Zn transport. In ad-
dition, the periplasmic metal transporter known as Fea1 or the
H43 protein, transports Fe, but not other metals (Cd, Cu, Co or Mn).
This Fea1 protein is only expressed under stress [i.e. high Cd level,
iron deficiency and high (4 3%) CO2 concentration]. One successful
way to enhance the metal binding capacity of this alga involves
the expression of synthetic genes encoding plasma membrane
anchored metal-binding proteins exposed to the periplasmic space
(Rajamani et al., 2007). Hanikenne et al. (2005) confirmed putative
metal transporters in two unicellular algal models, the green alga
Chlamydomonas reinhardtii and the red alga Cyanidioschizon
merolae.
5.1.3. The ion exchange concept
As mentioned earlier, the cell walls of microalgae consist
mainly of polysaccharides, proteins, and lipids, with several
functional groups [e.g., carboxyl (–COOH), hydroxyl (–OH), phos-
phate (–PO3), diphosphorus trioxide (–P2O3), amino (–NH2), sulf-
hydryl (–SH), amide, primary amine-group, aromatic-compound,
halide-group and aliphatic alkyl-group], that confer an overall
negative charge to the cell surface, and concomitantly a high
binding affinity for metal cations via counter ion interactions,
thereby endorsing metal exchange via ion exchange mechanisms
(Dönmez et al., 1999; Mehta et al., 2002; Monteiro et al., 2012; He
and Chen, 2014). González-Dávila (1995) supposed that during the
interaction of metal ions with proteins on biological surfaces,
these metal ions become coordinated to the complex forming
groups on the algal surface. However, in natural seawater, most of
these sites are bonded with protons at low pH or with calcium,
magnesium and sodium at high pH. Now, when metal ions such as
Cu2 þ , Mn2 þ , Zn2 þ , Ni2 þ , Cd2 þ , Fe3 þ and Pb2 þ are present, the
previously bound protons and metals are released and the later
metal ions (Cu2 þ , Mn2 þ , Zn2 þ , Ni2 þ , Cd2 þ , Fe3 þ , and Pb2 þ ) are
sorbed on the alga. But, in the presence of anionic complexing
ligands (exudates and natural organic materials), the adsorption
characteristics of metals can change significantly; for e.g. some
metal–ligand complexes are strongly bound at surfaces while
others form non-adsorbing complexes in solution, where ligands
compete with the surface for coordination of metal ions. Apart
from occupying surface sites and probably blocking the adjacent
sites, the adsorption of ligands onto the surface, alters the surface
charge. Besides, metal ions could either directly form complexes
with the surface, or could attach via previously bound ligands, or
may form complexes in solution, and can cause desorption of
metals from the surface (González-Dávila, 1995). Crist et al. (1990,
1992) supportingly state that in natural waters, the sorption pro-
cess (adsorption plus assimilation) is an ion exchange process with
the same amount of metal assimilated as the sum of Na þ , Ca2 þ ,
Mg2 þ and H þ released.
Several studies on electron microscopy, including X-ray energy-
dispersive analysis showed that most sites for metal sorption are
located on the cell surfaces, and adsorption via ion exchange ap-
pears to be the main contribution (i.e. up to 90%) to the total
amount of metal ion uptake by microalgal cells (Chojnacka et al.,
2005; Tiantian et al., 2011; Monteiro et al., 2012; Maznah et al.,
2012).
5.1.4. Physical adsorption
Physical adsorption is a reversible process, independent of
metabolism, and has several advantages. It is demarcated as a
process where the metal ion in solution binds to polyelectrolytes
present in microbial cell walls through electrostatic interactions:
Van der Waals forces, covalent bonding, redox interaction, and
biomineralization, to achieve electroneutrality (Perpetuo et al.,
2011). However, the physical adsorption encompassing a process,
wherein the metal ions are attracted by the negative potential of
the cell wall, is a pH dependent process. With increasing pH,
generally numerous sites (acetamide chitin, structural poly-
saccharides, phosphate and amino groups of nucleic acids, amino
and carboxyl groups of proteins and hydroxyl groups of poly-
saccharides) are replaced by negative charges that increase the
attraction of metallic cations and their adsorption to the cell sur-
face. Kuyucak and Volesky (1988) report uranium, cadmium, zinc,
copper and cobalt sorption by dead biomasses of algae occurring
through electrostatic interactions between the metal ions in so-
lutions, and, the cell walls of the cell. Similarly, in the alga C.
vulgaris, electrostatic interactions occur during copper biosorption
(Aksu et al., 1992); here, the physical adsorption occurs with the
aid of van der Waals forces.
5.1.5. Complexation
Any combination of cations with molecules or anions contain-
ing free electron pairs (bases) is termed coordination or complex
formation; this could be either electrostatic (i.e. coulombic) or
covalent in character. Davis et al. (2003) explained that in case of
complex formation, the HM cation which is bound, is often the
central atom, and is distinguished from the anions or molecules
with which it forms a coordination compound, the ligand(s). This
complexation is, however, divided into two types: (i) inner-sphere
complexation referring to the interacting ligand which is close or
adjacent to the metal cation; and (ii) outer-sphere complexation,
referring to ions of opposite charge that are attracted and ap-
proach each other within a critical distance and effectively form
what is termed an ion pair. In outer-sphere complexes, the metal
ion or the ligand or both generally retain their coordinated water
when the complex is formed, i.e. the metal ion and the ligand are
most often separated by one or more water molecules. However, in
the microalgal context, Perpetuo et al. (2011) visualized a “extra-
cellular complexation” or “coordination” occurring due to
 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352
electrostatic attraction between a metallic ion chelating agent and
a polymer that can be excreted by a microorganism, that is viable
or not (this could involve biosurfactants, polysaccharides, proteins
and nucleic acids). The authors concluded that these chelating
agents contain pairs of electrons that present electrostatic attrac-
tion, and when they cling to the metallic ions, there is no electron
transfer. However, the final structure has the electric charge of the
sum of individual charges of the participants of the complex. Aksu
et al. (1992) also noted that apart from adsorption, copper bio-
sorption in C. vulgaris takes place through the formation of co-
ordination bonds between metals and amino and carboxyl groups
of cell wall polysaccharides. Perpetuo et al. (2011) mention the
role of microbial organic acids (e.g., citric, oxalic, gluonic, fumaric,
lactic and malic acids), which may chelate toxic metals, thereby
resulting in the formation of metallo-organic molecules; these
organic acids help in the solubilization and leaching of metal
compounds from their surfaces. Metals may also be biosorbed or
complexed by carboxyl groups found in microbial polysaccharides
and other polymers. According to Gadd (1990) both ionic and
covalent binding are involved in biosorption, wherein proteins and
polysaccharides (present in the algal cell walls and extracellular
materials) have an important role.
5.1.6. Precipitation
At low pH the active sites of the cell wall are associated with
protons, restricting the approach of metal cations, and thus resulting
in a repulsive force. Therefore, as the pH increases, the number of
sites (acetamide chitin, structural polysaccharides, phosphate and
amino groups of nucleic acids, amino and carboxyl groups of proteins
and hydroxyl groups of polysaccharides) that are replaced by nega-
tive charges increases. This leads to attraction of the metallic cations
and their subsequent adsorption on the cell surface. As the solubility
of metallic ions decreases and subsequently their bioavailability is
reduced, precipitation occurs (Perpetuo et al., 2011).
Precipitation could be either dependent or independent of the
cellular metabolism: (i) In case of metabolism dependent pre-
cipitation, the metal removal is generally associated with an active
defense system of the microorganisms; here on exposure to a toxic
metal, the microbe produces certain compounds that favor the
precipitation process; and (ii) The precipitation not dependent on
the cellular metabolism may be a consequence of the chemical
interaction between the metal and the cell surface, wherein the
various biosorption mechanisms mentioned above can take place
simultaneously (Ahalya et al., 2003). Ballan-Dufrancais et al. (1991)
reported Cd2 þ precipitation in the vacuole of T. suecica.
5.1.7. Metallothioneins
Metallothioneins were first characterized by Vallee and co-
workers in the late 1950s in studies that were extended thereafter
(Stillman, 1995). Metallothionein proteins, products of mRNA
translation, are characterized as low molecular weight (6–7 kDa),
cysteine-rich, metal-binding proteins. The induction of me-
tallothioneins (MTs) as well as metallothionein-like proteins
(MTLPs), in aquatic organisms, has been recognized as a potential
biomarker of HM toxicity and bioaccumulation (Won et al., 2008).
In contrast to the mechanism used by eukaryotes, prokaryotic cells
employ ATP consuming efflux of HMs or enzymatic change of
speciation to achieve detoxification of metals. Reports also em-
phasize the role of (i) enzymatically synthesized short-chain
polypeptides named phytochelatins (class III metallothioneins),
and (ii) gene-encoded proteins; class II metallothioneins (class III
MT or MtIII) in case of algae–metal interactions (Perales-Vela et al.,
2006). These metallothioneins are structurally diverse, low-mo-
lecular-weight, cysteine-rich, polypeptides that complex “soft”
metal ions in thiol clusters. Several researchers confirm the pre-
sence and synthesis of class III MT in algae (Robinson, 1989a;
339
Stokes et al., 1977; Gekeler et al., 1988; Gaur and Rai, 2001).
Likewise, in vitro studies show that longchain MtIII can bind HMs
in a stable complex (Mehra et al., 1995; Perales-Vela et al., 2006).
According to Robinson (1989a), these molecules chelate toxic
trace metals, such as Cd, thereby reducing the concentration of
cytotoxic, free-metal ions; besides, some MT's are believed to be
involved in zinc and copper homeostasis. In fact, HMs such as
As3 þ , Ni2 þ , Cd2 þ , Ag þ , Bi3 þ , Pb2 þ , Zn2 þ , Cu2 þ , Hg2 þ and Au2 þ ,
induce class III MT biosynthesis, both in vivo and in vitro, in sev-
eral microalgae for e.g. S. subspicatus, S. bacillaris, S. tenue, and
Thalassiosira weissflogii (Robinson, 1989a, b; Ahner and Morel,
1995; Knauer et al., 1997, 1998; Pawlik-Skowrońska et al., 2003a, b,
2004; Perales-Vela et al., 2006).
5.1.7.1. Phytochelatins (PCs). While studying metal exposure to al-
gae, one should definitely consider that the mechanisms may in-
volve sequestering toxic metals by HM-binding Cys-rich proteins,
such as class II metallothioneins and non-translationally synthe-
sized polypeptides sometimes described as class III metallothio-
neins (phytochelatins-PCs or MtIII). Phytochelatins (PCs) are small
metal-binding peptides with molecular weights ranging from 2 to
10 kDa; they are synthesized by the constitutive enzyme named
phytochelatin synthase (Torres et al., 2008). Their synthesis from
glutathione, homo-glutathione, hydroxymethyl-glutathione or
g-glutamylcysteine (Hayashi et al., 1991) is catalyzed by a trans-
peptidase named phytochelatin synthase (the constitutive en-
zyme) which requires post-translational activation by HMs (Grill
et al., 1989; de Knecht et al., 1995; Klapheck et al., 1995; Chen et al.,
1997). All higher plants and most algae possess the capacity to
synthesize PCs (Gekeler et al., 1989; Ahner et al., 1995). Cobbett
and Goldsbrough (2002) carried out kinetic studies indicating that
the synthesis of PCs occurs within minutes, independent of de
novo protein synthesis. A wide range of metals and metalloids
such as Cd, Ag, Pb, Cu, Hg, Zn, Sn, Au, and As, can assist activation
of phytochelatin synthase (PCS) both in vivo and in vitro (Grill
et al., 1987; Chen et al., 1997; Torres et al., 2008). For e.g. Cd and Pb
induces PCn synthesis in P. tricornutum (Scarano and Morelli,
2002); further, both these elements are capable to form stable
complexes with PCn with n¼ 3–6. Pawlik-Skowrońska (2003a)
stated that the periphytic green alga S. tenue produced high
amounts of novel phytochelatin-related peptides on adaption to
high zinc concentrations. Moreover, Pawlik-Skowrońska (2001)
earlier reported PC production in freshwater algae Stigeoclonium in
response to HMs contained in mining water. In the same way,
increased synthesis of PCs has also been reported for Trebouxia
erici in response to excess Cd or Cu, but, in this case, Cd was a more
potent activator of PCs synthesis, and was even able to induce the
synthesis of PCs with longer (more stable) chains, up to PC5
(Bačkor et al., 2007). Wei et al. (2003) also reported that the ad-
dition of Cd, Cu and Zn to microalgal assemblages resulted in a by
3.6, 1.8, and 3.2 fold increase in PC production respectively, while
metal combinations such as “Cd þZn”, and “Cu þZn” caused a si-
milar 2–3 fold increase.
5.1.8. Sequestration and compartmentalization in the vacuole
Garnham et al. (1992) investigated removal of three metals (Zn,
Co, and Mn) by C. salina, and observed higher concentrations of
metal in the vacuole, rather than the cytosol; this phenomen un-
folds: (i) a possible mechanism of “regulation of the free metal ion
concentration” in the cytosol, and, (ii) a probable mechanism of
detoxification (Monteiro et al., 2012). According to Perales-Vela
et al. (2006), the metal-MtIII complex ends up in the vacuole of the
cell; based on microscopical and X-ray analyses, they indicated the
transport of metals complexed with MtIII into the vacuole of algae.
Heuillet et al. (1986) observed electron dense materials inside
the vacuoles of the microalga Dunaliella bioculata; this material
340 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352
contained cadmium and sulfur (in ratios between 2 and 2.4).
Perales-Vela et al. (2006) cited similar reports comprising the
green alga T. suecica exposed to Cd2 þ (Ballan-Dufrancais et al.,
1991), and, the diatom Skeletonema costatum which accumulated
Cd2 þ and Cu2 þ in the vacuole of cells having a sulfur/metal ratio
of 1.5 (Nassiri et al., 1997). In yet another study on three fresh
water microalgal isolates [Phormidium ambiguum (Cyanobacter-
ium), Pseudochlorococcum typicum and S. quadricauda var quad-
rispina (both Chlorophyta)], tested for tolerance and removal of
mercury (Hg2 þ ), lead (Pb2 þ ) and cadmium (Cd2 þ ) in aqueous
solutions, Shanab et al. (2012) reported that dark spherical elec-
tron dense bodies were accumulated in the vacuoles of algal cells
exposed to Pb. However, Shanab et al. (2012) assert that the me-
chanism of metal deposition inside the vacuoles or cytoplasm also
contributes towards HM tolerance. Here the cytoplasmic metal
concentrations are minimized by binding or complexing the metal
ions with phytochelatin or in the form of metallo-sulfur, metallo-
iron or metallo-phosphate complexes in the cytosol, and are car-
ried into the vacuoles where the acidic pH displaces the metal,
allowing the peptide to return to the cytosol. In the vacuole, the
metal would thereby be sequestered by organic acids that are
usually present in high concentration here. Overall the aforesaid
mechanism could be considered as a cellular protection mechan-
ism, or, a detoxification mechanism (Shanab et al., 2012).
5.1.9. Polyphosphate bodies in algae
Detoxification of metals in a microalgae could be achieved ei-
ther by: (i) binding to specific intracellular compounds and/or
transport to specific cellular compartments such as vacuoles or
polyphosphate bodies, or (ii) efflux of metals back into solution by
active transport (Monteiro et al., 2012); moreover, association of
divalent cations with polyphosphate and corresponding trafficking
into vacuoles is known to occur.
Dwivedi (2012) opined that initially metal ions are adsorbed to
the cell surface very quickly just in a few seconds or minutes
(physical adsorption), and thereafter these ions are transported
slowly into the cytoplasm in a process called chemisorption; they
precisely ascertained the function of polyphosphate bodies in
metal uptake. These polyphosphate bodies are also termed acid-
ocalcisomes or electron-dense vacuoles (Ruiz et al., 2001). The
formation of polyphosphate bodies facilitates metal accumulation
(which leads to potentially high metal toxicity) and storage (which
leads to lower toxicity when present in an inert form) (Wang and
Dei, 2006). As high polyphosphate content appears to be a dis-
tinctive microalgal trait, the polyphosphate bodies can be a means
of HM sequestration. Acknowledging the fact that polyphosphate
bodies enable storage of certain nutrients in microalgae, Dwivedi
(2012) proposed that these polyphosphate bodies could also se-
quester metals such as Ti, Pb, Mg, Zn, Cd, Sr, Co, Hg, Ni and Cu,
thereby performing two different functions viz. providing a “sto-
rage pool” for metals and enabling a “detoxification mechanism”.
For e.g. the alga Scenedesmus obliquus accumulated increased
amounts of Cd and Zn with higher phosphorus concentrations,
whereas selenium (Se) accumulation was found to be inhibited.
5.1.10. Sequestration to the chloroplast and mitochondria
Shanab et al. (2012) witnessed excessive accumulation of starch
around the pyrenoids (organelles within the chloroplasts of algae)
of three microalgal species exposed to mercury (Hg2 þ ), lead
(Pb2 þ ) and cadmium (Cd2 þ ) in aqueous solutions. This clearly
accounted for the possibility that HMs could find their way to
other organelles in a microalgal cell. In an earlier study, Perales-
Vela et al. (2006) provided an evaluation of the role of chloroplast
and mitochondria of microalgae in response to HMs. They cited
Mendoza-Cózatl et al. (2004) who studied Euglena gracilis with a
high tolerance to Cd2 þ , and high Cd2 þ accumulating capacity;
however, they mention that this organism lacked specialized re-
servoir organelles such as plant-like vacuoles. Similarly, the pre-
sence of Cd2 þ inside the chloroplast has also been reported for
Chlamydomonas reinhardtii (Nagel et al., 1996). Soldo et al. (2005)
found Oocystis nephrocytioides exposed to Cu2 þ to accumulate
high concentrations of Cu2 þ in the thylakoids and pyrenoids. They
conclude that localization of Cu2 þ suggests interaction of Cu2 þ
with ligands localized in the chloroplast. Alternatively, Cu2 þ might
have been transported from the cytosol to the chloroplast as a
Cu2 þ –ligand complex (Perales-Vela et al., 2006).
Mendoza-Cózatl and Moreno-Sánchez (2005) reported E. gra-
cilis to accumulate Cd2 þ inside the chloroplast. Avilés et al. (2003)
stated that Hg2 þ pretreated heterotrophic cells of E. gracilis ex-
posed to Cd2 þ accumulated some of the metal in the mitochon-
dria. Mendoza-Cózatl et al. (2004) explained that the presence of
MtIII and Cd2 þ in Euglena chloroplast and mitochondria, may be
due to either of the following processes: (i) MtIII are synthesized
in the cytosol where they sequester Cd2 þ ; the Cd–MtIII complexes
are subsequently transported into the chloroplast and mitochon-
dria, (ii) MtIII are synthesized inside the organelle where they bind
to Cd2 þ , which are transported as free ions and then form HMW
complexes, or (iii) both processes co-exist and MtIII are synthe-
sized in the three cellular compartments (Perales-Vela et al.,
2006).
5.1.11. Other mechanisms of heavy metal remediation
Along with the MtIII mechanism, Perales-Vela et al. (2006) also
takes into account several other mechanisms, i.e. they quote ex-
clusion mechanisms as an alternative that alga possess in order to
be in equilibrium with HMs in their environment. Among other
HM encountering mechanisms, Pistocchi et al. (2000) noted that a
few diatom and dinoflagellate groups capable of producing MtIII
and exocellular polysaccharides (macromolecules which provide
an effective adsorbing barrier against HMs) were more resistant to
HM stress. One report highlights sexual reproduction as a response
to severe HM shock in Scenedesmus spp. (Abd-El-Monem et al.,
1998), while another study showed that Scenedesmus incrassatulus
can respond to metal stress by expressing phenotypic plasticity
that may allow these cells to survive in a hostile environment such
as in the presence of Cd2 þ and Cu2 þ (Peña-Castro et al., 2004).
Perales-Vela et al. (2006) provided an example of the green alga,
Chlamydomonas reinhardtii, wherein Hg2 þ was not chelated by
MtIII, but by glutathione. This indicated that glutathione played
numerous roles, not only as the cGlu–Cys donor for MtIII, but also
as a detoxifying molecule itself, employing direct chelation (Howe
and Merchant, 1992). According to Satoh et al. (1999), unlike other
members of the genus Tetraselmis that produce MtIII producers,
Tetraselmis tetrathele intriguingly did not produce MtIII despite the
glutathione-pool depletion under Hg2 þ stress. In their study, they
reported that Tetraselmis tetrathele used a novel tripeptide, Arg-
Arg-Glu possessing a Hg2 þ -scavenging capacity that was re-
sponsible for the detoxification mechanism.
In addition, Perales-Vela et al. (2006) emphasized that few al-
gae respond to HMs by the production of proline (Pro).
5.2. Biosorption by biomass
Non-viable microalgae have been known for their ability to
eliminate HM ions in a short time by biosorption in an un-
complicated manner, and they pose no toxicity problems. Their
sorption capacity is only slightly less than their live counterparts.
In addition, their low price makes them economical (Sandau et al.,
1996b). The mechanisms associated with metal biosorption by
microorganisms are complex, depending on the metal ionand the
biological system, and include extracellular and intracellular metal
binding (Aksu, 1998). The mechanism of metal ion binding, in
 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352 341

particular, depends on the species of metal ion, the algal species,  ability for multi-metal removal as well as cleansing of mixed
chemical composition of the metal ion solution, prevailing en- wastes comprising HMs
vironment, and several other factors. A great deal of attention has  high affinity and high efficiency
been focused on the use of non-living algal biomass as a potential  fewer supplementary expensive reagents are required (these
industrial tool for the extraction of toxic metal ions from waste- reagents usually cause disposal and space problems)
waters and mining effluents. Aksu (1998) highlighted that differ-  does not require growth media and nutrients
ences in algal taxonomy, variation in cell structure, the type of  active over a wide range of physicochemical conditions in-
metal ion and its manner of binding, as well as, the chemical cluding temperature, pH, and presence of other ions such as
composition of the metal ion solution, could influence the metal calcium and magnesium
binding.  moderate capital investment and low operational costs
There are several reports available on various perspectives of  involves the use of naturally abundant renewable bioresources
metal sorption by microalgae; for e.g. Wilke et al. (2006), in si- which are produced economically
multaneous sorption processes, studied 30 algae, and reported the  ecofriendly (it not only reduces the level of risk of hazardous
following order of selective sorption: Pb 4Ni 4Cd 4Zn. Tüzün waste, but also evades the use of harmful chemical technologies
et al. (2005) described algal affinity and selectivity for metal ions, which could cause secondary pollution)
wherein Chlamydomonas reinhardtii was reported to have max-  metal removal system is not subjected to metal toxicity lim-
imum Hg(II), Cd(II), and Pb(II) ions biosorption capacities itations, and
(72.2 70.67, 42.6 70.54 and 96.3 70.86 mg g  1 dry biomass, re-  significant recovery of HMs from the biomass is feasible (i.e.
spectively). Here, the affinity order for algal biomass was Pb(II) desorption of the HM and reuse of algal biomass).
4Hg(II) 4Cd(II); where the maximum biosorption capacity of
microalgae for Hg(II), Cd(II) and Pb(II) ions were 72.2 70.67,
Sorption of HMs using dead biomass of microalgae has gained
42.6 70.54 and 96.3 70.86 mg g  1 dry biomass.
much popularity due to the ease of handling. Various pH values (2-
Several metallic anion complexes and oxoanions appear to bind
9) have been studied for metal removal by non-living microalgae
electrostatically to algal cells, although covalent bonding is cer-
(Table 1). Most researchers have focused on cadmium, copper and
tainly possible. Certain reports suggest algal biosorption of metal
lead removal by dead biomass. In addition, various mechanisms of
ions such as aluminum (III), copper (II), lead (II), and cobalt (II) to
uptake have been proposed; a few of the mechanisms detailed
occur via an ion-exchange process with metal cations competing
above for living microalgae, also hold true for HM uptake by bio-
with protons for negatively charged binding sites on the cell wall.
mass (for e.g. the significance of functional groups).
The binding sites could be carboxyl groups, or sulfates associated
with polysaccharides and proteins. Likewise, Gardea-Torresdey
5.2.1. Significance of functional groups in heavy metal uptake
et al. (1990) indicated that algal biosorption of metal ions appears
Gao et al. (2010) in their study on glow discharge electrolysis
to occur via an ion-exchange process with metal cations compet-
plasma emphasized the importance of functional groups (e.g. hydro-
ing with protons for negatively charged binding sites on the cell
xyl-, thio-, iminodiacetic- and amine groups) in absorption. They sta-
wall. Michalak and Chojnacka (2010), affirmed that Ca (II), Na (I), K
ted that nitrogen-containing functional groups are among the most
(I), and Mg (II) ions were released from the biomass after bio-
effective functionalities in the adsorption or removal of HM ions.
sorption of Cu (II), Mn (II), Zn (II), and Co(II) ions, indicating that
Similarly, for any commercially viable metal recovery or re-
ion exchange was a key mechanism in the biosorption of metal
mediation technology, it is essential that the functional groups of
ions by Vaucheria spp. biomass.
biomaterials responsible for metal uptake are identified and their
Contrarily, Lau et al. (1999) conceptualized the process of bio-
contribution to overall metal binding capacity are quantified
sorption as the sorption or complexation of dissolved metals by
(Rayson and Williams, 2011). In biological systems, hard ions form
dead or metabolically inactive biomass where the biosorption
stable bonds with OH  , HPO42  , CO32 þ , R–COO  , and QCQO
ability of the biomass relates to the anionic nature of their cell
(all of these groups include oxygen atoms); while the soft ions
wall. The authors reported that anionic groups such as carboxyl,
form very strong bonds with CN  , R–S  , -SH  , -NH2  , and imi-
phosphate, pyruvate, amide, as well as, extracellular poly-
dazole (i.e. groups containing N and S atoms) (Baldrian and Gabriel
saccharides, aminophosphates, and lipoproteins are able to bind
2003). This implies that hard ions (smaller in size) such as Fe, Zn,
metal ions by electrostatic forces. In addition, Aksu (1998) also
Mn would prefer to bind with OH  , HPO42  , CO32 þ , R–COO  , and
reported that biosorption of metal ions by algal biomass arose
QCQO, while soft ions (with a larger diameter) such as Cu, Pb
from the coordination of the ions to different functional groups on
and Cd would bind to CN  , R–S  , –SH  , –NH2  , and imidazole.
algal cells including amino, carboxylic, sulfhydryl, phosphate, thiol
Although researchers have attempted to determine the binding
groups and sulfate groups, provided by polysaccharide building
mechanism of metal ions to algal surfaces, it is necessary to es-
blocks of the algal cell walls; amino groups and the nitrogen and
tablish which chemical groups (especially present on the algal cell
oxygen of the peptide bond (provided by proteins); or other
wall), are responsible for binding to different metal ions. Reports
groups such as carboxyl oxygen and sulfate. Aksu (1998) stated
suggest that the biosorption, or binding, of metal ions by algal
that biosorption of HM ions in algae, was better than precipitation
biomass arises from the coordination of the ions to different
in terms of ability to adjust to changes in pH and HM concentra-
functional groups in or on the algal cell. These coordinating groups
tions, and, it was better than ion exchange and reverse osmosis in
(provided by proteins, lipids, and carbohydrates) include amino,
terms of sensitivity to the presence of suspended solids, organics,
thioether, sulfhydryl, carboxyl, carbonyl, imidazole, phosphate,
and the presence of other HMs; they further mention that only ion
phenolic, hydroxyl, and amide moieties (Gardea-Torresdey et al.,
exchange could compete with biosorption in terms of residual HM
1990). In the algal context, Crist et al. (1988) studied the interac-
concentrations.
tions of metals and protons with Vaucheria spp., Spirogyra spp.,
Compared to traditional HM removal technologies, biosorption
and Oedogonium spp., and indicated that metals adsorb to algal
by non-living microalgae have the following advantages:
surfaces by electrostatic attraction to negative sites such as car-
boxylated anions of polygalacturonic acid.
 capacity to treat large volumes rapidly Carboxyl groups are known for their high selectivity for protons
 high selectivity and specificity for particular HMs (Kratochvil and Volesky, 1998). Gardea-Torresdey et al. (1990)
342 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352

studied the carboxyl groups of biomass of five different algal
species, and reported that carboxyl groups on algal cells are re-
sponsible for a great portion of copper (II) and aluminum (III)
binding, and that they play an inhibitory role in gold (III) binding.
Likewise, Kratochvil and Volesky (1998) suggested that metal
uptake typically depends on pH due to the weakly acidic carboxyl
groups R–COOH of algal and fungal cell wall constituents as the
probable sites for ion exchange. Moreover, esterifications of car-
boxyl groups in the biomass of the fresh water algae C. vulgaris
(Cho et al., 1994), Chlorella pyrenoidosa and Cyanidium caldarium
(Gardea-Torresdey et al., 1990) support this notion. Esterification
of carboxyl groups present in the cell walls generally result in a
decrease in metal binding capacity; thereby indicating their role in
metal binding.
In an effort to generalize the main functional groups involved in
metal binding in microalgae, Tiantian et al. (2011) tabulated and
confirmed maximum involvement of –COOH, followed by –NH2, –
OH, –SO3H, and also –P2O3 groups. Rajamani et al. (2007) elaborated
the role of carboxylate, sulfate groups involved in pH-dependent
binding of cadmium, and specifically mention about the role of sul-
fate present in the glycoproteins of the cell wall in metal binding.
González et al. (2011) also confirmed that functional groups con-
taining O-, N-, S-, or P-, participate directly in the binding of certain
metals; they also specified the carboxyl-, hydroxyl-, sulfate and
amino groups in the algal cell wall polysaccharides, act as binding
sites for metals.
Reports also authenticate the contribution of other functional
groups present in the cells and the cell walls of algae and fungi,
such as the strongly acidic sulfate groups R–OSO3  . Lefebvre et al.
(2007) described the ability of cyanobacterial strains (Limnothrix
planctonica, Synechococcus leopoldiensis and Phormidium limnetica)
to convert Hg2 þ into elemental mercury Hg° and meta-cinnabar
(β-HgS) under controlled pH and aerated conditions. They attrib-
uted this to the interaction with metal binding sulfhydryl protein
as an intermediate step in the metal sulfide synthesis. Further-
more, Lengke et al. (2006) investigated gold bioaccumulation by
the cyanobacterium Plectonema boryanum from gold (III)-chloride
solutions and confirmed the role of thiol compounds in the re-
duction mechanism of gold (III) to metallic gold by this organism.
Essa and Mostafa (2013) studied metal bioprecipitation and tested
the efficiency of three cyanobacterial isolates (S. platensis, Nostoc
muscorum, and Anabaena oryzae) to precipitate some HMs (Hg2 þ ,
Cd2 þ , Cu2 þ and Pb2 þ ). FT-IR studies showed the existence of –OH
groups in the metal precipitate produced by algal isolates, while –
NH groups were identified only in the metal precipitates produced
by Nostoc muscorum and A. oryzae. That particular study of Essa
and Mostafa (2013) highlighted a novel approach for HM bior-
emediation through the transformation of these metals into ni-
trogen complexes and/or hydroxide complexes via using the cul-
ture biogas produced by some cyanobacterial species. In general,
reports indisputably suggest the involvement of –COOH groups in
the binding of metals such as Pb, Cu, Zn, Ni, Cd, Cr, Co, Fe, Au and U
(Ting et al., 1995; Baldrian and Gabriel, 2003; Davis et al., 2003;
Chojnacka et al., 2005; Uchimiya et al., 2012; Flouty and Este-
phane, 2012); whereas, the –OH group could bind with Pb, Cr, Cd,
Cu, (Sheng et al., 2004; Chojnacka et al., 2005; Flouty and Este-
phane, 2012; Greene et al., 1986; Flouty and Estephane, 2012).
The incidence of different binding groups on biomass surface in-
volved in metal ion complexation has been well described by
Nurchi and Villaescusa (2011). These authors proposed the higher
to lower affinity ranking of the groups as follows: carbox-
ylate4aromatic ring4 hydroxyl4amine 4phosphate4carbonyl
4thiol 4amide 4sulfonate. A schematic representation of a few
mechanisms, witnessed in HM exposed microalgae, are shown in
Fig. 1; however, it is rather difficult to clearly decipher and ela-
borate mechanisms and technologies separately for live micro-
algae and their biomass because a number of processes overlap.
6. Factors affecting heavy metal remediation
Although microalgal cells have several self-defense mechan-
isms to survive in metal-containing media, various factors influ-
ence the removal of metals. Precisely speaking, HM toxicity to
aquatic organisms can be affected by several biotic and abiotic
factors. The biotic factors include: tolerance, size and life stages,
species, surface area, depending on the algal division, and nutri-
tion related to the test organisms. Abiotic factors include ionic size,

Fig. 1. Schematic representation of several mechanisms of heavy metal translocation, sequestration, and uptake in living (Left), as well as, non-living (Right, brown-shaded)
microalgae; including Men+-Metal ion, L-liquid (Men++L represents metal ion in liquid); Metal-ion transporters (such as NRAMP, CTR, ZIP and FTR); Phytochelatin bio-
synthesis pathway, PC complexes and enzymes involved in the PC synthesis (GCS- glutamyl–cysteinyl synthase, GS- Glutathione synthase, PCS- phytochelatin synthase); AA-
Amino Acids; OA-Organic Acids; LMW PC-MeC - Low Molecular Weight Phytochelatin Metal Ion Complexes; HMW PC-MeC - High Molecular Weight Phytochelatin Metal Ion
Complexes; MTP - Metallothionein Protein; SA-surface adsorption; P- Precipitation; IE- Ion Exchange; CC- Complexation and Chelation and PD- Passive diffusion (modified
from Perales-Vela et al., 2006; Toress et al., 2008; Monteiro et al., 2012 and Blaby-Haas and Merchant, 2012). (For better interpretation of the color figure, the reader could
refer the web version of this article).
 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352
atomic weight or reduction potential of the metal, organic sub-
stances, pH, temperature, salinity and hardness, inorganic ligands,
interactions, and others (Wang, 1987; Dönmez et al., 1999). Mon-
teiro et al. (2012) stated that irrespective of the nature of cell/
metal interactions, sorption of metals by microalgae is affected by
several parameters, such as temperature, pH, metal concentration
and speciation, and the presence of other metals as extrinsic
factors; biomass concentration (in either living or dead status),
regeneration (and possibility for reuse), and pretreatment as
intrinsic factors. In addition, it is paramount to note that the
presence of amino acids, organic matter, humic acids, fulvic acid,
EDTA, and NTA can complex with HMs and render them unavailable.
Further, hard waters are also known to decrease metal toxicity; in
contrast, the presence of certain ions like calcium, magnesium and
phosphorus can alleviate the toxicity of metals (Rai et al., 1981).
According to Arıc et al. (2005), at pH 2, heat-treated and acid-
treated biomass of Chlamydomonas reinhardtii, more efficiently
removed Cr6 þ , as compared to the non-living (unmodified) bio-
mass (Table 1). However, as metal removal varies with the type of
microalgae, and, the prevalent physical and environmental factors,
one cannot generalize that any pretreated microalgae would have
superior metal removal capacities as compared to its native form.
6.1. Biotic factors
6.1.1. Species
The metal sensitivity, uptake and remediation capacities vary
with each alga, i.e. it differs with genus and species. For instance,
Monteiro et al. (2011b) reported effects of Cd upon growth of S.
obliquus and Desmodesmus pleiomorphus, and their corresponding
EC50 values as 0.058 and 1.92 mg l  1 respectively for Cd.
In contrast, even the algae belonging to the same group may
have a different adsorption capacity. In fact, algae belonging to the
same genus but varying in their species, respond differently to
HMs; for example, the freshwater green microalgae, C. miniata, C.
vulgaris, and C. reinhardtii are reported to remove divalent HMs
(viz. Hg, Cd, Pb, Ni, Cu and Zn), while the trivalent metals (viz. Fe
and Cr) were removed by C. vulgaris and S. platensis; on the other
hand, C. miniata and C. vulgaris removed the hexavalent cation Cr
(González et al., 2011).
6.1.2. Tolerance capacity
Metals break the oxidative balance of the algae, inducing an-
tioxidant enzymes, such as superoxide dismutase (SOD), glu-
tathione peroxidase (GPX) and ascorbate peroxidase (APX) (Ar-
unakumara and Xuecheng, 2008); consequently, the amount of
oxidized proteins and lipids in the algal cells could designate the
severity of the stress. Tolerance is an important mechanism by
which an organism reacts to an adverse environment. Certain al-
gae have acquired successful adaptation mechanisms after ex-
posure to pollutants (Stockner and Antia, 1976). However, the
metal tolerance capacity of each algae vary; in this regard, Wong
and Beaver (1980) compared two species of algae, stating that the
green alga Chlorella fusca was commonly found in lakes with high
metal concentrations, while another green alga Ankistrodesmus
bibraianum was very sensitive to metals.
Algal tolerance to HM is highly dependent upon the defense
response against possible oxidative damages; with regard to re-
ducing HM toxicity, numerous mechanisms have been proposed
for reducing HM toxicity in organisms. These include the pro-
duction of HM binding factors and proteins (metallothionein, GSH,
and phytochelatin conjugates), exclusion of toxic HMs from cells
by ion-selective metal transporters and excretion or compart-
mentalization (Arunakumara and Xuecheng, 2008; Hu et al., 2001;
Gharieb and Gadd, 2004). Arunakumara and Xuecheng (2008)
suggested that algal tolerance to HM depends on defense
343
responses against oxidative damage, exudation capacity of che-
lating compounds, active efflux of metal ions by primary ATPase
pumps and reduced uptake (Gaur and Rai, 2001). Correspondingly,
Priyadarshni et al. (2011) comply that microalgae preferentially
possess a mechanism of peptide production that enables them to
bind HMs. These organometallic complexes are further partitioned
inside vacuoles to facilitate an appropriate control of the cyto-
plasmic concentration of HM ions, thereby preventing or neu-
tralizing their potential toxic effects.
Reports suggest that phytoplanktons that are initially accli-
mated to low concentration of pollutants could be conditioned to
accept levels several fold higher by repeated exposure (Wang,
1987). Subsequent to chronic exposure to Cu, C. vulgaris are known
to have developed tolerance, thereby, showing an increased ability
to prevent Cu enrichment (Lindestrom, 1980). Silverberg et al.
(1976) reported Scenedesmus spp. grown in media containing
1 mg l  1 copper to become copper-tolerant, wherein they con-
tained 0.76 mg Cu g  1 dry weight; these authors also reported
that the non-tolerant Scenedesmus spp. failed to grow in media
containing Cu Z0.15 mg l  1.
6.1.3. Biomass concentration
Several authors have studied the effect of increasing biomass
concentration on metal removal. Monteiro et al. (2012) observed
that the amount of metals removed by microalgae from solutions
was apparently improved by increasing the biomass concentra-
tion, and this may be attributed merely to a higher number of
available metal-binding sites.
Contrarily, in a few cases a decreased metal removal is often
reported at very high biomass levels. This may be explained by the
partial aggregation of biomass (that reduces the effective surface
area available for sorption), as well as, by a decrease of the average
distance between available adsorption sites. It is also possible that
under the conditions of higher biomass concentrations, a screen
effect (caused by the dense outer layer of cells) could probably
occur. Thereby the binding sites are blocked from metal ions, re-
sulting in lower metal removal per unit biomass (Bishnoi et al.,
2004). In case of dead Spirogyra species, copper uptake decreases
when the biosorbent concentration is increased (40.5 g l  1)
(Bishnoi et al., 2004). Similarly, a marked reduction in Pb2 þ uptake
(from 121 to 21 mg g  1) by Spirulina maxima was reported when
biomass concentration increased from 0.1 to 20 g l  1 (Gong et al.,
2005).
Therefore, increasing the biomass level is only feasible to a
certain extent, in order to obtain a better metal uptake. However,
increasing the biomass concentration beyond a certain threshold
could probably cause a decrease in the level of metal binding per
unit cell mass (Esposito et al., 2001).
6.1.4. Size and volume of microalgae
The size and the life stage of an organism are known to have an
important effect on its sensitivity to metal toxicity. Needless to say,
in case of the algae are of the same species, when the HM quantity
remains constant, and the microalgal population size is increased,
there would definitely be a reduced toxic effect on each of the
individual microalgae.
Size is a fundamental factor in the ecology of algae because
their biochemical composition, metabolism, growth and loss pro-
cesses, are all strongly size-dependent. Small algae achieve higher
rates of photosynthesis and have higher specific growth rates, and,
also a faster transport of nutrients per unit of biomass (Hein et al.,
1995). The smallest microalgae, having the largest surface to vo-
lume ratios, are often the most effective in sequestering metals
(Khoshmanesh et al., 1997); for e.g. small cells (with large surface
area to volume ratios) are reported to be more sensitive to copper
than larger species (Quigg et al., 2006). In a comparative study,
344 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352
Levy et al. (2007) reported Micromonas pusilla having the smallest
surface area was approximately twice as tolerant as compared to
Minutocellus polymorphus, while, the dinoflagellate Heterocapsa
niei, the largest species tested, was one of the most sensitive to
copper. However, smaller organisms should be more sensitive to
copper because of the higher cell surface to volume ratio; more-
over, small cells may also recover faster due to their rapid growth
rates (Levy et al., 2007).
Nonetheless, the size of the inoculum can also influence the
toxicity of HMs; in this context, Truhaut et al. (1980) studied Cd
toxicity to C. vulgaris, and clearly unveiled that the volume of the
inoculum was of great importance.
6.2. Abiotic factors influencing metal removal
6.2.1. pH
pH can affect the solubility and toxicity of heavy metals in the
water; it is perhaps the most important parameter influencing
metal adsorption by microalgal biomass (Brinza et al., 2007; Jeba
kumar et al., 2013; Indhumati et al., 2014). It influences the spe-
ciation of metals in solution and algal tolerance (Brinza et al.,
2007); particularly, it influences both, cell surface metal binding
sites, and, metal chemistry in water. Peterson et al. (1984) pro-
vided evidence of pH-dependent metal toxicity in their report on
algae. A brief summary of the literature elucidating optimal pH for
the uptake of seven common toxic metals appears in Table 1.
The pH dependence of metal uptake is closely related to the
metal chemistry in solution as well as the acid–base properties of
various functional groups on the microalgal cell surface (Monteiro
et al., 2012). At low pH, cell wall ligands could be closely associated
with the hydronium ions H3O þ , thereby restricting the approach
of metal cations as a result of the repulsive force. Nonetheless, as
the pH increases, more ligands such as carboxyl, phosphate, imi-
dazole and amino groups would be exposed (these carry negative
charges), and subsequently an attraction of positive charged me-
tallic ions via a process of biosorption onto the cell surface ensues
(Dönmez et al., 1999). Monteiro et al. (2012) also opine that at low
pH, functional groups are associated with H þ ions, thus hamper-
ing the positively charged metal ions from binding (because of
repulsive forces). They state that as the pH increases, those func-
tional sites become deprotonated, therefore their negative charges
increase, and this facilitates binding to metal cations to a higher
extent. In brief, as the pH decreases the cell surface becomes more
positively charged, reducing the attraction between biomass and
metal ions. Thus, higher pH results in the facilitation of metal
uptake since the cell surface are more negatively charged. These
phenomena are of particular importance in binding via ionic at-
traction. For example with an increasing pH from 1.0 to 7.0, the
copper sorption by Spirogyra biomass is reported to have increased
from 31 to 86% (Bishnoi et al., 2004). Further, as pH increased from
4 to 7, the HM (Cu, Cd, and Zn) binding capacity of the blue-green
alga Chroococcus paris has been reported to increase (Les and
Walker, 1984).
At higher pH levels, precipitation of most metals tends to oc-
cur; this normally decreases the extent of metal removal, hence, it
is necessary to determine the optimal pH for algae–metal inter-
actions. For example, at pH values higher than the optimum pH
(3.5–4.0), enhanced Zn toxicity was reported in the alga Hormi-
dium spp. (Hargraves and Whitton, 1976). Aksu (2001) reported
highest Cd (II) uptake capacity of C. vulgaris to occur at pH 4.0 (at
20 °C; with an initial Cd (II) ion concentration of 200 mg l  1), and
state that further increase in pH caused a decline in the metal
uptake capacity.
Brinza et al. (2007) summarize several researchers on micro-
and macroalgae, stating that metal solubility function by pH var-
iations has to be a criterion in choosing the type of biomass (dead
or live macro/micro-algae) and understanding its uptake affinity
and mechanism; they state that at pH o 3 uptake capacity de-
creases because hydrogen and metal ions are in competition for
the binding sites, while above pH 6.5, heavy metals tend to pre-
cipitate as hydroxides, thus only a low amount of heavy metals
remain in solution complexing with ligands.
Rai et al. (1981) stated that at low pH, the availability of HMs to
algae greatly increased; hence, there would be elevated toxicity to
algae. Studies conducted by Crist et al. (1981) and Aksu (2001)
suggest that zero-point charge, or the isoelectric point, was found
at pH 3.0 for the algal biomass. Above this pH, the algal cells would
have a net negative charge, and the ionic state of ligands would be
conducive to promote reactions with metal ions. This would lead
to electrostatic attractions between positively charged cations
such as Cd2 þ and negatively charged binding sites causing the
rapid rise in the binding efficiency at lower pH (4.0). Shengjun and
Holcombe (1991) showed that 0.12 M HCl rinsed Chlorella had a
better Ni2 þ and Co2 þ adsorption.
Several functional groups are available for binding metal cations
at distinct pH ranges, i.e. at pH 2–5, carboxyl groups dominate, but
at pH 5–9 they are joined by phosphate groups, whereas at pH 9–
12, carboxyl, phosphate, and hydroxyl (or amine) groups would also
be suitable (Chojnacka et al., 2005; Monteiro et al., 2012). The car-
boxyl group having a wide range of pH susceptibility possesses the
ability to chelate different types of metals, while, the amino and
hydroxyl groups play a predominant role (at high pH), this holds
true for binding of Pb to Spirulina maxima (Gong et al., 2005).
A comprehensive effort of Hassett et al. (1981) reveals that the
influence of pH on metal accumulation by algae is quite species-
specific (Wilde and Benemann, 1993). Nevertheless, the effect of
pH on HM toxicity is very complex as it primarily depends on the
metal species, for e.g. Starý et al. (1983) reported that the uptake of
Zn and Cd by alga S. obliquus was a linear function of pH in the
range of 5–9, whereas, Hg uptake was pH independent.
6.2.2. Ionic strength
In an attempt to detail bioremediation of HM by algae, Dwivedi
(2012) observed increasing removal efficiency, with decreasing
ionic strength. At a particular pH, the number of functional groups
is fixed, however the sites available for metal ion uptake decrease
with increasing ionic strength; as a result, the ion removal would
be less at higher ionic strength (Dwivedi, 2012). In case of bior-
emediation of HMs by algae, the presence of high concentrations
of monovalent cations (Na þ and K þ ) causes an increase in ionic
strength of a wastewater; this, in turn, decreases metal biosorption
capacity of the biomass (Mehta and Gaur, 2005). Chen et al. (1997)
evaluated copper sorption by calcium alginate, and showed that
when the ionic strength decreases (from 0.5 to 0.005 mol l  1), the
removal efficiency increases (from 80% to 95%), while high ionic
strength is found to decrease the extent of biosorption; they hy-
pothesized the competition for the functional groups between
metal ions and other ions had played an important role in ad-
sorption. Schiewer and Wong (2000), studying effects of ionic
strength on metal uptake by algae, mentioned that there was a
decrease of proton binding with increasing ionic strength and pH,
and there was an increase of Cu and Ni binding with increasing pH
and decreasing ionic strength.
Though most reports on ionic strength influencing HM algal
uptake mainly focus on macroalgae, one could not rule out the
possibility of similar mechanisms and interpretations in micro-
algae. However, indepth investigations need to be focused on this
field.
6.2.3. Salinity and hardness
Salinity affects the uptake of HM with differing optimal salinity
values for different metals. For example, in case of the alga S.
 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352
bacillaris, 2.5% salinity was optimal for Cd and 20% salinity for Cu;
essentially high salt concentrations can severely limit metal
binding by algae (Wilde and Benemann, 1993). D. salina showed
maximum cadmium uptake when NaCl concentrations ranged
between 0.5 and 1.0 M; but, at NaCl concentrations4 1.0 M, con-
siderably less cadmium uptake occurred (Rebhun and Ben-Amotz,
1986). Wang (1987) reviewed several reports indicating that metal
(Ni, Zn, Sn, Cu, and Cd) toxicity decreases as chloride or salinity
increases.
Besides, Wang (1987) also reported that decreased high alka-
linity/hardness content caused a decrease in the toxicity of Cd, Cu,
Hg, and Zn. Here, two possible mechanisms were indicated viz. the
complexation of metal ions with carbonates, and the competition
between metal ion and Ca/Mg ions.
6.2.4. Temperature
Temperature affects a number of factors that are important for
the metal ion biosorption. These include the stability of the metal
ion species, the ligands and the ligand complex, as well as, the
solubility of the metal ions. Temperature exerts an important ef-
fect on metal speciation, because most chemical reaction rates are
highly sensitive to temperature changes (Elder, 1989). Lau et al.
(1999) provided a detailed understanding of temperature in their
study; in general, higher temperature favors greater solubility of
metal ions in a solution and hence weakens the biosorption of
metal ions. Moreover, in a thermodynamic context, biosorption
would be favored by high temperature if the binding is en-
dothermic but weakened if it is exothermic. Basically, metal–car-
boxylate interactions are endothermic, whereas metal-amine in-
teractions are exothermic. Besides, the relative occurrence and
contribution of the carboxylate or amine ligands in the cell wall or
at the cell surface, also determines the effect of high temperature
on metal sorption.
Unfortunately, studies available on the effect of temperature on
metal removal by microalgae are not fully consistent, and do not
match with each other sometimes; moreover, the effect of tem-
perature could also vary with the type of metal. Few authors point
out an increase in metal removal extent with increasing temperature,
while others claim a reduced uptake. For instance, Aksu (2002) ob-
served an increasing adsorption of Ni2 þ by the dry biomass of C.
vulgaris with rising temperature. Contrarily, the same author in a
previous study (Aksu, 2001) reported increase in temperature (from
20 to 50 °C), caused a decreased cadmium(II) biosorption capacity
(from 85.3 to 51.2 mg g  1). Gupta and Rastogi (2008) reported a
lower adsorption of Cd2 þ by Oedogonium spp. at higher tempera-
tures, but certain reports show that temperature has no effect on
metal sorption (Monteiro et al., 2012). However, Lau et al. (1999)
stated that temperature has a wide spectrum effect on metal bio-
sorption, but its influence is lower than the impact of pH.
6.2.5. Metal speciation
The toxic effect of trace metals on aquatic organisms frequently
depends on the species of the metallic ion which in turn may be
determined by the pH, or, the varieties of the complexing agents
found in natural waters (Crist et al., 1981). Precisely, the binding of
metal cations onto microalgae depends on their form (speciation)
and charge in solution, which again depends on pH (Monteiro
et al., 2012). Pagnanelli et al. (2003) also reported several effects of
metal speciation on HM sorption by Sphaerotilus natans. Doshi
et al. (2007) reported live Spirulina spp. accumulated 304 mg g  1
Cr3 þ , while it accumulated 333 mg g  1 Cr6 þ (Table 1).
Despite the fact that metals could occur under a variety of
chemical forms (free ions, complexes with inorganic/organic li-
gands, and adsorbates on particulate phases) in wastewaters, the
free metal ions in the solution, are the most toxic form for living
organisms at large, and generally bind the furthest to microalgae.
345
This was ellucidated by Rodea-Palomares et al. (2009), who ob-
served that the presence of Zn and Cd as free ions in solution was
toxic to a bioluminescent cyanobacterial bioreporter. Reinfelder
and Chang (1999) studied speciation and microalgal bioavailability
of inorganic silver, and stated that silver accumulation in aquatic
organisms is primarily attributed to the bioavailability of the free
Ag ion (Ag þ ). However, there is a lack of data that combines
measurements of metal speciation and its effects on microorgan-
isms. Therefore, the exact idea regarding the effect of metal spe-
ciation on algae under field conditions remains elusive (Meylan
et al., 2003).
6.2.6. Effect of combined metals
In any toxicological study, it is important to consider combined
effects of all toxic constituents present in a test sample. Generally,
wastewaters discharged into natural waters frequently carry more
than one toxic or potentially toxic substance. The combined toxic
effects of all the constituents are often not equivalent to the
summation of all individual effects, i.e. it could be greater or less.
There is a possibility of joint actions that are commonly sum-
marized into: (1) synergism, where the combined toxic effect of a
mixture is greater than the sum of the individual toxicities;
(2) antagonism, where the combined toxic effect of a mixture is
less than the sum of the individual toxicities; and, (3) non-inter-
active, or additive, where the combined effect is identical to the
sum of the individual toxicities. Many industrial wastewaters
contain high levels of more than one metal, e.g. mixtures of Cr, Ni,
Cd, and Zn are found in effluents of electroplating operations.
Therefore, examination of the effects of metal cations in multi-
metal solutions is more representative of actual environmental
problems than are single metal studies (Monteiro et al., 2012).
Although studies on metal mixtures are important, there are
scarcely any reports available on combined effects.
Wang (1984) observed all the three aforesaid interactions in
case of Fe and Zn in different sources of algal samples. The author
stated that several factors affected the interactions, such as metal
species, test organisms, and lethal vs. sub-lethal concentrations.
Even more, the most studied mixture of Cd and Zn was reported to
have synergistic effects in some organisms and antagonistic effects
in others. The interaction between metal ions appears to be in-
tricate, without a set pattern. Considering the competitive inter-
actions between the HM and the adsorption binding sites on the
cell surface, Monteiro et al. (2012) cited several studies on mix-
tures of metals/co-ions in solution using microalgal biomass.
Wong et al. (1978) tested 10 metals (As, Cd, Cr, Cu, Fe, Hg, Ni, Pb,
Se, and Zn) that were not toxic to algae when presented in-
dividually, and found that their mixtures at the same concentra-
tion were strongly inhibitive. Fraile et al. (2005) revealed that the
presence of Cd2 þ decreased the uptake of Zn2 þ in a competitive
manner. Further, Aksu and Dönmez (2006) described competitive
adsorption of Cd2 þ and Ni2 þ in C. vulgaris.
On the other hand, Lau et al. (1999) explained the antagonistic
effect of metal biosorption in a mixed metal system to be due to
competition for some common adsorption sites. They also clarified
that the presence of one metal simply prevented the adsorption of
the other due to steric and electrostatic effects. Therefore, it could
be stated that the preferential binding was related to the relative
strength of the interaction between types of metal ions and the
types of biomass. For e.g. Cu was generally preferred to Ni by C.
miniata and C. vulgaris due to the stronger binding strength of Cu
by the biosorbent; Cu has a larger ionic radius than Ni which fa-
vored a more covalent nature of interaction between the metal ion
and the ligands particularly the carboxylate groups of biomass.
This enhanced the binding strength of the metal to the ligand of
the biomass (at approximately 30 mg l  1 Ni concentration);
therefore, there was virtually no Ni biosorption. However, a
346 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352
substantial increase in the Ni concentrations (300 mg l  1) caused
masking of the adsorption sites and reduced the Cu sorption.
In another perspective, Wang (1987) stated that phosphate ions
can form non-dissolved metal-phosphate species and thus reduce
metal toxicity; however, the sulfate ion could either compete with
chromate ion or could form precipitates with Ba ion.
7. Potential microalgal species for metal remediation
The magnitude of the metal ion binding capacity may be due to
the properties of the metal sorbate (e.g. ionic size, atomic weight,
or reduction potential of the metal), and the properties of the al-
gae (e.g. structure, functional groups and surface area, depending
on the algal division, genera, and species) (Dönmez et al., 1999).
Microalgae have been discussed in extenso for HM removal from
effluents and wastewater (Mallick, 2002), and countless algal
strains have shown aptness for HM removal, however, most of the
surveys are based on batch growth of the microalgal species. Based
on the compilation of reports in Table 1, the microalga C. re-
inhardtii, C. vulgaris, Scenedesmus spp., and Chlorococcum spp.
could be outlined as potential candidates that could be commer-
cialized for various metal remediation technologies. On the other
hand, the cyanobacteria Lyngbya spiralis, Tolypothirx tenuis, Stigo-
nema spp., Phormidium molle, Aphanothece halophytica, C. paris,
and Spirulina also hold a great potential for HM remediation
(Tüzün et al., 2005).
8. Strategic approaches in heavy metal remediation
8.1. Immobilized microalgae and metals
The use of metabolically active immobilized microalgae is
particularly an attractive option for remediation of metal ions at
low concentrations, especially for detoxification and metal re-
covery processes. Immobilized algal systems have been tested by
several researchers for their efficiency in HM removal (Hameed
and Ebrahim, 2007). The pros and cons of using immobilized algae
have been discussed by several researchers (Darnall et al., 1986).
In a elaborate report, de-Bashan and Bashan (2010) tabulated
pollutant removal by immobilized algae, and mentioned the use of
several immobilizing materials (carrageenan, alginate, poly-
urethane foam, surface of Ulva spp., surface of Sargassum spp., si-
lica gel, Luffa cylindrica sponge, milk casein þglutaraldehyde, al-
ginate, agar, polyurethane foam, polyurethane, polyvinyl alcohol,
polyacrylamide, polysulphone and epoxy resin), and essentially
report removal of metals (Cd, Cs, Pb, Co, Mn, Cu, Au, Fe, Ni, Hg, Zn,
U, and their mixtures) using immobilized microalgae and cyano-
bacteria (Table 1). Based on several reports, Hameed and Ebrahim
(2007) summarized more than 14 algal species for their potential
in HM removal, and narrated the efficacy of C. vulgaris cells im-
mobilized in alginate as a suitable system to remove a wide range
of HMs. Aksu et al. (1998) reported the total Cu(II) removal by Ca-
alginate gel immobilized C. vulgaris was higher than that of agar-
ose- C. vulgaris systems over longer time periods. Akhtar et al.
(2003, 2008) proposed the use of C. sorokiniana immobilized on
the biomatrix of vegetable sponge of L. cylindrica to be suitable for
Cd and Cr(III) removal.
Apart from the type of immobilizing material, researches on
the dimension of the beads are also undertaken in this aspect.
Mehta and Gaur (2005) emphasized that the size of immobilized
beads is a crucial factor for use of immobilized biomass in bio-
sorption processes. Volesky (2001) recommended that beads
should range between 0.7 and 1.5 mm, corresponding to the size
of commercial resins meant for removing metal ions.
8.2. Transgenic microalgae
In order to further enhance the HM specificity and binding
capacity of microalgae, transgenic approaches have been devel-
oped recently with an objective to use microalgae for HM treat-
ment especially in wastewaters and sediments (Rajamani et al.,
2007). These strategies encompass the overexpression of enzymes
whose metabolic products ameliorate the effects of HM-induced
stress, and the expression of high-affinity, HM binding proteins on
the surface, and in the cytoplasm of transgenic cells (Rajamani
et al., 2007). For example, the eukaryotic microalga Chlorella spp.
DT, transformed with the Bacillus megaterium strain MB1 merA
gene that encodes mercuric reductase (MerA), is shown to mediate
the reduction of Hg2 þ to volatile elemental Hg°; here the merA
gene was successfully integrated into the genome of transgenic
strains and functionally expressed to promote the removal of Hg2 þ
(Huang et al., 2006).
However, in order to preclude these transgenic algae with good
metal remediation capacities, it is essential to have clever en-
gineering and biological considerations. The release of live trans-
genic microalgae with enhanced metal binding skills into the en-
vironment could accelerate the biogeochemical cycling of heavy
metals and their accumulation in the food chains, which could be
potentially harmful; thus the alternate strategy would be to use
nonviable transgenic algae with enhanced binding potential and
selectivity. Thus, even though there is a potential market for these
better performers in bioremediation, one should also consider the
risk of unleashing these transgenic forms into the environment.
8.3. Metal desorption
Due to their special cell wall structure, high HM-removal ca-
pacity, and ease of desorption, algae have been considered as ideal
biological adsorbents in various restoration technologies (Lu et al.,
2006). Besides, the metal sorbed on microalgal biomass can then
be desorbed with the aid of an appropriate eluant or desorbing
solution, thereby allowing a reuse of biomass in multiple sorption–
desorption cycles.
Apart from having desorption efficiency it is necessary that the
desorbing agent should preserve the biosorption capacity of the
sorbent, i.e. the desorbing agent should not cause irreversible
physical or chemical changes (or damage) to the biomass (Mon-
teiro et al., 2012). The most commonly used methods of metal
recovery from microalgal biomass comprise changing the pH of
the solution (Monteiro et al., 2012). Generally, lowering the pH
causes displacement of metal cations (back to the solution) by
protons that were abstracted from the binding sites, thus, allowing
recovery of those cations. Inorganic acids have maximum HM
elution efficiency, followed by inorganic salts, chelating agents,
and organic acids (Vannela and Verma, 2006). Chojnacka et al.
(2005) suggest the superiority of nitric acid (as compared to 0.1 M
EDTA, 0.1 M HNO3 and deionized water), in the removal of Cr3 þ ,
Cd2 þ , and Cu2 þ using Spirulina spp. However, Monteiro et al.
(2012) mentioned the widespread use of the inorganic acid HCl,
which exhibits a high capacity to desorb metals from biomass.
However, these authors stated that HCl decreases the metal
sorption ability of biosorbents by inflicting damage upon metal
binding sites, including hydrolysis of polysaccharides on the sur-
face of the cell wall when applied in sequential cycles.
8.4. Recycling of microalgal biomass
Among the techniques used for algal recovery, the use of the
flocculating agent chitosan is more popular for a large number of
microalgal species. Flocculating agents generally avoid tedious and
expensive centrifugation in microalgal harvesting. The second
 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352
method is “chemical attachment” wherein living cells are at stake,
because chemical interaction damages their cellular surface and so
drastically reduces viability. Thirdly, ion binding could be em-
ployed, which though not harmful is less effective and involves pH
and ionic strength of the surrounding medium. Finally, the gel
entrapment via synthetic polymers (e.g., polyacrylamide, photo
cross-linkable resins, silica gel, or polyurethanes) or natural
polysaccharides (e.g., agar, carrageenan, or alginate) are also re-
commended. However, the use of natural polysaccharides would
be preferable due to their lower toxicity to biomass, and alginate is
the most common support for microalgae (Monteiro et al., 2012).
9. Algal biosorbent technologies currently in use
There is phenomenol interest in biological processes for metal
removal/recovery from contaminated environments. As evidenced,
microalgae have a remarkable ability to take up and accumulate
metals from the surrounding environment (Monteiro et al., 2012).
There are several commercially available technologies comprising
the use of microalgae either solely or along with other organisms.
A few of them are detailed here.
The AlgaSORB sorption process (Bio-recovery Systems Inc.,
Cincinnati, OH), which uses algae to remove HM ions from aqu-
eous solutions, consists of dead algal cells immobilized in a silica
gel polymer (Kuyucak and Volesky, 1990). There are several vari-
eties of AlgaSORB available, for instance the algae-silica prepara-
tion (AlgaSORB-scy) which is deliberately made from the natural
dead cells of the blue-green cyanobacterial alga, Scytonema, and is
used for Arsenic(III) removal (Prasad et al., 2006). The AlgaSORB
technology comprising biomass of C. vulgaris immobilized in silica
and polyacrylamide gels, adsorbs metals from diluted solution
with concentrations between 1–100 mg l  1; moreover, it is known
to undergo more than 100 biosorption desorption cycles (Oliveira
et al., 2011). The use of inactivated microalgal biomass, in the form
of biotraps (algaSORBVR) has been used as a commercial ad-
sorbent material for the removal of metals from industrial ef-
fluents (Monteiro et al., 2012).
Yet another biosorbent BIO-FIX, made up of a variety of bio-
masses including Sphagnum peat moss, algae (Ulva spp., and
Spirulina), yeast (Saccharomyces cerevisiae), bacteria, and/or aqua-
tic flora (Lemna spp.) immobilized in high density polysulfone, has
also gained attractiveness (Garnham 1997; Vijayaraghavan and
Yun, 2008; http://www.slideshare.net/sjcc/environmental-pro-
blems-and-health-risk-presentation). The cost of the BIO-FIX
process (partially based on algal biomass) is comparable with lime
precipitation; moreover, the metals recovered from the BIO-FIX
could offer some more income (Garnham, 1997; Wang and Chen,
2009). The metal affinity of BIO-FIX is as follows: Al3 þ 4Cd2 þ 4
Zn2 þ 4 Mn2 þ , apart from this it has a much lower affinity for
Mg2 þ and Ca2 þ (Gupta et al., 2000).
The company B. V. SORBEX, Inc. (Canada) has produced a series
of biosorbents based on different types of biomaterials, including
microalgae (C. vulgaris) and macroalgae (S. natans, Ascophyllum
nodosum, Halimeda opuntia, Palmyra pamata, and Chondrus cris-
pus). These biosorbents are effective over a range of pH values and
solution conditions, and can biosorb a wide range of metal ions
(Wang and Chen, 2009). Travieso et al. (2002) detailed yet another
aspect using living microalgal biomass entailing a reactor con-
taining immobilized cells (BIOALGA) that met with commercial
success; using this bioreactor with S. obliquus, a maximum re-
moval of 94.5% of Co was achieved (after 10 days of exposure to
3000 mg l  1 solutions).
Selected strains of microalgae, purposefully cultivated and
processed for specific bioremoval applications could have metal-
removal potential suitable in several environmental applications
347
(de-Bashan and Bashan, 2010). Algae-based biotechnologies that
are used for the removal of pollutants like metals as well as in-
organic nutrients (Adey et al., 1996; Hoffmann, 1998; Toumi et al.,
2000; Perales-Vela et al., 2006) include: (i) Algal Turf Scrubber
(ATS) and (ii) High Rate Algal Ponds (HRAP). These technologies
are proven to have higher Cu2 þ removal rate per unit volume per
day (Oswald, 1988; Toumi et al., 2000; Perales-Vela et al., 2006).
The ATS comprises suspended biomass of common green algae
(Chlorella, Scenedesmus, Cladophora), cyanobacteria (Spirulina, Os-
cillatoria, Anabaena) or consortia of both (Craggs et al., 1996). It has
been tested for treating polluted underground waters for the ef-
ficient removal of HMs and chlorinated and aromatic organic
compounds (Adey et al., 1996). Toumi et al. (2000) reported that
the greater metal removal capacity of the HRAP could be due to
the high pH achieved as a result of algal photosynthesis that en-
hances metal precipitation. In yet another study, Phillips et al.
(1995) reported that a consortium of algae and cyanobacteria ef-
fectively decreased excessive Mn concentrations to an en-
vironmentally safe level. Prasad et al. (2006) have also examined
arsenic removal using this Scytonema-based biosorbent.
Although the abilities of various microalgae to adsorb high
concentrations of metals are known and their suitability as can-
didates for efficient and commercially feasible wastewater bior-
emediation strategies have been proven, there is a lack of com-
mercialization of technologies in this field. Currently, there is a
rising demand to develop microalgal technologies based on mi-
croalgae such as Chlamydomonas reinhardtii (having the ability to
sequester a wide range of HMs including: copper, zinc, lead, cad-
mium, cobalt, nickel, mercury, silver, and gold) (Cai et al., 1995;
Rajamani et al., 2007) and other microalgal strains (C. vulgaris,
Scenedesmus spp., and Chlorococcum spp.) and cyanobacteria (L.
spiralis, T. tenuis, Stigonema spp., P. molle, A. halophytica, C. paris,
and Spirulina spp.), which are potentially suitable for Hg(II), Cd(II)
and Pb(II) removal in aqueous solutions (Tüzün et al., 2005).
10. Conclusion and future avenues
The implementation of meticulous legislation worldwide and
the increase in demand for metal resources has motivated en-
ormous growth in diversified avenues of biosorbent research. Al-
gae, show capabilities for accumulating, adsorbing and recovering
HMs, are well-studied, for their potentiality, mechanism of metal
accumulation, modeling of metal adsorption, and genetic manip-
ulation. They exhibit constitutive mechanisms for removal of free
metal ions from water, thereby both detoxifying and remediating
the water in question. Microalgae, particularly, possess several
appreciable mechanisms to sequester HM ions, and hence are
demarcated as promising biosorbents. Numerous reports suggest
their superiority to various commercial and traditional physico-
chemical methods, and their usefulness in large-scale remediation
technologies as well as in removal of metals from effluents. Several
biotic and abiotic parameters influencing the effectiveness of a
microalgal remediation technology include: strain, tolerance, size
and life stages, the type of metal and concentration, its size, atomic
weight, as well as, other environmental parameters (pH, tem-
perature, salinity and hardness, etc.). Now a days, significant ad-
vances in technology appear propose the improvement of biomass
containment by immobilization techniques, as well as systematic
utilization of transgenic microalgae. Bioremoval capabilities of
microalgae have been studied extensively worldwide and their
advantages have been projected genuinely; this has resulted in
their utility in various commercial, environmental and technolo-
gical applications. However, it is essential to deliberate on various
avenues of microalgal remediation technologies as ecofriendly
alternatives for a better environment.
348 K. Suresh Kumar et al. / Ecotoxicology and Environmental Safety 113 (2015) 329–352
Microalgal remediation capabilities offer advantages over other
biological materials in some conceptual process schemes. Micro-
algae are known to perform well at low levels of contaminants;
they do not generate toxic sludge, are easy to culture and main-
tain, have a good binding affinity (due to relatively high specific
surface area and net negative charge), and, are suitable for small-
scale as well as large-scale remediation strategies. Apart from
averting the adverse consequences of HMs in the environment,
microalgae quintessentially provide an ecofriendly means of car-
bon dioxide mitigation; however, the main advantages of micro-
alga-mediated sorption include its efficacy and low-cost. Despite
these advantages, there are scarce microalgal technologies with
commercial success as yet. Therefore, efforts need to be focused on
removal and recovery of HMs using microalgae; this would include
the development of innovative technologies, or improvising the
existing ones. A holistic multidisciplinary approach involving
chemists, engineers and biologists would be beneficial in this re-
gard. With increasing availability of knowledge regarding im-
proved selectivity, better shelf-life, efficacy, specificity, economic
viability and a convincing market, there is bound to be a huge
market for these eco-friendly biosorbents in the future.
Acknowledgments
We thank Prof. Martin Podhurst (Sangmyung University,
Republic of Korea) for language correction. This research was fi-
nancially supported by the National Research Foundation of Korea,
South Korea (NRF-2012R1A2A2A02012617) and by the research
fund of Hanyang University, South Korea (HY-2014-P).
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