tmp1085 TMP

Ecotoxicology and Environmental Safety 113 (2015) 329352
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
Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 80424, Taiwan, ROC
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
b
c
art ic l e i nf o
a b s t r a c t
Article history:
Received 25 June 2014
Received in revised form
3 December 2014
Accepted 8 December 2014
Biotechnology of microalgae has gained popularity due to the growing need for novel environmental
technologies and the development of innovative mass-production. Inexpensive growth requirements
(solar light and CO2), and, the advantage of being utilized simultaneously for multiple technologies (e.g.
carbon mitigation, biofuel production, and bioremediation) make microalgae suitable candidates for
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
with their ability to grow and concentrate heavy metals, ascertains their suitability in practical applications of waste-water bioremediation. Heavy metal uptake by microalgae is afrmed to be superior to
the prevalent physicochemical processes employed in the removal of toxic heavy metals. In order to
evaluate their potential and to ll in the loopholes, it is essential to carry out a critical assessment of the
existing microalgal technologies, and realize the need for development of commercially viable technologies 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.
Keywords:
Microalgae
Heavy metals
Bioremediation
Mechanism
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
Hazardous effects of heavy metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
Conventional vs. novel approaches in heavy metal remediation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
Microalgae and their potential in metal remediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332
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 ux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
Corresponding author. Fax: 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) 329352
5.2.1.
Signicance of functional groups in heavy metal uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
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 inuencing 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
6.
1. Introduction
Heavy metals (HMs) occur as natural constituents of the earth
crust and soil. Although there is no clear denition of a HM, in
most cases density is the dening factor; conventionally, HMs are
dened 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 ve 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 relatively high density and is toxic or poisonous at low concentrations
(http://www.lenntech.com/processes/heavy/heavy-metals/heavymetals.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 unknown 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 concentrations [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) categorized 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 unabated agrarian practices, have caused global concern worldwide.
Increasing urbanization and industrialization have made HMs
reach alarming toxic levels in the environment; thereby suggesting HM enrichment in many ecosystems to be strongly related to
human activities (Lasat, 2000; Herrera-Estrella and Guevara-Garcia, 2009). Various reports elucidate the variegated traditional uses
of HMs (Jrup, 2003; Torres et al., 2008), their use in several industrial (Wang and Chen, 2009) and agricultural practices (Brower
et al., 1997), as well as, their indiscriminate disposal in the environment (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 metallurgical industry, chemical industry, textile printing and dyeing
zincication, plastics, paint and tire manufacturing, power plants,
gasoline and fossil fuel combustion, battery, petroleum rening,
cement and ceramic production, steel production, agrochemical
and animal feed industries, electroplates, paper and pulp, alloy
preparation, viscose rayon yarn and ber production (Frstner and
Wittmann, 1983; Olade, 1987; Guo, 1994; Periasamy and Namasivayam, 1996; Cheng, 2003; Freitas et al., 2008; http://pacicenvironment.org/downloads/GCA%20IT%20Campaign%20Report%
20Phase%20One.pdf).
Several metallic species mobilized and released into the environment by various technological activities by humans, persist
indenitely 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 challenging 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-purication ability of an aquatic body (Khan
et al., 2008).
2. Hazardous effects of heavy metals

The center for Hazardous Substance Research (US) clearly categorized HMs as individual metals and metal compounds that can
impact human health (http://www.engg.ksu.edu/chsr/les/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
to humans). Though the adverse health effects of HMs are known

since a long time, exposure to HMs continues and is even increasing in some regions (Jrup, 2003). HMs are key environmental 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 disposal of industrial and domestic wastes (containing heavy metals)
into aquatic ecosystems, threatens the inhabiting aquatic organisms (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 environment (Khan et al., 2008). Even if they are present in dilute
undetectable quantities, their recalcitrance and consequent persistence in water bodies implies that, through natural processes
such as biomagnication, 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 dened maximum contaminant level for certain 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 effective, economic/affordable, and consistent; moreover, it should
effectively reduce HM concentrations to environmentally acceptable levels, and be applicable to eld conditions such as efuents
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
sulde precipitation), chemical oxidation or reduction, lime coagulation, ion exchange (using resins, starch xanthate, etc.), reverse
osmosis, solvent extraction, evaporation recovery, cementation,
adsorption (involving use of activated carbon), electrodeposition, 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 1100 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 metallurgical 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 remediation provide incomplete metal removal (Volesky, 1990), require 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 effective 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 scientic community is under
tremendous pressure to develop new, innovative, cost-effective,
efcient and sustainable methods for the removal of toxic substances from aquatic bodies as well as wastewaters. There is a
growing impulse for the production of cheaper adsorbents to replace costly wastewater treatment methods; thereby non-conventional HM remediation technologies have been gaining immense popularity. Researchers have intensied their efforts in
developing suitable technologies for (i) prevention of HM pollution, (ii) decreasing HMs concentrations to a lower level (for e.g. by
decreasing the efux of HMs into the receiving bodies such as
rivers, sewer and lake, etc.), and, (iii) removal of HMs from contaminated 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, highefciency 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 microbial 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 differences in selectivity and afnity 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 exopolysaccharides), 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 efcacy of live organism vs. dead biomass has been extensively debated. For example, Rayson and Williams (2011) studied the plant, Datura innoxia, and reported that non-living biomaterials exhibit high capacity, rapid binding, and selectivity towards HMs. They correspondingly state that functional groups of the lipids, carbohydrates, 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 considerable versatility with respect to metals as they efciently 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 lamentous
fungi possess a high potential of accumulating HMs from aqueous
solutions, the direct application of living fungal cells as biosorbents for HMs is unfavorable. Further, derivatives of fungal
332
biomass (puried cell wall fractions, whole or heat killed mycelium) are known to possess a moderate degree of regenerability,
and superior HM remediation ability compared to commercial ionexchange 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-sulde precipitates at a large-scale (PeralesVela et al., 2006). However, its main drawbacks include: long residence time (weeks), the need for a continuous supply of organic
substrates and large steel bioreactors (White et al., 1997; PeralesVela 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
metalmicrobe 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 afnity between the metallic species or its ionic forms and the binding sites on the molecular structure of the cellular membrane, cell-wall and capsule.
On another stance, algae as renewable natural biomass exhibit
different afnities toward different metals, and, are therefore, very
important candidates, employed as biosorbent materials (Mallick,
2002; Doshi et al., 2006). Macro- and microalgae exhibit constitutive mechanisms for the removal of free metal ions from
waters, thereby detoxifying and remediating the water in question. 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, Scenedesmus, and Cladophora), cyanobacteria (Spirulina, Oscillatoria,, and
Anabaena), or a consortia of both. Khan et al. (2008) reported
Chlorella vulgaris to have Cd (II) removal efciency while Sphagnum moss peat could remove Cu (II) and Ni (II). In addition, PrezRama et al. (2002) report cadmium removal by living marine microalga 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 efuent water
and valuable biomass (which in turn could have diverse applications 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 photosynthetic 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). Researchers have globally emphasized on the advantages of using
microalgae in metal biosorption. The benets include: rapid metal

uptake capability, time and energy saving, eco-friendly, user-friendly,
year round occurrence, ease of handling, recyclable/ reusable, lowcost, faster growth rate (as compared to higher plants), high efciency, 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 efcacy, microalgae
enable easy recovery of HMs involving a few simple desorption
chemicals. Live algal biomass needs minimum nutrients and environmental conditions, while dead biomass does not require specic
nutrients or oxygen; moreover, they could remove HMs from multimetal 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 efciently sequester HMs, and
mention about the transgenic approaches used to enhance the HM
binding efciency of microalgae including the use of uorescent HM
biosensors developed using transgenic Chlamydomonas.
Microalgal afnities 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 microand 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: Chlamydomonas reinhardtii, Chlorella salina, Chlorella sorokiniana, C. vulgaris, Chlorella miniata, Chlorococcum spp., Cyclotella cryptica,
Lyngbya taylorii, Phaeodactylum tricornutum, Porphyridium purpureum, Scenedesmus abundans, Scenedesmus quadricauda, Scenedesmus 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 organisms, preferentially produce peptides capable of binding HMs.
These peptide molecules bind the HMs forming organometallic
complexes, which are further positioned inside vacuoles to facilitate 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 microalgae. According to this table, reports comprising cadmium removal
generally encompass studies on Cd2 , and the pH range studied varied from 4 to 8 (Table 1). Moreover, the microalgae C. reinhardtii,
S. platensis, as well as, various Scenedesmus spp., Tetraselmis spp. and
Chlorella spp. efciently contribute to Cd removal. However, live cells
of Planothidium lanceolatum are reported to take up signicant quantities of Cd2 (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 chromium (i.e. Cr3 , Cr O72 and Cr6 ) studied, more emphasis has been
laid on its hexavalent form (Cr6 ), wherein Chlorella and Spirulina spp.
as good contenders, and, a wide pH range (28.2) has been investigated. Among the several studies on chromium remediation using
live, dead, heat-treated and acid-treated forms of microalgae, remarkable 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 Cu2 (Table 1), where, the most
333
Table 1
Heavy metal removal efciency of some microalgae.
Metal
Speciation
Organism
pH
Type of biomass
Metal uptake (mg/g)
Reference
Country
Cd
Cd2
AER Chlorella
AER Porphyridium
AER Spirulina
Aulosira fertilissima
Calothrix parietina TISTR 8093
Chaetoceros calcitrans
Chlamydomonas reinhardtii
Chlorella homosphaera
Chlorella pyrenoidosa
Chlorella sorokiniana
Chlorella sorokiniana
Chlorella sp. HA-1
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris BCC 15
Chlorella vulgaris CCAP211/11B
Cyclotella cryptica
Desmodesmus pleiomorphus
Desmodesmus pleiomorphus (ACOI 561)
Desmodesmus pleiomorphus (L)
Hydrodictyon reticulatum
Isochrysis galbana
Phaeodactylum tricornutum
Phormidium spp.
Pithophora odeogonia
Planothidium lanceolatum
Porphyridium cruentum
Porphyridium purpureum
Pseudochlorococcum typicum
Scenedesmus abundans
Scenedesmus abundans
Scenedesmus acutus IFRPD 1020
Scenedesmus obliquus
Scenedesmus obliquusCNW-N
Scenedesmus subspicatus
Spirogyra hyalina
Spirogyra insignis
Spirogyra neglecta
Spirulina platensis
Spirulina platensis
Spirulina platensis
Spirulina platensis
Spirulina platensis
Spirulina platensis TISTR 8217
Spirulina spp.
Spirulina spp.
Synechocystis sp.
Tetraselmis chuii
Tetraselmis chuii
Tetraselmis chuii
Tolypothrix tenuis TISRT 8063
Oscillatoria angustissima
Spirogyra hyalina
Spirulina spp.
Chlorella miniata
Chlorella miniata
Chlorella miniata
Chlorella spp.
Chlorella spp.
Spirulina
Spirulina spp.
37
37
37
5
7
8
5.5
5.5
6
6
6
Non-living
Non-living
Non-living
Non-living
Non-living
Live
Cell wall
Without cell wall
Ca-alginate
Immobilized
Non-living
6.87.0
5
5
Non-living
Immobilized
7.74
7.55
7.28
14.57
79
1055.27
5.75
3
28.9
79.7
42.6
2.3
8.4
2.8
33.5
192
21.6
86.6
62.3
33.72
12.45
8.41
2.6
76
62
22.24
58.6
85.3
61.2
7.2
0.02
1.24
9.6
13.07
275.51
8.84
0.42
5.48
574
0.64
110
11.4
60.8
24.4108.5
7.29
18.18
22.9
27.95
12.08
8.06
357
44.56
47.89
70.92
98.04
36.63
0.46
0.463
199.83
13.46
292.6
210.54
90
0.89
1.3
15.32
12.82
0.01
14.17
28.72
41.12
98
9.62
304
167
Sandau et al., 1996a

Singh et al., 2007
Inthorna et al., 2002
Sjahrul and Arin, 2012
Mace and Welbourn, 2000
Bayramolu et al., 2006
Tzn et al., 2005
Munoz and Guieysse, 2006
Akhtar et al., 2003
Akhtar et al., 2003
Chen et al., 2012
Aksu and Dnmez, 2006
Aksu, 2001
Klimmek et al., 2001
Schmitt et al., 2001
Monteiro et al., 2011c
Monteiro et al., 2010
Singh et al., 2007
Sbihi et al., 2012
Wang et al., 1998
Singh et al., 2007
Sbihi et al., 2012
Shanab et al., 2012
Chen et al., 2012
Chen et al., 2012
Kumar and Oommen, 2012
Romera et al., 2007
Singh et al., 2007
Solisio et al., 2008
Murugesan et al., 2008
Murugesan et al., 2008
Rangsayatorn et al., 2004
Chojnacka et al., 2004
Chen et al., 2012
Tiantian et al., 2011
Sjahrul and Arin, 2012
da Costa and de Franca, 1998
da Costa and de Franca, 1998
Mehta and Gaur, 2005
Han et al., 2006
Han et al., 2006
Han et al., 2006
Doshi et al., 2008
Akhtar et al., 2008
Doshi et al., 2007
Doshi et al., 2007
Germany
Germany
Germany
India
Thailand
Indonesia
Canada
Canada
Turkey
Turkey
Turkey
Sweden
Sweden
Sweden
Pakistan
Pakistan
Taiwan
Turkey
Portugal
Germany
Germany
Germany
Sweden
Thailand
Thailand
Germany
Portugal
Portugal
Portugal
India
Morocco
Germany
USA
India
Morocco
Germany
Germany
Egypt
Portugal
Taiwan
Thailand
Portugal
Portugal
Taiwan
Germany
India
Spain
India
Germany
Germany
Italy
India
India
Thailand
Thailand
Thailand
Poland
Taiwan
China
Indonesia
Brasil
Brasil
Thailand
Canada
Canada
India
India
Poland
Hong Kong
Hong Kong
Hong Kong
India
Pakistan
India
India
Co
Co
Cr
Cr3
4
4
4
6
37
6.87.0
7
7
6
Non-living
Non-living
Non-living
Non-living
6
5
5
7
37
6
7
7.88
Non-living
Non-living
Non-living
Non-living
Live
Live
Non-living
Live
Non-living
Non-living
Non-living
Live
Non-living
Non-living
Live
Live
Non-living
4
4
5
6
6
6
5
6
37
78
6
7
47
7.5
7
5.5
5.5
4
7.5
3
4
4.5
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Live
Immobilized
Alginate immobilized
Non-living
Silica-immobilized
Non-living
Live
Live
Live
Non-living
Non-living
Cell wall
Without cell wall
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Live
Non-living
334
Table 1 (continued )
Metal
Speciation
Cr6
Cr2O7
Cu
Cu2
2
Organism
Spirulina sp.(HD-104)
Chlamydomonas angulosa
Chlorella vulgaris
Chlorella spp.
Chlorella vulgaris
Dunaliella sp. 1
Dunaliella sp. 2
Nostoc muscorum
Oscillatoria nigra
Oscillatoria tenuis
Phormidium bohneri
Spirulina
Spirulina spp.
Synechocystis spp.
Ulothrix tenuissima
Chlorella spp.
Anabaena cylindrica
Anabaena spiroides
Asterionella formosa
Asterionella formosa
Aulacoseira varians
Aulacoseira varians
Ceratium hirundinella
Ceratium hirundinella
Chlorella fusca
Chlorella miniata
Chlorella pyrenoidosa
Chlorella spp.
Chlorella spp.
Chlorella spp.
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Closterium lunula
Cyclotella cryptica
Eudorina elegans
Eudorina elegans
Isochrysis galbana
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis spp.
Phormidium spp.
Scenedesmus quadricauda
Spirogyra insignis
Spirogyra neglecta
Spirulina
Spirulina platensis
Spirulina platensis
Spirulina platensis
Spirulina spp.
pH
Type of biomass
Metal uptake (mg/g)
Reference
Country
8.2
2
2
2
2
Live
Non-living
Non-living native
Heat-treated
Acid-treated
Non-living
306
5.32
18.2
25.6
21.2
23
9.62
23.6
58.3
45.5
22.92
1.86
7.35
8.55
15.6
333
143
19.2
4.56
104
226
12.62
8.73
1.1
0.53
2.29
3.03
21.77
2.3
5.75
6.42
7.54
3.2
23.26
2.4
33.4
220
108
16.14
37.6
34.89
40
48.17
76.71
63.08
1.8
10.9
7.5
18.72
3.63
4.26
0.5
26.28
3.96
2.13
08.72
0.11
8.21
2.47
0.003
1.67
10.1
23.08
134.32
0.27
20
1.8
13.28
2.8
19.3
40.83
389
10.33
10
0.85
100
Doshi et al., 2008

Dwivedi et al., 2010
Arc et al., 2005
Arc et al., 2005
Arc et al., 2005
Dnmez et al., 1999
Akhtar et al., 2008
Aksu and Kutsal, 1990
Dnmez and Aksu, 2002
Dnmez and Aksu, 2002
Gupta and Rastogi, 2008
Dnmez et al., 1999
Doshi et al., 2007
Doshi et al., 2007
Dnmez et al., 1999
Doshi et al. 2008
Doshi et al., 2008
Tien et al., 2005
Tien et al., 2005
Tien et al., 2005
Tien et al., 2005
Tien et al., 2005
Tien et al., 2005
Singh et al., 2007
Tien et al., 2005
Tien et al., 2005
Dnmez et al., 1999
Lau et al., 1999
Yan and Pan, 2002
Maznah et al., 2012
Doshi et al., 2006
Doshi et al., 2008
Romera et al., 2006
Romera et al., 2006
Dnmez et al., 1999
Romera et al., 2006
Dnmez et al., 1999
Dnmez et al., 1999
Lau et al., 1999
Tien et al., 2005
Tien et al., 2005
Yan and Pan, 2002
Tien et al., 2005
Tien et al., 2005
Singh et al., 2007
Sbihi et al., 2012
Tien et al., 2005
Tien et al., 2005
Singh et al., 1998
Wang et al., 1998
Singh et al., 2007
Sbihi et al., 2012
Dnmez et al., 1999
Yan and Pan, 2002
Dnmez et al., 1999
Romera et al., 2007
Singh et al. 2007
Doshi et al. 2007
Dnmez et al., 1999
Nalimova et al., 2005
Doshi et al., 2007
India
India
Turkey
Turkey
Turkey
Turkey
Pakistan
Turkey
Turkey
Turkey
India
India
India
India
Turkey
India
India
Turkey
India
India
India
Taiwan
Taiwan
Taiwan
Taiwan
Taiwan
Taiwan
India
Taiwan
Taiwan
Canada
Canada
Turkey
Hong Kong
China
Malaysia
India
India
Spain
Turkey
Spain
Turkey
Spain
India
India
Turkey
Germany
Turkey
Hong Kong
Taiwan
Taiwan
China
Germany
Taiwan
Taiwan
India
Morocco
Taiwan
Taiwan
India
Germany
USA
India
Morocco
Germany
Turkey
China
Germany
Turkey
Spain
India
India
Germany
Turkey
Russia
India
4
2
2
3
8.2
8.2
8.2
2
2
8.2
4.05.0
4.05.0
4.05.0
4.05.0
4.05.0
4.05.0
5
4.05.0
4.05.0
5.5
5.5
6
6
7
7
2
4
4
4.5
4.5
4.5
4.5
5
6
6
6
4.05.0
4.05.0
7
6
4.05.0
4.05.0
5
4.05.0
4.05.0
9.2
6
5
5
7
6
4.5
7
6
4
4
5
6
6
9
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Live
Non-living
Non-living
Non-living
Non-living
Live
Live
Live
Live
Non-living
Live
Non-living
Non-living
Live
Non-living
Cells with Cell wall
Cells without cell wall
Live
Non-living
Live
Immobilized
Live
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Free
Immobilized
Non-living
Non-living
Non-living
Live
Non-living
Live
Non-living
Live
Non-living
Non-living
Live
Live
Non-living
Non-living
Non-living
Non-living
Non-living
Live
Non-living
Non-living
Live
Non-living
Non-living
Non-living
Non-living
Live
Non-living
Live
Non-living
335
Metal
Speciation
Fe
Fe3
Hg
Hg2
Ni
Pb
Ni
Pb2
Organism
Spirulina spp.
Synechocystis spp.
Chlorella vulgaris
Microcystis sp.
Cyclotella cryptica
Spirogyra hyalina
Spirulina spp.
Arthrospira (Spirulina) platensis
Chlorella miniata
Chlorella miniata
Chlorella spp.
Chlorella spp.
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Phormidium spp.
Spirogyra insignis
Spirogyra neglecta
Spirulina
Spirulina spp.
Spirulina spp.
Spirulinasp.(HD-104)
Synechocystis spp.
Anabaena osaquae
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Cyclotella cryptica
Microcystis novacekii
Oscillatoria laete-virens
Phormidium spp.
Spirogyra hyalina
Spirogyra insignis
pH
4.5
2
9.2
7
6
6
6
7
7
4
4
4
7
7
4
7.5
7
5.05.5
5
5.5
5.5
6
7.4
4.5
4.5
4.5
4.7
5
5
5
5
6
7.4
5.05.5
5
5
5
5
6
5
7.5
5
55.5
5
7
5
6
6
4
4
6
55.5
7
7
6
5
5
5
6
5
5
6
7
7
6
5
Type of biomass
Live
Non-living
Non-living
Non-living
Non-living
Ca-alginate immobilized
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Live
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Cell wall
Without cell wall
Non-living
Live
Live
Non-living
Free
Immobilized
Non-living
Live
Non-living
Immobilized
Non-living
Non-living
Live
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Live
Non-living
Non-living
Live
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Live
Non-living
Non-living
Non-living
Non-living
Live
Non-living
Non-living
Non-living
Non-living
Metal uptake (mg/g)
Reference
Country
0.271
576
23.4
24.52
0.03
19
35.9
106.6
72.2
18
16
11.92
0.51
0.51
15.13
20
9.2
35.71
1.34
27
20.78
4.16
0.4
0.63
20.37
1.37
122
183
58.4
59.29
111.41
24.06
15.4
15.6
28.6
42.3
12.06
0.64
29.29
13.86
5.7
11.81
18.7
17.5
26.3
1378
0.19
515
1108
15.8
70
102.56
31.12
45
96.3
230.5
380.7
97.38
90
17.13
131.36
127
39
36.68
24
80
21.6
1.49
13.6
71.13
0.32
4.49
90
38.71
31.25
51.5

Doshi et al., 2008
Dnmez et al., 1999
Romera et al., 2006
Singh et al., 1998
Tzn et al., 2005
Shanab et al., 2012
Schmitt et al. 2001
Kumar and Oommen 2012
Chojnacka et al. 2004
Ferreira et al., 2011
Singh et al., 2007
Lau et al., 1999
Wong et al., 2000
Doshi et al., 2006
Doshi et al., 2008
Aksu and Dnmez, 2006
Al-Rub et al., 2004
Al-Rub et al., 2004
Al-Rub et al., 2004
Dnmez et al., 1999
Lau et al., 1999
Wong et al., 2000
Singh et al., 2007
Wang et al., 1998
Singh et al., 2007
Dnmez et al., 1999
Romera et al., 2007
Singh et al., 2007
Doshi et al., 2007
Doshi et al., 2007
Doshi et al., 2008
Dnmez et al., 1999
Arunakumara et al., 2008
Singh et al., 2007
Tzn et al., 2005
Singh et al., 2007
Ribeiro et al., 2010
Miranda et al., 2012
Wang et al., 1998
Singh et al., 2007
Shanab et al., 2012
Romera et al., 2007
Poland
India
Turkey
Spain
India
Thailand
Turkey
Turkey
Turkey
Thailand
Thailand
Germany
Germany
Germany
Egypt
Thailand
Germany
India
Poland
Thailand
Brazil
India
Canada
Canada
Hong Kong
Hong Kong
India
India
Turkey
India
India
Germany
U.A.E
U.A.E
U.A.E
Turkey
Hong Kong
Hong Kong
Brazil
India
USA
India
Turkey
Spain
India
India
Poland
India
India
Turkey
China
Brazil
India
Thailand
Turkey
Turkey
Turkey
Germany
Turkey
Germany
Brazil
Thailand
Thailand
Germany
India
Brazil
India
Germany
USA
India
Germany
Egypt
Thailand
Germany
India
Spain
336
Metal
Zn
Speciation
Zn2
Organism
pH
Type of biomass
Metal uptake (mg/g)
Reference
Country
Spirogyra neglecta
Spirogyra spp.
Spirulina (Arthrospira) platensis
Spirulina maxima
Spirulina maxima
Spirulina platensis
Stigeoclonium tenue
Stigeoclonium tenue
Synechocystis spp.
Chlorella homosphaera
Chlorella spp.
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Cyclotella cryptica
Desmodesmus pleiomorphus
Euglena gracilis
Isochrysis galbana
Phormidium spp.
Scenedesmus quadricauda
Spirogyra insignis
Spirogyra neglecta
Spirulina platensis
Spirulina spp.
Stigeoclonium tenue
Stigeoclonium tenue
5
5
7
5.5
5.5
6
6.8
8.2
7
4
4
6
5.05.5
6
5
Immobilized
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
Live
Non-living
Non-living
Non-living
Live
Non-living
Non-living
Non-living
Non-living
Live
Non-living
Non-living
Non-living
Non-living
Non-living
Non-living
90.19
140.84
188
32
42
16.97
0.86
0.38
155.63
31
33.21
19.15
15.6
28.5
24.19
24.5
6.42
43.41
242.9
360.2
7.5
3.7
0.3
14.52
9.4
8.98
118.66
2.01
22.3
6.67
5.03
72.06
21.1
31.51
7.36
0.17
0.88
0.77
Singh et al., 2007

Gupta and Rastogi, 2008
Arunakumara et al., 2008
Gong et al., 2005
Gong et al., 2005
Pawlik-Skowroska, 2003b
Tiantian et al., 2011
Singh et al., 2007
Maznah et al., 2012
Singh et al., 2007
Sbihi et al., 2012
Wang et al., 1998
Singh et al., 2007
Sbihi et al., 2012
Omar, 2002
India
India
China
China
China
Germany
Poland
7
5.05.5
5
Non-living
Non-living
Live
Intact biomass
Pretreated biomass
Non-living
Non-living
Non-living
Live
Non-living
Non-living
Non-living
6
5
5
7
6
6
6
5
6
7.5
6.8
8.2
used organism was C. vulgaris in various forms (non-living, living, free,

and immobilized), and the range of pH tested includes pH 47.
However, procient 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 , C. vulgaris and C. reinhardtii proved their potentiality,
while the pH range studied was 47.5. Immobilized C. reinhardtii
showed remarkable removal capacities (106.6 mg g 1; Bayramolu
et al., 2006). The metals nickel and zinc have been studied in their
divalent form with pH ranges of 4.57.5 and 57.5, respectively. Potential 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 Pb2 removal abilities. In addition,
immobilized cells of C. reinhardtii are also reported to have signicant
lead removal (380.7 mg g 1; Bayramolu 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 tabulation, it could be stated that live as well as immobilized microalgae 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

Romera et al., 2007
Singh et al., 2007
Sandau et al., 1996b
China
Thailand
Brazil
India
Spain
Malaysia
Germany
Turkey
Germany
Brazil
Germany
Portugal
Sweden
India
Morocco
Germany
USA
India
Morocco
Germany
Portugal
Egypt
Germany
Spain
India
Germany
Poland
Poland
metabolic dependence (Gadd, 1990). In particular, the mechanisms

by which microorganisms remove metals from solutions include:
(i) extracellular accumulation/precipitation which can be facilitated 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 mechanisms 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 biomass or biomass products. This rst process (comprising bioaccumulative uptake) forms the principle for waste detoxication processes (for e.g. biological uidized beds employing continually
growing biolms). 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
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 removed Cd was adsorbed on algal cell surfaces. They claimed a
faster removal of Cd by dried cells rather than by living cells.
Nevertheless, Prez-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
modied 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 rapid (passive) removal of metals by the cell, occurring at the cell
surface, and (ii) a much slower one that occurs inside the cell.
The rst process (i.e. the passive removal), is non-metabolic,
rapid, and essentially reversible, occurring in both living and nonliving cells; here, HM ions are adsorbed to functional groups
present on the cell surface by electrostatic interactions. However,
these groups differ in their afnity for metal and specicity
binding. This process includes physical adsorption, ion exchange,
chemisorption, coordination, complexation, chelation, microprecipitation, entrapment in the structural polysaccharide network, and diffusion through the cell wall and membrane (Monteiro 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 irreversible. Moreover, as most of the HMs are hydrophilic, their transport through the partially lipophilic biological membrane surrounding the cell is mediated by specic 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 efux 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 toxicity; 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 resistance mechanisms in microalgae and claimed that, complexation and microprecipitation are the most efcient mechanisms,
even though ion exchange dominates.
Reports on utilization of live microalgae for metal remediation
suggest various specic pH values for efcient 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 cytoplasm, is the cell wall, which acts as the rst 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 rst 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;
Dnmez et al., 1999; Monteiro et al., 2012). Crist et al. (1981) exclusively 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 coordination bonding with metallic ions like Cu2 . However, such
bond formation could be accompanied by the displacement of
protons dependent in part on the extent of protonation, as determined 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; phosphate, PO3; amino, NH2; and sulfhydryl, SH) that confer a net
overall negative charge to the cell surface, and concomitantly a
high binding afnity for metal cations via counterion interactions.
Chojnacka et al. (2005) conrmed 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 ux
Membrane transport of the metal ions into the cytoplasm of the
cell is a vital process. Particularly, the plasma membrane metal
338
transporters are pivotal in the interaction of algae with their environment. These membrane transporters represent the rst line
of defense during cellular perturbations occurring during HM exposure. Analysis of the algal metal transporter repertoires could
provide insight into a fundamental aspect of algal biology. According 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 ResistanceAssociated Macrophage Proteins), ZIP (Zrt-, Irt-like Proteins), FTR
(Fe TRansporter) and CTR (Cu TRansporter) families. The assimilative 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 deciency. 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 transporters, which provide metal for organelle-localized metal-dependent proteins. When present in the membranes of the secretory pathway, Group B transporters when present in the membranes of the secretory pathway mediate the exocytosis of excess
metal. This group includes members from the CDF (Cation Diffusion Facilitator), P1B-type ATPases, FPN (FerroPortiN) and Ccc1 (Ca
(II)-sensitive Cross-Complementer 1)/ VIT1 (Vacuolar Iron Transporter 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 transporters 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 addition, 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 deciency 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) conrmed 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), phosphate (PO3), diphosphorus trioxide (P2O3), amino (NH2), sulfhydryl (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 afnity for metal cations via counter ion interactions,
thereby endorsing metal exchange via ion exchange mechanisms
(Dnmez et al., 1999; Mehta et al., 2002; Monteiro et al., 2012; He
and Chen, 2014). Gonzlez-Dvila (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 , Mn2 , Zn2 , Ni2 , Cd2 , Fe3 and Pb2 are present, the
previously bound protons and metals are released and the later
metal ions (Cu2 , Mn2 , Zn2 , Ni2 , Cd2 , Fe3 , and Pb2 ) 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 signicantly; for e.g. some
metalligand 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 (Gonzlez-Dvila, 1995). Crist et al. (1990,
1992) supportingly state that in natural waters, the sorption process (adsorption plus assimilation) is an ion exchange process with
the same amount of metal assimilated as the sum of Na , Ca2 ,
Mg2 and H released.
Several studies on electron microscopy, including X-ray energydispersive analysis showed that most sites for metal sorption are
located on the cell surfaces, and adsorption via ion exchange appears 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 polysaccharides, phosphate and amino groups of nucleic acids, amino
and carboxyl groups of proteins and hydroxyl groups of polysaccharides) are replaced by negative charges that increase the
attraction of metallic cations and their adsorption to the cell surface. 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 solutions, 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 containing 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 approach 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 extracellular complexation or coordination occurring due to
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 attraction, and when they cling to the metallic ions, there is no electron
transfer. However, the nal 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 biosorption in C. vulgaris takes place through the formation of coordination 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 negative 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 precipitation, 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 precipitation in the vacuole of T. suecica.
5.1.7. Metallothioneins
Metallothioneins were rst characterized by Vallee and coworkers in the late 1950s in studies that were extended thereafter
(Stillman, 1995). Metallothionein proteins, products of mRNA
translation, are characterized as low molecular weight (67 kDa),
cysteine-rich, metal-binding proteins. The induction of metallothioneins (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 efux of HMs or enzymatic change of
speciation to achieve detoxication of metals. Reports also emphasize 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 algaemetal interactions (Perales-Vela et al.,
2006). These metallothioneins are structurally diverse, low-molecular-weight, cysteine-rich, polypeptides that complex soft
metal ions in thiol clusters. Several researchers conrm the presence 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 , Ni2 , Cd2 , Ag , Bi3 , Pb2 , Zn2 , Cu2 , Hg2 and Au2 ,
induce class III MT biosynthesis, both in vivo and in vitro, in several microalgae for e.g. S. subspicatus, S. bacillaris, S. tenue, and
Thalassiosira weissogii (Robinson, 1989a, b; Ahner and Morel,
1995; Knauer et al., 1997, 1998; Pawlik-Skowroska et al., 2003a, b,
2004; Perales-Vela et al., 2006).
5.1.7.1. Phytochelatins (PCs). While studying metal exposure to algae, one should denitely consider that the mechanisms may involve sequestering toxic metals by HM-binding Cys-rich proteins,
such as class II metallothioneins and non-translationally synthesized polypeptides sometimes described as class III metallothioneins (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 transpeptidase named phytochelatin synthase (the constitutive enzyme) 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 n 36. Pawlik-Skowroska (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-Skowroska (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
(Bakor et al., 2007). Wei et al. (2003) also reported that the addition 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 Zn, and Cu Zn caused a similar 23 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 unfolds: (i) a possible mechanism of regulation of the free metal ion
concentration in the cytosol, and, (ii) a probable mechanism of
detoxication (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
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 (Ballan-Dufrancais et al.,
1991), and, the diatom Skeletonema costatum which accumulated
Cd2 and Cu2 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 (Cyanobacterium), Pseudochlorococcum typicum and S. quadricauda var quadrispina (both Chlorophyta)], tested for tolerance and removal of
mercury (Hg2 ), lead (Pb2 ) and cadmium (Cd2 ) in aqueous
solutions, Shanab et al. (2012) reported that dark spherical electron dense bodies were accumulated in the vacuoles of algal cells
exposed to Pb. However, Shanab et al. (2012) assert that the mechanism 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, metalloiron or metallo-phosphate complexes in the cytosol, and are carried 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 mechanism, or, a detoxication mechanism (Shanab et al., 2012).
however, they mention that this organism lacked specialized reservoir organelles such as plant-like vacuoles. Similarly, the presence of Cd2 inside the chloroplast has also been reported for
Chlamydomonas reinhardtii (Nagel et al., 1996). Soldo et al. (2005)
found Oocystis nephrocytioides exposed to Cu2 to accumulate
high concentrations of Cu2 in the thylakoids and pyrenoids. They
conclude that localization of Cu2 suggests interaction of Cu2
with ligands localized in the chloroplast. Alternatively, Cu2 might
have been transported from the cytosol to the chloroplast as a
Cu2 ligand complex (Perales-Vela et al., 2006).
Mendoza-Czatl and Moreno-Snchez (2005) reported E. gracilis to accumulate Cd2 inside the chloroplast. Avils et al. (2003)
stated that Hg2 pretreated heterotrophic cells of E. gracilis exposed to Cd2 accumulated some of the metal in the mitochondria. Mendoza-Czatl et al. (2004) explained that the presence of
MtIII and Cd2 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 ; the CdMtIII complexes
are subsequently transported into the chloroplast and mitochondria, (ii) MtIII are synthesized inside the organelle where they bind
to Cd2 , which are transported as free ions and then form HMW
complexes, or (iii) both processes co-exist and MtIII are synthesized in the three cellular compartments (Perales-Vela et al.,
2006).
5.1.9. Polyphosphate bodies in algae

Detoxication of metals in a microalgae could be achieved either by: (i) binding to specic intracellular compounds and/or
transport to specic cellular compartments such as vacuoles or
polyphosphate bodies, or (ii) efux of metals back into solution by
active transport (Monteiro et al., 2012); moreover, association of
divalent cations with polyphosphate and corresponding trafcking
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 acidocalcisomes 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 distinctive 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 sequester metals such as Ti, Pb, Mg, Zn, Cd, Sr, Co, Hg, Ni and Cu,
thereby performing two different functions viz. providing a storage pool for metals and enabling a detoxication 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.
5.1.11. 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 exclusion 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 dinoagellate 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 and Cu2 (Pea-Castro et al., 2004).
Perales-Vela et al. (2006) provided an example of the green alga,
Chlamydomonas reinhardtii, wherein Hg2 was not chelated by
MtIII, but by glutathione. This indicated that glutathione played
numerous roles, not only as the cGluCys 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 stress. In their study, they
reported that Tetraselmis tetrathele used a novel tripeptide, ArgArg-Glu possessing a Hg2 -scavenging capacity that was responsible for the detoxication mechanism.
In addition, Perales-Vela et al. (2006) emphasized that few algae respond to HMs by the production of proline (Pro).
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 ), lead
(Pb2 ) and cadmium (Cd2 ) in aqueous solutions. This clearly
accounted for the possibility that HMs could nd their way to
other organelles in a microalgal cell. In an earlier study, PeralesVela et al. (2006) provided an evaluation of the role of chloroplast
and mitochondria of microalgae in response to HMs. They cited
Mendoza-Czatl et al. (2004) who studied Euglena gracilis with a
high tolerance to Cd2 , and high Cd2 accumulating capacity;
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 uncomplicated 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
particular, depends on the species of metal ion, the algal species,

chemical composition of the metal ion solution, prevailing environment, and several other factors. A great deal of attention has
been focused on the use of non-living algal biomass as a potential
industrial tool for the extraction of toxic metal ions from wastewaters and mining efuents. Aksu (1998) highlighted that differences in algal taxonomy, variation in cell structure, the type of
metal ion and its manner of binding, as well as, the chemical
composition of the metal ion solution, could inuence the metal
binding.
There are several reports available on various perspectives of
metal sorption by microalgae; for e.g. Wilke et al. (2006), in simultaneous sorption processes, studied 30 algae, and reported the
following order of selective sorption: Pb 4Ni 4Cd 4Zn. Tzn
et al. (2005) described algal afnity and selectivity for metal ions,
wherein Chlamydomonas reinhardtii was reported to have maximum Hg(II), Cd(II), and Pb(II) ions biosorption capacities
(72.2 70.67, 42.6 70.54 and 96.3 70.86 mg g 1 dry biomass, respectively). Here, the afnity order for algal biomass was Pb(II)
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,
42.6 70.54 and 96.3 70.86 mg g 1 dry biomass.
Several metallic anion complexes and oxoanions appear to bind
electrostatically to algal cells, although covalent bonding is certainly possible. Certain reports suggest algal biosorption of metal
ions such as aluminum (III), copper (II), lead (II), and cobalt (II) to
occur via an ion-exchange process with metal cations competing
with protons for negatively charged binding sites on the cell wall.
The binding sites could be carboxyl groups, or sulfates associated
with polysaccharides and proteins. Likewise, Gardea-Torresdey
et al. (1990) indicated that algal biosorption of metal ions appears
to occur via an ion-exchange process with metal cations competing with protons for negatively charged binding sites on the cell
wall. Michalak and Chojnacka (2010), afrmed that Ca (II), Na (I), K
(I), and Mg (II) ions were released from the biomass after biosorption of Cu (II), Mn (II), Zn (II), and Co(II) ions, indicating that
ion exchange was a key mechanism in the biosorption of metal
ions by Vaucheria spp. biomass.
Contrarily, Lau et al. (1999) conceptualized the process of biosorption as the sorption or complexation of dissolved metals by
dead or metabolically inactive biomass where the biosorption
ability of the biomass relates to the anionic nature of their cell
wall. The authors reported that anionic groups such as carboxyl,
phosphate, pyruvate, amide, as well as, extracellular polysaccharides, aminophosphates, and lipoproteins are able to bind
metal ions by electrostatic forces. In addition, Aksu (1998) also
reported that biosorption of metal ions by algal biomass arose
from the coordination of the ions to different functional groups on
algal cells including amino, carboxylic, sulfhydryl, phosphate, thiol
groups and sulfate groups, provided by polysaccharide building
blocks of the algal cell walls; amino groups and the nitrogen and
oxygen of the peptide bond (provided by proteins); or other
groups such as carboxyl oxygen and sulfate. Aksu (1998) stated
that biosorption of HM ions in algae, was better than precipitation
in terms of ability to adjust to changes in pH and HM concentrations, and, it was better than ion exchange and reverse osmosis in
terms of sensitivity to the presence of suspended solids, organics,
and the presence of other HMs; they further mention that only ion
exchange could compete with biosorption in terms of residual HM
concentrations.
Compared to traditional HM removal technologies, biosorption
by non-living microalgae have the following advantages:
capacity to treat large volumes rapidly

high selectivity and specicity for particular HMs
341
ability for multi-metal removal as well as cleansing of mixed

wastes comprising HMs
high afnity and high efciency

fewer supplementary expensive reagents are required (these
reagents usually cause disposal and space problems)
does not require growth media and nutrients

active over a wide range of physicochemical conditions in

cluding temperature, pH, and presence of other ions such as

calcium and magnesium
moderate capital investment and low operational costs
involves the use of naturally abundant renewable bioresources
which are produced economically
ecofriendly (it not only reduces the level of risk of hazardous
waste, but also evades the use of harmful chemical technologies
which could cause secondary pollution)
metal removal system is not subjected to metal toxicity limitations, and
signicant recovery of HMs from the biomass is feasible (i.e.
desorption of the HM and reuse of algal biomass).
Sorption of HMs using dead biomass of microalgae has gained

much popularity due to the ease of handling. Various pH values (29) have been studied for metal removal by non-living microalgae
(Table 1). Most researchers have focused on cadmium, copper and
lead removal by dead biomass. In addition, various mechanisms of
uptake have been proposed; a few of the mechanisms detailed
above for living microalgae, also hold true for HM uptake by biomass (for e.g. the signicance of functional groups).
5.2.1. Signicance of functional groups in heavy metal uptake
Gao et al. (2010) in their study on glow discharge electrolysis
plasma emphasized the importance of functional groups (e.g. hydroxyl-, thio-, iminodiacetic- and amine groups) in absorption. They stated that nitrogen-containing functional groups are among the most
effective functionalities in the adsorption or removal of HM ions.
Similarly, for any commercially viable metal recovery or remediation technology, it is essential that the functional groups of
biomaterials responsible for metal uptake are identied and their
contribution to overall metal binding capacity are quantied
(Rayson and Williams, 2011). In biological systems, hard ions form
stable bonds with OH , HPO42 , CO32 , RCOO , and QCQO
(all of these groups include oxygen atoms); while the soft ions
form very strong bonds with CN , RS , -SH , -NH2 , and imidazole (i.e. groups containing N and S atoms) (Baldrian and Gabriel
2003). This implies that hard ions (smaller in size) such as Fe, Zn,
Mn would prefer to bind with OH , HPO42 , CO32 , RCOO , and
QCQO, while soft ions (with a larger diameter) such as Cu, Pb
and Cd would bind to CN , RS , SH , NH2 , and imidazole.
Although researchers have attempted to determine the binding
mechanism of metal ions to algal surfaces, it is necessary to establish which chemical groups (especially present on the algal cell
wall), are responsible for binding to different metal ions. Reports
suggest that the biosorption, or binding, of metal ions by algal
biomass arises from the coordination of the ions to different
functional groups in or on the algal cell. These coordinating groups
(provided by proteins, lipids, and carbohydrates) include amino,
thioether, sulfhydryl, carboxyl, carbonyl, imidazole, phosphate,
phenolic, hydroxyl, and amide moieties (Gardea-Torresdey et al.,
1990). In the algal context, Crist et al. (1988) studied the interactions of metals and protons with Vaucheria spp., Spirogyra spp.,
and Oedogonium spp., and indicated that metals adsorb to algal
surfaces by electrostatic attraction to negative sites such as carboxylated anions of polygalacturonic acid.
Carboxyl groups are known for their high selectivity for protons
(Kratochvil and Volesky, 1998). Gardea-Torresdey et al. (1990)
342
studied the carboxyl groups of biomass of ve different algal

species, and reported that carboxyl groups on algal cells are responsible 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 RCOOH of algal and fungal cell wall constituents as the
probable sites for ion exchange. Moreover, esterications of carboxyl 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. Esterication
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
conrmed 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 specically mention about the role of sulfate present in the glycoproteins of the cell wall in metal binding.
Gonzlez et al. (2011) also conrmed that functional groups containing O-, N-, S-, or P-, participate directly in the binding of certain
metals; they also specied 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 ROSO3 . Lefebvre et al.
(2007) described the ability of cyanobacterial strains (Limnothrix
planctonica, Synechococcus leopoldiensis and Phormidium limnetica)
to convert Hg2 into elemental mercury Hg and meta-cinnabar
(-HgS) under controlled pH and aerated conditions. They attributed this to the interaction with metal binding sulfhydryl protein
as an intermediate step in the metal sulde synthesis. Furthermore, Lengke et al. (2006) investigated gold bioaccumulation by
the cyanobacterium Plectonema boryanum from gold (III)-chloride
solutions and conrmed the role of thiol compounds in the reduction mechanism of gold (III) to metallic gold by this organism.
Essa and Mostafa (2013) studied metal bioprecipitation and tested

the efciency of three cyanobacterial isolates (S. platensis, Nostoc
muscorum, and Anabaena oryzae) to precipitate some HMs (Hg2 ,
Cd2 , Cu2 and Pb2 ). FT-IR studies showed the existence of OH
groups in the metal precipitate produced by algal isolates, while
NH groups were identied 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 bioremediation through the transformation of these metals into nitrogen complexes and/or hydroxide complexes via using the culture 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 Estephane, 2012); whereas, the OH group could bind with Pb, Cr, Cd,
Cu, (Sheng et al., 2004; Chojnacka et al., 2005; Flouty and Estephane, 2012; Greene et al., 1986; Flouty and Estephane, 2012).
The incidence of different binding groups on biomass surface involved in metal ion complexation has been well described by
Nurchi and Villaescusa (2011). These authors proposed the higher
to lower afnity ranking of the groups as follows: carboxylate4aromatic 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 difcult to clearly decipher and elaborate mechanisms and technologies separately for live microalgae and their biomass because a number of processes overlap.
6. Factors affecting heavy metal remediation

Although microalgal cells have several self-defense mechanisms to survive in metal-containing media, various factors inuence 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 nutrition 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 biosynthesis pathway, PC complexes and enzymes involved in the PC synthesis (GCS- glutamylcysteinyl synthase, GS- Glutathione synthase, PCS- phytochelatin synthase); AAAmino 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 (modied
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 gure, the reader could
refer the web version of this article).
atomic weight or reduction potential of the metal, organic substances, pH, temperature, salinity and hardness, inorganic ligands,
interactions, and others (Wang, 1987; Dnmez et al., 1999). Monteiro 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 Arc et al. (2005), at pH 2, heat-treated and acidtreated biomass of Chlamydomonas reinhardtii, more efciently
removed Cr6 , as compared to the non-living (unmodied) biomass (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
(Gonzlez et al., 2011).
6.1.2. Tolerance capacity
Metals break the oxidative balance of the algae, inducing antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and ascorbate peroxidase (APX) (Arunakumara 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 algae have acquired successful adaptation mechanisms after exposure 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 reducing HM toxicity, numerous mechanisms have been proposed
for reducing HM toxicity in organisms. These include the production 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 compartmentalization (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 chelating compounds, active efux 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 cytoplasmic concentration of HM ions, thereby preventing or neutralizing their potential toxic effects.
Reports suggest that phytoplanktons that are initially acclimated 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 contained 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 concentration, 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, resulting 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 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 denitely 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 processes, are all strongly size-dependent. Small algae achieve higher
rates of photosynthesis and have higher specic 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 volume 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
Levy et al. (2007) reported Micromonas pusilla having the smallest

surface area was approximately twice as tolerant as compared to
Minutocellus polymorphus, while, the dinoagellate 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; moreover, small cells may also recover faster due to their rapid growth
rates (Levy et al., 2007).
Nonetheless, the size of the inoculum can also inuence 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 inuencing 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 inuencing
metal adsorption by microalgal biomass (Brinza et al., 2007; Jeba
kumar et al., 2013; Indhumati et al., 2014). It inuences the speciation of metals in solution and algal tolerance (Brinza et al.,
2007); particularly, it inuences both, cell surface metal binding
sites, and, metal chemistry in water. Peterson et al. (1984) provided 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 acidbase 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 , 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, imidazole and amino groups would be exposed (these carry negative
charges), and subsequently an attraction of positive charged metallic ions via a process of biosorption onto the cell surface ensues
(Dnmez et al., 1999). Monteiro et al. (2012) also opine that at low
pH, functional groups are associated with H ions, thus hampering the positively charged metal ions from binding (because of
repulsive forces). They state that as the pH increases, those functional 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 attraction. 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 occur; this normally decreases the extent of metal removal, hence, it
is necessary to determine the optimal pH for algaemetal interactions. For example, at pH values higher than the optimum pH
(3.54.0), enhanced Zn toxicity was reported in the alga Hormidium 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 microand macroalgae, stating that metal solubility function by pH variations has to be a criterion in choosing the type of biomass (dead
or live macro/micro-algae) and understanding its uptake afnity

and mechanism; they state that at pH o 3 uptake capacity decreases because hydrogen and metal ions are in competition for
the binding sites, while above pH 6.5, heavy metals tend to precipitate 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 and negatively charged binding sites causing the
rapid rise in the binding efciency at lower pH (4.0). Shengjun and
Holcombe (1991) showed that 0.12 M HCl rinsed Chlorella had a
better Ni2 and Co2 adsorption.
Several functional groups are available for binding metal cations
at distinct pH ranges, i.e. at pH 25, carboxyl groups dominate, but
at pH 59 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 carboxyl 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
inuence of pH on metal accumulation by algae is quite speciesspecic (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. Star 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 59, 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 efciency, with decreasing
ionic strength. At a particular pH, the number of functional groups
is xed, 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 bioremediation of HMs by algae, the presence of high concentrations
of monovalent cations (Na and K ) 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 efciency increases (from 80% to 95%), while high ionic
strength is found to decrease the extent of biosorption; they hypothesized the competition for the functional groups between
metal ions and other ions had played an important role in adsorption. 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 inuencing HM algal
uptake mainly focus on macroalgae, one could not rule out the
possibility of similar mechanisms and interpretations in microalgae. However, indepth investigations need to be focused on this
eld.
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.
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, considerably 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 alkalinity/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 effect 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 endothermic but weakened if it is exothermic. Basically, metalcarboxylate interactions are endothermic, whereas metal-amine interactions 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 temperature 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) observed an increasing adsorption of Ni2 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 by Oedogonium spp. at higher temperatures, 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 biosorption, but its inuence 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 , while it accumulated 333 mg g 1 Cr6 (Table 1).
Despite the fact that metals could occur under a variety of
chemical forms (free ions, complexes with inorganic/organic ligands, 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 observed 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 ). However, there is a lack of data that combines
measurements of metal speciation and its effects on microorganisms. Therefore, the exact idea regarding the effect of metal speciation on algae under eld 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 summarized 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-interactive, 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 efuents of electroplating operations.
Therefore, examination of the effects of metal cations in multimetal 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 intricate, without a set pattern. Considering the competitive interactions between the HM and the adsorption binding sites on the
cell surface, Monteiro et al. (2012) cited several studies on mixtures 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 individually, and found that their mixtures at the same concentration were strongly inhibitive. Fraile et al. (2005) revealed that the
presence of Cd2 decreased the uptake of Zn2 in a competitive
manner. Further, Aksu and Dnmez (2006) described competitive
adsorption of Cd2 and Ni2 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 claried
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 favored 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
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 algae (e.g. structure, functional groups and surface area, depending
on the algal division, genera, and species) (Dnmez et al., 1999).
Microalgae have been discussed in extenso for HM removal from
efuents 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. reinhardtii, C. vulgaris, Scenedesmus spp., and Chlorococcum spp.
could be outlined as potential candidates that could be commercialized for various metal remediation technologies. On the other
hand, the cyanobacteria Lyngbya spiralis, Tolypothirx tenuis, Stigonema spp., Phormidium molle, Aphanothece halophytica, C. paris,
and Spirulina also hold a great potential for HM remediation
(Tzn et al., 2005).
8.2. Transgenic microalgae

In order to further enhance the HM specicity and binding
capacity of microalgae, transgenic approaches have been developed recently with an objective to use microalgae for HM treatment 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-afnity, 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 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
(Huang et al., 2006).
However, in order to preclude these transgenic algae with good
metal remediation capacities, it is essential to have clever engineering and biological considerations. The release of live transgenic microalgae with enhanced metal binding skills into the environment 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. Strategic approaches in heavy metal remediation
8.3. Metal desorption
8.1. Immobilized microalgae and metals
Due to their special cell wall structure, high HM-removal capacity, 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 efciency 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 (Monteiro 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 efciency, 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 ,
Cd2 , and Cu2 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 inicting damage upon metal
binding sites, including hydrolysis of polysaccharides on the surface of the cell wall when applied in sequential cycles.
The use of metabolically active immobilized microalgae is

particularly an attractive option for remediation of metal ions at
low concentrations, especially for detoxication and metal recovery processes. Immobilized algal systems have been tested by
several researchers for their efciency 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, polyurethane foam, surface of Ulva spp., surface of Sargassum spp., silica gel, Luffa cylindrica sponge, milk casein glutaraldehyde, alginate, 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 cyanobacteria (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 efcacy of C. vulgaris cells immobilized in alginate as a suitable system to remove a wide range
of HMs. Aksu et al. (1998) reported the total Cu(II) removal by Caalginate gel immobilized C. vulgaris was higher than that of agarose- 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 biosorption 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.
8.4. Recycling of microalgal biomass

Among the techniques used for algal recovery, the use of the
occulating 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
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 employed, 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 recommended. 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 aqueous solutions, consists of dead algal cells immobilized in a silica
gel polymer (Kuyucak and Volesky, 1990). There are several varieties of AlgaSORB available, for instance the algae-silica preparation (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 1100 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 adsorbent material for the removal of metals from industrial efuents (Monteiro et al., 2012).
Yet another biosorbent BIO-FIX, made up of a variety of biomasses including Sphagnum peat moss, algae (Ulva spp., and
Spirulina), yeast (Saccharomyces cerevisiae), bacteria, and/or aquatic ora (Lemna spp.) immobilized in high density polysulfone, has
also gained attractiveness (Garnham 1997; Vijayaraghavan and
Yun, 2008; http://www.slideshare.net/sjcc/environmental-problems-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 afnity of BIO-FIX is as follows: Al3 4Cd2 4
Zn2 4 Mn2 , apart from this it has a much lower afnity for
Mg2 and Ca2 (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 crispus). 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 containing immobilized cells (BIOALGA) that met with commercial
success; using this bioreactor with S. obliquus, a maximum removal 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 specic bioremoval applications could have metalremoval 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 inorganic 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 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, Oscillatoria, Anabaena) or consortia of both (Craggs et al., 1996). It has
been tested for treating polluted underground waters for the efcient 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 enhances metal precipitation. In yet another study, Phillips et al.
(1995) reported that a consortium of algae and cyanobacteria effectively decreased excessive Mn concentrations to an environmentally 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 candidates for efcient and commercially feasible wastewater bioremediation strategies have been proven, there is a lack of commercialization of technologies in this eld. Currently, there is a
rising demand to develop microalgal technologies based on microalgae such as Chlamydomonas reinhardtii (having the ability to
sequester a wide range of HMs including: copper, zinc, lead, cadmium, 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 (Tzn et al., 2005).
10. Conclusion and future avenues

The implementation of meticulous legislation worldwide and
the increase in demand for metal resources has motivated enormous growth in diversied avenues of biosorbent research. Algae, show capabilities for accumulating, adsorbing and recovering
HMs, are well-studied, for their potentiality, mechanism of metal
accumulation, modeling of metal adsorption, and genetic manipulation. 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 physicochemical methods, and their usefulness in large-scale remediation
technologies as well as in removal of metals from efuents. Several
biotic and abiotic parameters inuencing 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, temperature, salinity and hardness, etc.). Now a days, signicant advances 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 technological applications. However, it is essential to deliberate on various
avenues of microalgal remediation technologies as ecofriendly
alternatives for a better environment.
348
Microalgal remediation capabilities offer advantages over other

biological materials in some conceptual process schemes. Microalgae are known to perform well at low levels of contaminants;
they do not generate toxic sludge, are easy to culture and maintain, have a good binding afnity (due to relatively high specic
surface area and net negative charge), and, are suitable for smallscale 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 carbon dioxide mitigation; however, the main advantages of microalga-mediated sorption include its efcacy 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 benecial in this regard. With increasing availability of knowledge regarding improved selectivity, better shelf-life, efcacy, specicity, 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 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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Ecotoxicology and Environmental Safety 113 (2015) 329352

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

Microalgae A promising tool for heavy metal remediation

Corresponding author. Fax: 82 31 416 6173.

2. Hazardous effects of heavy metals

to humans). Though the adverse health effects of HMs are known

3. Conventional vs. novel approaches in heavy metal

have moderate or no metal selectivity (Antunes et al., 2003), need

4. Microalgae and their potential in metal remediation

microalgae in metal biosorption. The benets include: rapid metal

Metal uptake (mg/g)

Sandau et al., 1996a

Metal uptake (mg/g)

Doshi et al., 2008

Metal uptake (mg/g)

Chojnacka et al., 2004

Metal uptake (mg/g)

Singh et al., 2007

used organism was C. vulgaris in various forms (non-living, living, free,

5. Metal removal mechanisms adapted by microalgae

Schmitt et al., 2001

metabolic dependence (Gadd, 1990). In particular, the mechanisms

resting cells. This resulting adsorption is quite rapid, occurs to a high

et al. (2012) stated that the algal resistance mechanism comprises

sorbed on the alga. But, in the presence of anionic complexing

electrostatic attraction between a metallic ion chelating agent and

contained cadmium and sulfur (in ratios between 2 and 2.4).

5.1.9. Polyphosphate bodies in algae

5.1.11. Other mechanisms of heavy metal remediation

5.1.10. Sequestration to the chloroplast and mitochondria

5.2. Biosorption by biomass

particular, depends on the species of metal ion, the algal species,

capacity to treat large volumes rapidly

ability for multi-metal removal as well as cleansing of mixed

high afnity and high efciency

does not require growth media and nutrients

cluding temperature, pH, and presence of other ions such as

Sorption of HMs using dead biomass of microalgae has gained

studied the carboxyl groups of biomass of ve different algal

Essa and Mostafa (2013) studied metal bioprecipitation and tested

6. Factors affecting heavy metal remediation

Levy et al. (2007) reported Micromonas pusilla having the smallest

or live macro/micro-algae) and understanding its uptake afnity

substantial increase in the Ni concentrations (300 mg l 1) caused

7. Potential microalgal species for metal remediation

8.2. Transgenic microalgae

8. Strategic approaches in heavy metal remediation

8.3. Metal desorption

8.1. Immobilized microalgae and metals

The use of metabolically active immobilized microalgae is

8.4. Recycling of microalgal biomass

method is chemical attachment wherein living cells are at stake,

9. Algal biosorbent technologies currently in use

(de-Bashan and Bashan, 2010). Algae-based biotechnologies that

10. Conclusion and future avenues

Microalgal remediation capabilities offer advantages over other

Pawlik-Skowroska, B., 2003b. Resistance, accumulation and allocation of zinc in

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