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Biosorption and removal of toxic heavy metals by metal tolerating bacteria

for bioremediation of metal contamination: A comprehensive review

Monika Priyadarshanee, Surajit Das

PII: S2213-3437(20)31035-6
DOI: https://doi.org/10.1016/j.jece.2020.104686
Reference: JECE 104686

To appear in: Journal of Environmental Chemical Engineering

Received Date: 19 August 2020

Revised Date: 16 October 2020
Accepted Date: 28 October 2020

Please cite this article as: Priyadarshanee M, Das S, Biosorption and removal of toxic heavy
metals by metal tolerating bacteria for bioremediation of metal contamination: A
comprehensive review, Journal of Environmental Chemical Engineering (2020),
doi: https://doi.org/10.1016/j.jece.2020.104686

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Biosorption and removal of toxic heavy metals by metal tolerating bacteria for
bioremediation of metal contamination: A comprehensive review

Monika Priyadarshanee and Surajit Das*

Laboratory of Environmental Microbiology and Ecology (LEnME), Department of Life

Science, National Institute of Technology, Rourkela- 769 008, Odisha, India

*Correspondence. Tel. +91661 2462684; Fax. +91661 2462022

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E-mail: surajit@nitrkl.ac.in or surajit@myself.com

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Graphical Abstract

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Highlights

 Bacteria are efficient, cost-effective adsorbents for heavy metal ions

 Different structures and components of bacteria aid in the metal uptake process
 Environmental parameters influence the metal uptake process
 Various kinetics and isotherm models are used to analyze the adsorption mechanism

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  Regeneration of adsorbents is useful for several cycles of biosorption of metals

Abstract
Heavy metal pollution caused due to the industrialization has been considered as a significant
public health hazard, and these heavy metals exhibit various types of toxicological
manifestations. Conventional remediation methods are expensive and also yield toxic by-
products, which negatively affect the environment. Hence, a green technology employing
various biological agents, predominantly bacteria, algae, yeasts, and fungi, has received more
attention for heavy metal removal and recovery because of their high removal efficiency, low
cost, and availability. However, bacterial biosorption is the safest treatment method for the

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toxic pollutants that are not readily biodegradable such as heavy metals. Metal biosorption by
bacteria has received significant attention due to a safe, productive, and feasible technology

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for the heavy metal-containing wastewater treatment. These metal tolerating bacteria can bind
the cationic toxic heavy metals with the negatively charged bacterial structures and live or
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dead biomass components. Due to the large surface area to volume ratio, these bacterial
biomasses efficiently act as the biosorbent for metal bioremediation under multimetal
conditions. This review summarizes the biosorption potentials of bacterial biomass towards
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different metal ions, cell wall constituent, biofilm, extracellular polymeric substances (EPS)
in metal binding, and the effect of various environmental parameters influencing the metal
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removal. Suitable mathematical models of biosorption and their application have been
discussed to understand and interpret the adsorption process. Furthermore, different
desorbing agents and their utilization in heavy metals recovery and regeneration of biosorbent
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have been summarized.

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Keywords: Heavy metals, biosorption, bacterial biomass, bioremediation, desorption,

adsorption kinetics
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Table of contents
1. Introduction
2. Treatment methods of heavy metal contamination
2.1. Conventional methods
2.2. Biological methods for treatment of contaminated effluents

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 2.3. Why biosorption is better alternative than other treatment methods?
3. Mechanisms of bacterial biosorption
3.1. Bacterial structure and components associated with biosorption
3.2. Mechanisms involved in heavy metal biosorption
4. Various bacterial biosorbents for toxic metal removal
4.1. Bacterial biomass in toxic heavy metal removal
4.2. Bacterial biofilm and EPS in metal removal
4.3. Modification of bacterial biosorbents
4.3.1. Physically modified bacterial biosorbents
4.3.2. Chemically modified bacterial biosorbents

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4.3.3. Biologically modified bacterial biosorbents
5. Biosorption of heavy metals under multimetal stress

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5.1. Biosorption of metals in the binary and ternary metal ion solution
5.2. Influencing parameters in bacterial uptake of heavy metals
6. Adsorption kinetics and isotherm models for evaluating the biosorption process
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6.1. Adsorption kinetics for the biosorption process
6.2. Various isotherm models for characterization of biosorption
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7. Desorption and recovery of heavy metal ions
7.1. Eluents used for desorption of heavy metals
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7.2. Estimation of heavy metals after desorption

8. Conclusion
9. Acknowledgements
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10. Declaration of interest statement

11. References
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1. Introduction
The term “heavy metal” denotes the group of metals and metalloids with an atomic
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density of more than 4 g/cm3, which is five times higher than water (Garbarino et al., 1995).
Various toxic heavy metals such as lead (Pb), chromium (Cr), zinc (Zn), cadmium (Cd), iron
(Fe), copper (Cu), manganese (Mn), and nickel (Ni) are released from different industrial
manufacturing or various catalytic processes through their water and solid waste discharges
(Ghaly et al., 2014). These toxic metals are persistent environmental contaminants because
they are non-degradable or non-destroyable and continuously accumulated in the food chain,
causing a severe threat to the environment (Tak et al., 2013). Unlike organic pollutants, heavy

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metals are not converted to simpler forms by chemical or biological methods. They can only
be transformed into less toxic species, e.g., Hg2+ to the less toxic form Hg0 (Dash and Das,
2014), As3+ to As5+ (Biswas and Sarkar, 2019), and Cr6+ to Cr3+ (Mohamed et al., 2020). The
natural environment has been polluted by the continuous accretion of heavy metals and
metalloids through discharges from the rapidly escalating industrial practices, ore mining,
dumping of elevated metal wastes, chemical residues of gasoline and paints, excessive use of
chemical fertilizers in cultivation land, sewage sludge, insecticides, pesticides, utilization of
wastewater for agricultural purpose, leftovers of coal combustion, spillage of organic
hydrocarbons, and atmospheric deposition (Zhang and Wang, 2020). The excessive mining of
ores results in the discharge of the metal-containing residues on the land surface, entering the

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soil system, and contaminating the surface and groundwater (Verma and Dwivedi, 2013).
From the soil and water, heavy metals are accumulated in the animal manure through the

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metal-contaminated food and water (Chen et al., 2020).
Heavy metals remain as natural constituents of the environment, and to a small extent,
they enter the body system along with food, air, and water and are bio-accumulated (Khan et
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al., 2015). In trace amounts, a few of the heavy metals such as Cu2+, Zn2+, Fe2+/Fe3+, etc. are
needed for various vital biological activities in human physiology, but toxicological
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manifestation arises in humans even at slightly higher concentrations (Batayneh, 2012). The
factors associated with metal toxicity are the dose, route of exposure, chemical species, and
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the age, gender, genetics, and nutritional status of exposed individuals. When the heavy
metals are consumed above the bio-recommended level, various harmful effects are noticed.
Some of the toxic heavy metals responsible for various types of ailment are Cd, Pb, As, Hg,
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Zn, Cu, and Al, causing gastrointestinal disorders, diarrhea, stomatitis tremor, and
hemoglobinuria (Duruibe et al., 2007). This may also include short-term memory loss,
mental-retardation, and gastrointestinal problems (Das et al., 2014). Toxicity is metal-specific
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and dose-dependent such as Pb causes oxidative stress due to imbalance of free radicals,
Cr(VI) disrupts DNA and proteins, Hg, and As are involved in the creation of toxic derivative
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of thiol or methyl groups, Cd bound to various proteins that are vital leading to their damage
and Fe causes corrosive effect and involved in peroxidation of lipid (Jaishankar et al. 2014).
These pollutants are cytotoxic at a low concentration and may lead to cancer in humans
(Dixit et al., 2015). Besides, these causes oxidative stress by forming reactive oxygen species
(ROS), which leads to base damage and the breakage of the DNA molecule (Singh et al.,
2019).

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 Various treatment methods have been applied for ages to overcome this
environmental problem. All the techniques, including the conventional and biological
methods, are up to some or large extent efficient in the remediation of toxic metals.
Applications of biological processes for cleanup of heavy metal polluted sites are more
advantageous than the chemical treatment methods in several aspects. Biosorption of toxic
metal ions by employing bacteria has been considered one of the most reliable and efficient
methods. Several studies are citing the biosorption behavior of different bacterial species
towards various heavy metals. This review article entails the prospects of biosorption by
bacterial biomass in single and in the multimetal environment. Besides, information about the
equilibrium and kinetics aspects of heavy metal biosorption has been listed. The applications

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of various desorbents for metal desorption and regeneration of biosorbents are also elaborated
in this review.

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2. Treatment methods of heavy metal contamination
Heavy metal contamination has been recognized as the most persistent and complex
environmental issue causing a severe hazard to the natural flora and fauna. Several methods
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have been employed for the handling of metals released with industrial effluents. Primarily,
these methods are categorized under two broad divisions, such as conventional and biological
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methods.
2.1. Conventional methods
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The conventional methods are based upon precipitation, adsorption, ion exchange,
reverse osmosis, solvent extraction, evaporation, membrane filtration, and electrochemical
technologies (Kurniawan et al., 2006; Azimi et al., 2017). Many of these techniques failed to
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decontaminate the polluted environment from the dreadful effect of the toxicants. These
techniques have certain disadvantages such as the production of secondary waste products,
high operating and maintenance cost, low efficiency, operational complexity, loss of solvents,
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difficulty in phase separation, the formation of emulsion, high energy requirements, and
incomplete metal removal (Lin et al., 2005; Joshi, 2017). Some of the conventional methods
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such as ion exchange mechanism (Erdem et al., 2004), reverse osmosis (Kurniawan et al.,
2006), electrodialysis (Sadrzadeh et al., 2009), ultrafiltration (Gunatilake, 2015), and
precipitation (Henneberry et al., 2011) involve high expenditure, inefficient removal,
generation of enormous sludge and secondary toxic waste products. Hence, an
environmentally feasible approach with high efficiency is essential for removing toxic metals
from the contaminated sites.
2.2. Biological methods for treatment of contaminated effluents

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 Bioremediation process is an approach for removing these toxic elements from the
polluted sites and/or transforming into less toxic form by applying live or dead microbes.
Microorganisms are omnipresent in the environment and play a pivotal role in the
biogeochemical cycles (Das et al., 2006). The bioremediation technique can be implemented
in in-situ and ex-situ methods. In the former process, the remediation is carried out in the site
of contamination by stimulating the growth of indigenous microbes by supplying adequate
nutrition or by application of engineered microbes (Kulshreshtha et al., 2014). The latter
process involves transporting contaminated soil and water from the polluted region to other
regions for treatment (Gao et al., 2018). The major advantages of the implementation of
biological methods for removing pollutants are no/less accumulation of secondary pollutants

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and cost-effectiveness. Biosorption of heavy metals by microbes is advantageous because of
the specificity and potential to function at low concentrations.

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2.3. Why biosorption is a better alternative than other treatment methods?
Biosorption can be defined as removing metal or metalloid species, compounds, and
particulates from solution by biological materials (Gadd, 1993). The removal of metal ions is
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carried out mainly by two primary mechanisms: metabolism independent and metabolism
dependent. Biosorption, a metabolically passive physiochemical process, involves biosorbent
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of biological origin to allow binding of sorbates, i.e., metal ions onto the biosorbent surface
(Mrvčić et al., 2012). However, the bioaccumulation of metals into the microbial cells is
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metabolism dependent (Javanbakht et al., 2014). To replace the conventional methods for
removing metals, biosorption based bioremediation has been proven as a potential approach
due to technical novelty and potential application in the industry (Beni et al., 2020). Various
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types of biosorbents have been developed from different raw biomass, such as bacteria (e.g.,
Bacillus thuringiensis) (Vijayaraghavan and Yun, 2008), fungi (e.g., Botrytis cinerae) (Akar
et al., 2005), and algae (e.g., Anabaena sphaerica) (Abdel-Aty et al., 2013). However, there
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are several advantages of microbial-based biosorption in the removal of metal ions, such as
the high metal removal efficiency because of their selectivity towards particular metal (Arief
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et al., 2008). Due to the small size of the microorganisms, a small amount of microbial
biomass provides a large surface area to volume ratio for adsorption (Zouboulis et al., 2004).
Most importantly, the microbial biosorbent could be eco-friendly, cost-effective, and the
chances of their metal recovery, as well as the revival, is high (Yu et al., 2020). Besides,
biosorption is an independent, reversible process, and both the live and dead biomass can be
used as biosorbent for metal uptake (Tabak et al., 2005; Vijayaraghavan and Yun 2008).

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 Most of the research on biosorption has been carried out to remove single-metal ion
(Kumari et al., 2017; Xu et al., 2020). However, comparatively less study has been done to
elucidate the biosorption performance in systems with multimetal ions released with
industrial discharges. Among the microorganisms, bacterial biomass can act as suitable
biosorbent because of the high efficiency and low cost. Due to various unique properties of
bacterial biomass, such as metal-binding functional groups, and exceptional sorption ability,
they can be effectively utilized in the bioremediation of toxic heavy metals. Additionally, due
to the reusable property of biosorbents the process is economically feasible and left minimal
waste.
3. Mechanisms of bacterial biosorption

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3.1. Bacterial structure and components associated with biosorption
Bacteria are the most abundant, ubiquitous unicellular microorganism and are also

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found as symbiont with other organisms. Bacteria possess a very simple cell structure lacking
a true nucleus and membrane-bound organelles. The most prominent bacterial structure is the
cell wall that provides structural integrity to the cell. Based on the cell wall composition,
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bacteria can be divided into two groups, Gram-positive bacteria that have a thick
peptidoglycan layer (20-80 nm) (Dijkstra and Keck, 1996) linked by amino acid bridges, and
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Gram-negative bacteria which have a thinner cell wall comprising of a single layer of
peptidoglycan (10-15 nm thick) and are not profoundly crosslinked. The peptidoglycan layers
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of Gram-positive bacteria are densely functionalized with anionic polyalcohols

called teichoic acids. Some of these are linked with lipid to form lipoteichoic acids, which act
as a bridge for connecting peptidoglycan to the cytoplasmic membrane by covalently linking
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lipids within the cytoplasmic membrane. The network formed by cross-linking of

peptidoglycan molecules wraps the bacterial cell in a protective covering. Teichoic acids
provide an overall negative charge to the cell due to phosphodiester bonds between the
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monomers of teichoic acid (Neuhaus and Baddiley, 2003). The phosphoryl and carboxyl
groups of secondary polymers and peptide chains, respectively, render the Gram-positive cell
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wall a negative charge, which helps in the sequestration of metal cations (Prescott et al.,
2002; Kumar et al., 2011). In the Gram-positive bacteria, the sites for metal interaction are
the phosphate group of lipopolysaccharides and on membrane phospholipids (Prescott et al.,
2002; Ansari, 2016). Generally, the metal removal by the bacteria involves binding metal
cations onto the functional groups of the cell wall, followed by internalization into the cell.
3.2. Mechanisms involved in heavy metal biosorption

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 The biosorption mechanism is a complex process that utilizes biosorbents for binding
of sorbates on to it. Many natural resources such as microbial biomass (bacterial, fungal,
algal biomass), agricultural waste, industrial by-products, etc. are used as biosorbent.
Biosorption can remove pollutants even in a significantly lower concentration and take out
heavy metals present in even ppb (parts per billion) concentration from a polluted
environment (Ahemad and Kibret, 2013). Several ways are adapted by the bacteria to take up
these metals and classified according to various criteria. Depending on cell metabolism,
biosorption is categorized into metabolism dependent and metabolism independent.
According to the metal absorption/accumulation location, biosorption can be classified as
extracellular precipitation, cell surface sorption, and intracellular accumulation. In the

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metabolism-dependent biosorption, intracellular accumulation of metals occurs through the
cell membrane, carried out by viable cells only (Mustapha and Halimoon, 2015). Metal

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uptake in metabolism independent biosorption occurs by physico-chemical interaction
between the functional groups on the surface of bacteria and the metal ions. Binding of metal
ions on to the cell surface of a bacterium in metabolism independent biosorption involves
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various mechanisms such as physical interaction (electrostatic or van der Waals interaction)
or chemical interaction (displacement of attached metal cations by ion exchange),
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complexation, diffusion, surface adsorption or by precipitation (Fig. 1).
Complexation involves associating two or more species, i.e., metal ions and the
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functional groups present on the bacterial cell surface. It is categorized into two different
types: monodentate and polydentate complex (Kanamarlapudi and Chintalpudi, 2018). In
monodentate complexes, the metal ion binds with the ligands via covalent bonds. It occupies
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the ligand's central position, but in polydentate complexes, more than one metal ion interacts
with the ligands. The Fourier-transformed infrared spectroscopy (FTIR) analysis of non-
living biomass of Kocuria palustris and Micrococcus luteus showed that carboxylate ions and
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functional groups such as amino and phosphate present on the bacterial cell surfaces
contribute to Cd binding by complex formation (Machalova et al., 2015). In the ion exchange
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mechanism, the exchange of binary metal cations occurs with the counter ion resides on the
surface of the biosorbent. An ion exchange process was identified for Pb(II) adsorption by
extracellular polymeric substances (EPS) of Klebsiella sp. J1 with the counter ions K+ and
Mg2+ (Wei et al., 2016).
The mechanism of metal binding by precipitation occurs by reaction of metal ions
with the functional groups present on the bacterial surface which results in insoluble organic
metal precipitates and remains attached to the microbial cells. Removal of some of the toxic

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heavy metals such as Ni(II), Cd(II), Cr(III), Cr(VI) and Co(II) from various industrial effluent
was carried out by Escherichia coli C90, that produces alkaline phosphatase to precipitate the
metal cations (Chaudhuri et al., 2013). Physical adsorption of metals on the microbial surface
takes place by van der Waals forces between the biomass and metal ions. Biosorption of Cd
and Cr ions from metal solutions was effectively done by applying Bacillus laterosporus and
Bacillus licheniformis (Zouboulis et al., 2004).
The transport of heavy metals across the cell membrane by living bacteria occurs in
two steps. The first step is independent of metabolism, which involves the attachment of
heavy metals to the cell surface. The second one is the internalization of metal ions across the
cell membrane (Das et al., 2008). This mechanism is similar to the uptake of some essential
ions such as Na2+, K+, Ca2+, etc. by the cells. Thus, the ion channels may be confused by the

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metal cations having identical charge and ionic radius associated with essential ions (Ahalya

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et al., 2003). The factors that influence biosorption mechanism are i) properties of the
biosorbents (i.e., living or non-living), ii) characteristics of the targeted metal, iii) type of the
binding sites involved for metal sequestration (biological ligand), iv) parameters involved in
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biosorption (temperature, pH, contact time, amount of sorbent and sorbate), and v) the
characteristics of metal solution and the presence of competing co-ions.
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4. Various bacterial biosorbents for toxic metal removal
4.1. Bacterial biomass in toxic heavy metal removal
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The biomass of the bacteria can be effectively used as biosorbents for heavy metal
removal from aqueous medium even at a significantly lower concentration (Luo et al., 2014;
Todorova et al., 2019). The living and dead biomass of bacteria has been used as an
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adsorbent to remediate toxic metals (Malkoc et al., 2016; Mohapatra et al., 2019). The
negative charge of the microorganisms is due to various anionic structures on their cell
surfaces that allow the binding of metal cations (Wang et al., 2014). Several bacterial species
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in mobilized and immobilized states can efficiently bio-accumulate various toxic metal ions
such as U, Cu, Pb, etc. from contaminated effluents. Since the mode of metal uptake by non-
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living or inactive cells is extracellular, the chemical functional groups of the cell wall
constituents play an important role in biosorption. It is crucial to recognize the functional
groups involved in metal binding on the surface of bacterial biomass.
The bacterial biosorbent is powered by several functional groups, including carboxyl,
imidazole, sulfydryl, amino, phosphate, sulfate, thioether, phenol, carbonyl, amide, and
hydroxyl groups that are involved in metal adsorption (Van derWal et al., 1997). These
functional groups could be essential to develop biosorbents by altering the surface active sites

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via grafting or interchange of functional groups. The negatively charged functional groups
available in the peptidoglycan, teichoic acids, and teichuronic acids of Gram-positive bacteria
and the peptidoglycan, phospholipids, and lipopolysaccharides of Gram-negative bacteria are
the components mainly responsible for the anionic nature and metal-binding affinity of cell
wall (Sherbert, 1978). Because of the anionic characteristic, carboxyl group actively takes
part in the binding of metal ions. Golab and Breitenbach (1995) observed that the bacterium
Streptomyces pilosus could effectively bind Cu ions due to the presence of carboxyl groups
on its cell wall peptidoglycan. Amine groups can also remove metal ions effectively because
they can chelate cationic metal ions as well as adsorb anionic metal ions through hydrogen
bonding or by electrostatic interaction. The negatively charged chromate ions are bound by

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amine groups present in the cell wall via electrostatic interaction (Volesky, 2007). Kang et al.
(2007) observed that the amine moieties of cell wall protonated at a lower pH and bound with

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the chromate ions via electrostatic interaction. Ziagova et al. (2007) also showed that the
amine groups of Staphylococcus xylosus and Pseudomonas sp. were responsible for the
binding of Cd and Cr ions via electrostatic attraction. An increase in pH of the medium
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increases the overall negative charge on the cell surface by deprotonating all the associated
functional groups, which favors the electrochemical attraction and adsorption of metal ions
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(Ledin et al., 1997; Claessens, et al., 2006). Besides, bacterial polymer such as extracellular
polymeric substances (EPS) associated with biofilm also has the metal binding affinity
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(Gawali Ashruta et al., 2014). Various functional groups of microbial cells and EPS that are
involved in metal binding are given in Table 1.
4.2. Bacterial biofilm and EPS in metal removal
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Biofilm can be defined as a cluster of microbial cells attached to a substratum and

enclosed within a matrix of extracellular polymeric substances. The bacteria that reside in the
biofilm environment are more vigorous and resistant to hostile conditions than free-living
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cells (Chakraborty and Das, 2014; Kumari and Das, 2019). Biofilm has high sorption ability,
and the cost of production is meager; hence, it attracts attention in wastewater treatment
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(Gadd, 2009). One of the significant components of biofilm is the EPS. This biofilm polymer
is enriched with various functional groups, which act as sorption sites for diverse chemical
substances, including toxic inorganic pollutants such as heavy metals (Dash and Das, 2016;
Kumari et al., 2017). The multicellular microbial communities and the biopolymer associated
with them act as a suitable carrier for adsorption of heavy metal ions. Treatment of metal-
contaminated water for removing toxic metal ions by employing bacterial biofilm includes
entrapment of metal ions within the biofilm (Fig. 2). The utilization of bacteria biofilm for

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adsorption of heavy metal ions has been reported in various studies. For example, Lameiras et
al. (2008) utilized the biofilm of Arthrobacter viscosus along with granular activated carbon
to remove Cr(VI) from aqueous medium. Biosorption of Cr(VI), along with other organic
contaminants, was observed by the application of Arthrobacter viscosus biofilm (Quintelas et
al., 2006). Biosorption of Cr(VI) was enhanced by the application of E. coli ASU 7 biofilm
supported on granulated activated carbon than the lyophilized biomass of the bacterium and
the sole granulated activated carbon (Gabr et al., 2009). The same bacterial biofilm was used
to remove Pb(II) and Fe(II) with 100% efficiency at lower concentrations of both the metals
(Quintelas et al., 2002). Black et al. (2014) proved that the biosorption efficiency of fixed
bacterial cells is higher than the suspended free cells towards the removal of Pb(II) and

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Cu(II). The Bacillus circulans biofilm was efficiently able to uptake 78% and 40% of Pb(II)
and Cr(VI), respectively, from 500ppm of each metal solution (Khanafari et al., 2008).

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Qureshi et al. (2001) prepared a filter with Pseudomonas aeruginosa strain CMG156 biofilm
over a flexible polyvinyl conduit and utilized it for Cu(II) removal. The filter was found to
remove 85% of the Cu from the solution. The Escherichia coli biofilm supported on kaolin
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was capable of eliminating various metals, and the order of biosorption followed the
sequence as Fe(III) > Cd(II) > Ni(II) > Cr(VI) (Quintelas et al., 2009). Similarly, the E.
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coli biofilm supported on NaY zeolite showed higher adsorption of Fe(III) followed by
Ni(II), Cd(II), and Cr(VI). The biofilm of Klebsiella sp. 3S1 entrapped on porous ceramic
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rings could remove 70 mg of Pb(II)/gm of bacterial biofilm with 99% regeneration efficiency
up to 4 cycles of adsorption process (Muñoz et al., 2016). In addition to the bacterial cells,
adsorption of metal to the EPS in biofilm is governed by various mechanisms and
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interactions.
EPS are localized at or outer surface of the bacterial cells and composed of various
organic macromolecules with high molecular weight compounds such as polysaccharides,
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proteins, nucleic acids, phospholipids, and other low molecular weight non-polymeric
components (Decho and Gutierrez, 2017). The binding of toxic metal ions by EPS has been
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reported by several studies (Bhaskar and Bhosle, 2006; Gutierrez et al., 2008). This method is
independent of metabolism and energy that involves the interaction of negatively charged
functional groups on the EPS layer and cationic metals. EPS is an essential component in the
formation and maintenance of microbial biofilm. The EPS of biofilm binds to the heavy
metal ions, facilitating their transport and enhancing metal toxicity. Several studies have
proved that EPS-covered cells have a better affinity for adsorption of heavy metals than the
EPS removed cells (Wei et al., 2011). Functional groups such as -OH, -COO, -NH, and C=O

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present in the EPS matrix help bind with the positively charged heavy metal ions and remove
them from the contaminated solution (Gerbino et al., 2011). Various functional groups like
amine, amide, sulfhydryl, and carboxyl groups of proteins and hydroxyl and phosphate
groups present in EPS carbohydrates directly impact metal complexation on the EPS matrix.
The hetero-polysaccharides are usually polyanionic compared to homo-polysaccharides due
to the presence of these active and ionizable functional groups (Pal and Paul, 2008). Various
mechanisms such as ion exchange, surface precipitation, complexation, and native adsorption
are involved in binding metal ions onto the surface functional groups of EPS (Fig. 3).
In the ion exchange mechanism, the charge density of metal cations is crucial for the
interchange of ions with negatively charged functional groups of EPS (Pardo et al., 2003).

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The pH of the adsorption medium influences the ionization state and adsorption characteristic
of several functional groups of EPS. The adsorption of metal ions is more at higher pH due to
less availability of H+ ions in the solution (Lopez et al., 2000). The uptake of Pb(II) ions by

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Klebsiella sp. J1 was observed by the exchange of ions between Pb2+ and the functional
groups of EPS. Pb2+ also competes with K+ and Mg2+ to bind with the anionic groups of the
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EPS matrix (Wei et al., 2016). The metal speciation and solubility in an aqueous medium are
predominantly influenced by specific parameters such as pH. In the surface precipitation
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mechanism, increasing the pH of the medium increases the precipitation of heavy metal ions
onto the surface of the EPS matrix (Comte et al., 2008). The precipitation of ferric ion was
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observed by the EPS of Paenibacillus jamilae (Pérez et al., 2008).

Further, the precipitation of Fe(III) was shown more at pH 6 in comparison to pH 3.
The complexation of toxic heavy metals with the anionic functional groups of EPS occurs via
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electrostatic and covalent bonds. In neutral solutions (pH 7), the carboxyl group acquires a
net negative charge that interacts electrostatically with positively charged heavy metal to
form organometallic complexes (Salehizadeh and Shojaosadati, 2003; Guibaud et al., 2005).
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The EPS of Bacillus thuringiensis PW-05 also exhibited a strong binding affinity for Hg(II)
(Dash and Das, 2016). Similarly, the extracted EPS from Bacillus licheniformis showed a
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higher binding affinity for Cu(II) and Zn(II). The binding of these metal ions with the
functional groups of EPS components is mediated by electrostatic interaction (Biswas et al.,
2020). Morillo et al. (2006) also showed the maximum complexation ability of Paenibacillus
jamilae EPS towards Pb (230 mg Pb/g of EPS).
Furthermore, the interaction of heavy metal ions with the bacterial biomass and
biofilm-EPS of diverse bacterial strain has been well elucidated by various sophisticated
instruments, including FTIR and Energy dispersive X-ray spectroscopy (EDX). EDX analysis

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of a biosorbent provides information about their chemical and elemental nature. EDX spectral
analysis was performed to study the adsorption of Pb(II), Cr(VI), and Cd(II) by the EPS of P.
pseudoalcaligenes NP103, the biomass of Bacillus pumilus, and the cells of Halomonas sp.
respectively (Kanagaraj et al., 2014; Rajesh et al., 2014; Kumari et al., 2017) (Fig. 4). The
entrapment of different heavy metals on the biofilm-EPS of various bacterial species has been
depicted through FTIR analysis (Fig. 5). The absorption peaks in spectral analysis indicate
the vibration frequency between the metal ions and functional groups. Broadly, two types of
vibrations are observed in FTIR analysis, such as stretching vibration and bending vibration
associated with change in bond lengths and bond angles. Thus, the FTIR spectral analysis
provides ideas regarding the occurrence, nature, and involvement of binding sites during the

of
biosorption process. The FTIR spectral data illustrated the interaction of the metal cations
such as Cd(II), Hg(II), and Pb(II) with various negatively charged functional groups (–SH,
P=O, –OH, N–H, C–O, etc.) on the biofilm-EPS of P. aeruginosa JP-11 (Chakraborty and

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Das, 2014), B. cereus BW-201B (Dash et al., 2017) and P. pseudoalcaligenes NP103
respectively (Kumari et al., 2017).
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The bacterial biosorbents such as biomass, biofilm, and EPS have been extensively
explored. They are effectively applied in biological metal remediation practices due to their
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rapid, competent, cost-effective, and feasible approach. Moreover, modifications of bacterial
biomass and utilization of biofilm-EPS enhance the net anionic charge, which can further
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improve the affinity and selectivity towards metal cations. The involvement of bacterial
biomass and biofilm-EPS in removing toxic metals has been well characterized in the
laboratory conditions. However, to exploit their efficiency in the field study, further
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development and up-scaling of the techniques are essential.

4.3. Modification of bacterial biosorbents
It was observed that the pristine biomass possesses low biosorption capacity as the
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sorption of heavy metals mainly takes place on the surface of the biomass. Thus, it would be
a practical approach for enhancing the biosorption capacity by increasing and/or activating
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the binding sites on the surface of the biosorbent. Studies have broadly exploited either
untreated or pre-treated bacterial biomass to enhance sorption efficiency for many metal ions
(Wierzba and Latała, 2010; Suazo-Madrid et al., 2011). To overcome the disadvantages
associated with untreated pristine biosorbent, it is recommended to modify the biosorbents by
pre-treatment methods to enhance their adsorption ability (Qin et al., 2020). Various ways are
used to modify the biosorbents, such as physical pre-treatment, chemical pre-treatment, and
biological pre-treatment (Fig. 6).

13
4.3.1. Physically modified bacterial biosorbents
The modification of bacterial biosorbent can be done by pre-treatment of the bacterial
biomass by physical means. The sorption ability of the bacterial biomass towards heavy metal
ions increases by autoclaving the biomass (Paul et al., 2012). This treatment method is
significant due to the application of high temperature and pressure that ruptures the cell wall
of bacteria, which exposes an additional number of binding sites for metal ion attachment. Du
et al. (2012) reported the sorption capacity of heat-treated Pseudomonas sp. strain DY1 and
found high sorption towards metal-complex dye compared to the live biomass. As a result of
heat treatment, the permeability of the cell wall increased, and intracellular proteins were
denatured, which contributed to the increased biosorption of metal-complex dye. The pre-

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treated biomass of Bacillus subtilis by autoclaving at 110°C for 10 min showed more
significant Ni(II) removal than the untreated biomass (Al- Gheethi et al., 2017). Pardo et al.

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(2003) observed that the freeze-dried biomass of Pseudomonas putida was efficient in the
removal of Cd(II), Cu(II), Pb(II), and Zn(II). The pre-treatment of Streptomyces rimosus
biomass using heat showed biosorption towards Zn(II) (Mameri et al., 1999). Similarly, Gabr
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et al. (2008) also observed the higher uptake of Pb(II) and Ni(II) by the pre-treated dead cells
of Pseudomonas aeruginosa ASU 6a. Several physical treatment methods, such as
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autoclaving, freeze-drying, and heating, are applied to modify biosorbents. However,
vigorous screening of these techniques should be carried out with different biomass to
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enhance their affinity towards metal adsorption.

4.3.2. Chemically modified bacterial biosorbents
There are several approaches for modifying the biosorbents by chemical means such
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as pre-treatment of the biosorbents, modification, improvement, and polymerization of the

binding sites. For enhancing the biosorption process, the biomass is commonly pre-treated
with chemicals like acid, alkaline, ethanol, and acetone (Gupta et al., 2015). From various
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studies, it has been observed that the biosorbents that were modified chemically show better
adsorption than unmodified ones (Wang and Chen, 2010; Zhao et al., 2015). The major
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factors that are influenced by chemical pre-treatment are the cellular components of the
biomass. Acid pre-treatment is advantageous over other treatment methods because it
removes impurities and ions that block the binding sites.
The lyophilized cells of Pseudomonas aeruginosa showed enhanced biosorption of
Cu2+ and Ni2+ after pre-treatment with NaOH or NH4OH or toluene. However, the uptake rate
by the same biomass was not enhanced by oven heating at 80ºC, autoclaving at 121ºC, acid,
detergent, and acetone treatments (Sar et al., 1999). Mao et al. (2013) pre-treated the

14
Corynebacterium glutamicum biomass with polyacrylic-acid and observed that the treated
biomass exhibited 3.2 times greater Cd uptake than the untreated biomass. Similarly, the
increased biosorption of the heavy metals was observed when the cells of Pseudomonas
putida 5-x pretreated with HCL for Ni(II) uptake (Wang et al., 2003) and cells of
Thiobacillus thiooxidans with NaOH for Zn(II) and Cu(II) removal (Liu et al., 2004). The
Micrococcus luteus IAM 1056 biomass after treatment with chloroform-methanol or
concentrated-KOH exhibited higher uptake of Cu(II) than the unprocessed biomass
(Nakajima et al., 2001). The uptake of As(III) and As(V) from aqueous solutions was
increased by the exposure of Staphylococcus xylosus biomass with ferric ions (Aryal et al.,
2010). The prior treatment of Bacillus subtilis biomass with supercritical carbon dioxide (SC-

of
CO2) increased the uptake rate of Ni(II) ions due to the inactivation of the bacterial cell by
SC-CO2, which further modified the bacterial cell membrane to enhance the affinity towards

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the heavy metal ions (Al- Gheethi et al., 2017). The immobilized cells of Micrococcus sp.
with calcium alginate (2%) and polyacrylamide gel (10%) beads enhanced Cu(II) removal by
61% (Wong et al., 2001).
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Although studies have been carried out to increase the active binding sites to improve
the biosorption, very little interest has been given to the modification of functional sites. For
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example, the sorption of As(V) by the biomass of Lactobacillus casei DSM20011 was
aminated to modify the negative charge of carboxylic groups into positive amino groups
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(Halttunen et al., 2007). It was shown by Vijayaraghavan and Yun (2007) that blocking of
carboxyl groups in the biomass of Corynebacterium glutamicum exhibited maximum
biosorption than the pristine biomass. The grafting of long polymer chains onto the biomass
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surface by direct attachment or polymerization of a monomer is one more effective method

for introducing active functional groups to the biomass surface (O’Connell et al., 2008). The
grafting of polyallylamine (PAA) on the surface of the biomass of Pseudomonas sp. Lk9
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introduces bilayer modification of the functional groups. This modified adsorbent enhanced
the adsorption rate of Pb(II), Cd(II), and Cu(II) by 4.96, 10.60, and 10.00 fold higher than
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that of the untreated biomass, respectively (Luo et al., 2014). The biomass of Staphylococcus
aureus was coated by phthalic acid along with iron oxide magnetite nanoparticle and utilized
for biosorption of toxic metal ions. The modified biomass exhibited greater removal of
Pb(II), Ni(II), and Cu(II) as compared to the pristine biomass (Mahmoud et al., 2016). The
chemical pre-treatment methods are extensively used for modifications of bacterial
biosorbents. The appropriate use of acids, alkalis and/or organic solvents can improve the
binding efficiency of the biosorbent with minimal damage.

15
4.3.3. Biologically modified bacterial biosorbents
Biosorbents without any modification generally lack specificity in metal-binding,
which may give rise to difficulties in retrieving and reprocessing the specific metal or metals.
However, biological modification is a potential alternative to improve the specificity and the
accumulating properties of the microbes (Yang et al., 2015). The application of genetic and
protein engineering in this regard may increase the affinity of binding sites on
microorganisms for the sorption of metal cations (Goyal et al., 2003). The biological
modifications make the adsorbent more selective for removing a specific metal ion and
provide higher resistance to environmental conditions.
In response to the metals, bacteria express cysteine-rich peptides, such as glutathione

of
(GSH) (Blindauer et al., 2011), phytochelatins (PCs) (Bellini et al., 2020), and
metallothioneins (MTs) (Murthy et al., 2011), which have the affinity for metal ion

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sequestration. GSH is prevalently found in bacterial species and protects from various
physiological stresses such as heavy metals. The GSH mutant Streptococcus pneumonia
strain exhibited sensitivity towards Cu(II), Cd(II), and Zn(II) (Potter et al., 2012). PCs act as
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metal chelators and are essential for binding as well as detoxification of heavy metals. The
cloned PC synthase gene from Anabaena sp. PCC7120 when transformed into E. coli
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genome, the bacterium exhibited resistance against Cd(II) and Cu(II) (Chaurasia et al., 2008).
Metallothioneins are the proteins with low molecular weight, and they have the unique
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property of metal chelation and detoxification of heavy metals. The expression of the bmtA
gene of Pseudomonas aeruginosa N6P6 was enhanced by many folds with an increase in
Pb(II) concentration from 200 to 800 ppm (Kumari and Das, 2019). The rate of accumulation
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of metals is also enhanced with the overexpression of MTs in bacterial cells, which in turn
provides a potential approach for the development of microbial-based biosorbents for the
remediation of heavy metal pollution (Mejáre and Bülow, 2001). Mauro and Pazirandeh
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(2000) demonstrated that the fungus MT was expressed in 1 to 12 repeats by fusion with the
protein which is responsible for the catabolism of maltodextrins in the periplasm of E. coli,
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and the Cd binding ability was enhanced by ten times with one repeat and a maximum sixty-
five times with nine repeats. The intracellular expression of MTs may block the recovery of
the biosorbents. Hence, the accumulated metals can not be simply released out of the cell.
This transport problem can be overcome by expressing MTs on the bacterial cell surface (Lin
et al., 2010). Besides, other metal-binding protein MerP originated from Bacillus cereus
RC607 and Pseudomonas sp. K-62 was overexpressed in Escherichia coli to remove heavy
metals like Cr(III), Zn(II), and Ni(II) (Kao et al., 2008).

16
 The biological modifications are the safest and highly selective method for metal ion
adsorption. Expression of the metal-binding proteins will help enhance the adsorption of
metal ions in a developed bacterial strain. However, biosorption of a particular metal by a
specific biosorbent depends on the number, availability, and chemical state of the binding site
and the interaction among the functional sites and metal ions. In addition, pre-treatment of
biosorbents improves their physical and chemical properties and surface active sites and
enhances the sorption efficiency by maintaining their mechanical stability. Pre-treatment
methods have been applied in the biosorption process to improve the sorption ability of
biosorbents in lab-based studies. However, the ways needed for pre-treatment of biosorbents
should be judicially selected for large scale applications.

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5. Biosorption of heavy metals under multimetal stress
The industrial effluents, ore mining sites, and groundwater generally contain a

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mixture of metals. Since most contaminated areas are polluted with multiple heavy metals,
multimetal resistant microbes have been recognized as the competent agent for the
biosorption (Giovanella et al., 2017; Choińska-Pulit et al., 2018). However, multimetal
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biosorption is affected by many factors such as the number of metals, their concentration,
metal combination, the order of addition, and contact time in the biosorption medium.
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5.1. Biosorption of metals in the binary and ternary metal ion solution
The presence of single toxic metallic species in a contaminated solution is rare, and
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the occurrence of multimetal ions often causes an interactive effect. The utilization of free-
living microorganisms and immobilized microorganisms for the simultaneous removal of
multimetals from the metal solution has gained attention recently. The occurrence of other
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metal ions in the solution alters the overall ability of metal-binding due to competition. A
comparative account of heavy metal removal by bacteria in single and in the multimetal
system is given in Table 2. The multimetal biosorption studies can lead to a better
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understanding of these complex systems and their behaviour.

In the binary or ternary metal ion solution, if the metal ions are common, they will
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compete for the same functional site on the cell surface (Gola et al., 2017). The presence of
more than one metal ion in untreated water is expected to cause interactive effects depending
on various factors, such as the number of competing metals for binding sites, the
concentration of metals, and the nature and the amount of biosorbent. Removal of individual
metal ion occurs preferentially over other metal ions, but the presence of more than one metal
ion in a solution declines the removal of preferably metal adsorbate (Alimohamadi et al.,
2005; Vijayaraghavan and Balasubramanian, 2015).

17
 In a bimetal solution of Pd(II) and Pt(IV), the E. coli biomass showed selective
binding behavior towards Pd(II). In the single metal solution of Pd(II) and Pt(IV), the
bacterium showed slightly higher biosorption of Pt(IV). However, in binary metal solution,
Pd(II) uptake rate was increased (Kim et al., 2015). The biosorption of both the metals in
single metal ion solutions was approximately the same, which shows that the binding sites for
both the metals were available in the bacterial biomass. It may be due to the more affinity of
Pd(II) towards the binding site present in the biomass. The results of various studies showed
intense competition between metal ions for the active site present on the surface of the
biosorbent. Park and Chon (2016) showed that the Exiguobacterim sp. efficiently adsorbed
Cd in comparison to Pb and Zn in individual single metal solutions, but in multimetal ion

of
solution, the bacterium preferentially adsorbed Pb followed by Cd and Zn (Pb > Cd > Zn).
Due to the greater binding affinity, the adsorption was shifted towards Pb(II) in a multimetal

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solution.
Some of the studies showed increasing biosorption order of the metal ions in binary
and ternary metallic solutions according to the increase in the ionic radius of the metal
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present in the solution. Masood and Malik (2015) reported the biosorption of Cr(VI), Cu(II),
and Ni(II) onto Acinetobacter sp. FM4 biomass from single, binary, and ternary metal
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solutions. From all the combinations of binary solutions (Cu2+-Ni2+, Cr6+-Cu2+, & Ni2+-Cr6+),
it was observed that the presence of one metal ion reduces the biosorption rate of the
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competitive metal ions. In the binary solution, a reduction in the biosorption rate of both the
metal ions was observed compared to their specific metal solution. The observed sorption
order in binary solution was Cr(VI)> Cu(II)> Ni(II) based on their increasing ionic radii.
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Similarly, in the ternary system, the other two metals in the solution inhibited the sorption
rate of the third metal, and the uptake rate of all the metals was lower with respect to their
sole metal ion solutions. The reason behind this may be due to the overlapping of adsorption
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sites of the individual metals. The biosorption of few metals favors the dominant ones present
in the multimetallic solution. Ziagova et al. (2007) showed the biosorption of Cd(II) and
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Cr(VI) by the bacterium Staphylococcus xylosus in both single and bimetallic solutions. In
the single metal solution, bacterial biomass efficiently adsorbed Cd as compared to Cr. But in
a binary mixture, the higher adsorption rate was shifted towards the dominant metal ion
present in the solution. Jiang et al. (2017) also reported similar results for the removal of
Pb(II) in the presence of Cd(II) and Zn(II).
A few investigations also reported better biosorption efficacy of dead bacterial
biomass than its living counterpart. Fan et al. (2014) used two Gram-negative bacteria

18
Ochrobactrum intermedium LBr and Cupriavidus metallidurans CH34, for the uptake of
Cu(II) and Cr(VI) from their respective metal solutions. Both the isolates showed reduced
biosorption efficiency by the living cells than their dead biomass. Similar results were also
observed by Huang et al. (2013) for the uptake of Cd by Bacillus cereus RC-1 and for
removal of Pb and Ni by Pseudomonas aeruginosa ASU 6a (Gabr et al., 2008). Dead cells of
Bacillus megaterium and Bacillus circulans efficiently removed Cr(VI) from the aqueous
solution than the living cells (Srinath et al., 2002). The biosorption study mediated by living
cells of the bacterium showed an altered affinity of binding towards heavy metals in different
stages of growth phases. The cells of Ochrobactrum intermedium LBr and Cupriavidus
metallidurans CH34 at the logarithmic growth phase showed a higher affinity towards

of
removing Cu and Cr than the cells of stationary phase (Fan et al., 2014). Daughney et al.
(2001) also showed more significant binding of Cd and Fe by the exponential phase culture

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of the Bacillus subtilis than stationary phase culture.
The occurrence of co-ions significantly affects the sorption efficiency of the
biosorbents towards a specific metal ion. As the biosorbents acquire a limited number of
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binding sites, it is obvious that the removal rate of a particular metal ion in a multimetal
system is less than the single metal system. Furthermore, each biosorbents have a different
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affinity towards various heavy metals that affects the sorption of metal ions in multimetal
solutions.
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5.2. Influencing parameters in bacterial uptake of heavy metals

In addition to the type, nature, and the chemical forms of the biosorbents, several
critical physico-chemical factors influence the process of biosorption (Park et al., 2010).
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Biosorption of metal is influenced by various factors such as pH, temperature, contact time
with metal, initial metal ion concentration, the dosage of biosorbent, and agitation speed.
i) pH: pH of the solution plays a vital role in the biosorption of heavy metal ions.
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Bacterial biomass contains weak acid and basic functional groups, contributing to the binding
and solubility of metal ions. Hence, pH influences the solution chemistry of the pollutants,
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chemical nature of metal ions, ionization state of functional groups on the surface of
biosorbent, and competition with coexisting ions in a solution (Vijayaraghavan and Yun,
2008; Özdemir et al., 2009; Liu et al., 2013). In the case of metal biosorption, the pH strongly
influences the speciation and availability of metal ions (Esposito et al., 2002; Dissanayake et
al., 2016). However, pH inhibits the dissolution of metal ions, which reduces the toxic effect
of heavy metals in the bioremediation process (Chen et al., 2020). An increase in pH of the
solution promotes electrochemical attraction and metal ion adsorption due to the rise in the

19
negative charge on the biomass surface (Huang et al., 2013). The optimum pH value favors
the highest metal sorption; usually, it ranges from 4 to 8 (Abdel-Monem et al., 2010; Lin et
al., 2012). It was reported that the uptake of Cu(II) ions by Micrococcus sp. increases with an
increase in pH (from 2 to 6) of the sorption medium. A similar result was obtained in the case
of Mesorhizobium amorphae CCNWGS0123 cells, where Cu removal was maximum at pH 5
(Mohamad et al., 2009).
At lower pH, H+ ions compete with Cu2+ ions for the active binding sites of the
bacterium. However, at higher pH value, more Cu2+ ions were available for the sorption site
of the cells (Wong et al., 2001). Similarly, the uptake of metals decreased with a decrease in
pH of the solution. When the pH is below 2, the removal of metals is least and insignificant.

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Similarly, the optimum pH for Cr(VI) removal by Streptococcus equissimilis is 2, and the
uptake rate increased with a decrease in the solution (from 7.5 to 2). This may be due to the

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occurrence of hydroxyl ions at higher pH of the medium, which interferes with the binding of
Cr ions for the negatively charged groups of the bacterial cells (Goyal et al., 2003). However,
the influence of pH on the biosorption rate does not follow a particular trend and varies with
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the nature of adsorbents (biomass) and adsorbates (metal ions). According to Aryal and
Liakopoulou-Kyriakides (2014), biosorption of Cr(III) by the Mycobacterium sp. strain Spyr1
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was increased with an increase in pH from 1 to 5, but the removal of Cr(VI) was decreased
with an increase in pH from 2 to 7. A similar result was observed in the case of Cr(III) and
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Cr(VI) removal using Mesorhizobium amorphae strain CCNWGS0123 biomass (Xie et al.,
2013). Different pH of the solution affects the biosorption process differently. The solution
with a lower pH value reduces the rate of metal uptake due to the competition for binding
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sites between H+ ions and metal ions. However, at higher pH, precipitation of metal ions
declines the sorption rate of heavy metals. Thus, the optimum pH of the sorption medium
favours the maximum biosorption of metal ions.
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ii) Temperature: Temperature influences several conditions that are crucial for the
metal ion biosorption. With the increase in temperature, liquid viscosity decreases, resulting
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in an increased rate of adsorbate diffusion across the outer periphery layer and in the
adsorbate particles (Al-Qodah, 2006). Generally, the rate of biosorption increases with an
increase in temperature because of the increase in kinetic energy and surface activity of the
solute. However, temperature above optimum can destroy the binding sites available for
metal ion binding (Aryal and Liakopoulou-Kyriakides, 2013). The sorption of Cr(VI) by
Streptococcus equissimilis was increased with an increase in temperature up to 40ºC. The
more metal removal at high temperatures is due to an increase in surface active sites on the

20
biosorbent (Goyal et al., 2003). The application of high temperature also causes deformation
of functional binding sites on the biosorbent, which in turn results in lesser removal of metal
ions such as Pb(II), Cd(II), and Zn(II) by Citrobacter strain MCM B-181 (Puranik and
Paknikar, 1999) and Cr(VI) by Bacillus licheniformis (Zouboulis et al., 2004). The uptake of
heavy metals by utilizing bacterial biomass is enhanced by increasing and decreasing the
temperature of the biosorption medium (Aryal et al., 2012). It was reported that the
temperature between 20°C to 35°C is suitable for the uptake of heavy metal ions such as
Cr(VI), Pb(II), and Cu(II) by Staphylococcus saprophyticus (Ilhan et al., 2004) and Pb(II),
Cu(II) and Cd(II) by Bacillus thuringiensis strain OSM29 (Oves et al., 2013). Depending
upon the endothermic and exothermic nature of the biosorption process, temperature affects

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the sorption efficiency of biomass. The temperature of the reaction medium above the
optimum range may hinder the biosorption process by inhibiting the growth of the microbes

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or by the destruction of the active binding sites on the biomass. Hence, the temperature
should be suitably opted depending on the organisms and the targeted metals.
iii) Dosage of biosorbent: The amount of biomass is an essential factor that affects
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the biosorption process for the uptake of heavy metal ions. Usually, the increase in
biosorption occurs by the increase in the concentration of biomass, which may be due to the
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rise in the number of binding sites involved in the attachment of heavy metal ions. However,
biosorption rate increases up to a certain limit; after that it decreases with an increase in the
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amount of biomass as evident in Staphylococcus xylosus biomass for As (Aryal et al., 2010)
and Mesorhizobium amorphae strain for Cr (Xie et al., 2013). Due to the electrostatic
interaction among the cells, it plays a significant role in metal removal. At equilibrium, the
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biomass at low cell density uptakes more metal ions than their high cell density (Rani et al.,
2010). Thus, the high concentration of biomass hinders the binding of metal ions onto the
surface of biosorbents. Besides, the generation of more biomass in biosorption practices is a
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costly approach, and the optimal dose estimation is essential from an economic point of view.
iv) Competing ions: The presence of other metal ions in the polluted water makes
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the application of biosorption complicated to treat the industrial effluents. Other metal ions
compete with the metal of interest for the binding site on the biosorbent and reduce the
specificity of the biosorbent by binding to the site which is meant for the metal of interest
(Sar et al., 2004). Biosorption of Cu(II) ion was reported to reduce by the presence of Zn(II)
in the binary metal solutions when Thiobacillus thiooxidans were utilized as biosorbent (Liu
et al., 2004). However, the presence of multiple co-ions had a relatively lesser effect on
Cr(VI) binding to the Mycobacterium sp. strain Spyr1 cells than the Cr(III) due to the high

21
selectivity of Cr(VI) ions than the Cr(III) (Aryal and Liakopoulou-Kyriakides, 2014). The
presence of competing ions up to a certain limit could not change the biosorption behavior of
the desired metal ion, but further increase in the concentration of co-ions may hamper the
uptake rate. The biomass of Arthrobacter sp. Sphe3 and Bacillus sphaericus can effectively
remove Cu(II) from a multimetal solution. The co-ions up to 25 ppm each did not affect the
Cu(II) removal, but a reduced uptake of Cu(II) ion was observed with an increase in the
concentration of competing ions (Aryal et al., 2012). It was also observed that cations
enhance the uptake of anionic species by increasing the binding of negatively-charged anions
(Gadd, 2009). However, in a metal-specific adsorption mechanism, biosorbents should have a
unique binding affinity towards the specific metal, and the presence of other metal ions in a

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medium adversely affects the biosorption of the desired metal ion.
v) Agitation: Agitation is an essential factor in the process of the biosorption

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mechanism. The increase in the uptake of metal was observed by minimizing mass transfer
resistance with the increase in agitation speed. Sometimes constant agitation can diminish
this mass transfer resistance. However, the physical structure of the biosorbent may be
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damaged by the effect of agitation (Park et al., 2010). The Mesorhizobium amorphae strain
CCNWGS0123 biomass showed enhanced removal of Cu(II) ions at moderate agitation i.e.,
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150×g, and a further increase in the speed of agitation reduced the Cu adsorption rate
(Mohamad et al., 2009). Streptomyces ciscaucasicus strain CCNWHX 72-14 also showed
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increased Zn(II) removal by increasing agitation speed from 60 to 90 rpm (Li et al., 2010).
Agitation provides uniform diffusion of metal ions to the active binding sites of the
biosorbent in the reaction medium, which in turn accelerates the biosorption process.
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vi) Nature of biosorbent: The nature of biosorbent and the binding sites available on
their surface, growth rate, modification by physical or chemical means, size and amount of
biosorbents are the crucial factors for biosorption of heavy metal ions (Gadd, 2009; Park et
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al., 2010; Li and Tao, 2015). The size of the biosorbent is crucial for biosorption, and small-
sized cells are more efficient in a short duration of equilibrium time due to the higher surface
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area. The age of the culture also has a certain effect on the removal efficiency of biosorbent.
It was observed that cell mass collected from 24 h of growth is more compact than the cell
mass collected from 48 h culture (Goyal et al., 2003). The biosorption process is also affected
by the composition of cell wall, extracellular polymeric substances, etc., and is influenced by
the growth condition of biomass, nutrient supplements, and age of the biomass.
vii) Contact time: To obtain maximum biosorption of metal ions from the aqueous
solution, optimum contact time between the biosorbent and adsorbate is essential. It has been

22
observed that the rate of metal uptake increases within a very short duration of contact with
the biosorbent, and later on, it becomes constant. After reaching equilibrium, the biosorption
rate of the metal does not increase significantly with time (Ozdemir et al., 2003; Colak et al.,
2011). The rate of Ni biosorption by Bacillus thuringiensis OSM29 was highest up to 30 min
due to the elevated binding of free metal ions with the active groups on biosorbent. However,
after some time, the rate of biosorption was reduced and attained equilibrium due to the
saturation of all the biomass binding sites with the metal ions (Oves et al., 2013). The dead
biomass of Bacillus cereus RC-1 also showed the highest removal of Cd(II) within 30 min of
contact time (Huang et al., 2013). Further, a very short duration of contact time was observed
in the case of Cu removal by using Arthobacter sp. Sphe3 and Bacillus sphaericus cells,

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where the maximum biosorption was obtained at 5 and 10 min of contact time by both the
bacterial biomass, respectively (Aryal et al., 2012). Similarly, freeze-dried biomass of

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Pseudomonas putida was able to remove more than 80% of Cd(II), Cu(II), Pb(II), and Zn(II)
ions within 10 min of contact time (Pardo et al., 2003). Around 90% removal of radionuclides
such as U(VI) and Th(VI) was observed by employing Pseudomonas sp. biomass at 10 min
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of contact time (Sar et al., 2004). In some cases, longer contact time is needed for better
absorption of metal ions. Guo et al. (2012) showed the highest biosorption of Cd ions was
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observed at 12 hr of contact time by utilizing Pseudomonas plecoglossicida biomass.
Contact time for different biosorbents varies from a few minutes to several hours with
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varying species of metal. The uptake of heavy metals by the adsorbents depends on several
factors such as biomass, metal ions, contact time, and environmental parameters. The reaction
medium can be adjusted at appropriate conditions, i.e., optimum pH, temperature, agitation,
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contact time, etc. to achieve maximum sorption. However, when the treatment of a
considerable volume of real industrial effluents is considered, it is quite complicated to
control the process.
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6. Adsorption kinetics and isotherm models for evaluating the biosorption process
The metal adsorption process is assessed by an efficient and precise model for
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analyzing the adsorption system. The adsorption process depends predominantly on two
essential aspects: adsorption kinetics and equilibrium isotherm. In addition to the adsorption
rate assessment, kinetics analysis also provides information about rate expressions
characteristic of possible reaction mechanisms. Besides, isotherm models explain the
correlation between the adsorbent state and the solute concentration in the adsorption medium
under the equilibrium condition. There are various models available for kinetic analysis and
isotherm data analysis.

23
6.1. Adsorption kinetics for the biosorption processes
Various studies in the adsorption of toxic metals interpret experimental results about
the biosorption efficiency of solute at equilibrium and their kinetics of adsorption. Further,
the data are analyzed following several models and/or empirical formulas. Kinetic models are
used to analyze the dynamics of biosorption processes in heavy metal removal and evaluate
its potential in the rate-controlling steps such as mass transport and chemical reaction
processes. Some kinetics models have been discussed below:
i) Pseudo-first-order kinetic model: The pseudo-first-order model was first
introduced by Lagergren, based on solid/liquid adsorption, and the equation refers to
intraparticle diffusion processes (Lagergren et al., 1898). It is generally expressed as:

of
Where k1 is the pseudo-first-order rate constant (mg g-1), qe is the amount of adsorbate at

ro
equilibrium, q is the amount of adsorbed solute, and t is the time at equilibrium. Ho and
McKay (1999) proposed the linear form of the equation by modifying the equation, t = 0, q =
0, which can be expressed as:
-p
re
The equation may also be represented as,
lP

When qe is evaluated from the experiment, the fractional uptake (F) (with respect to
equilibrium) at time t can be expressed as:
na

Then, k1 can be represented as,

ur

For fitting the data to the above equation, the equilibrium adsorbate (qe) must be
known. In most studies, the pseudo-first-order kinetic equation is not appropriate for the
Jo

experimental design, which has a more extended contact time range. However, it is suitable
for the narrow range of sorption time i.e., 20 to 30 min of the initial sorption process (Gerente
et al., 2007). In the case of pseudo-first-order rate constant, when the value of q increases and
comes near to the value of qe, the value of (qe-q) becomes less significant. So, the value of
ln(qe-q) becomes larger and unreliable when q is equal to the qe value. This phenomenon
reduces the accuracy of first-order rate constant (k1) in assessment of adsorption rate.

24
 The pseudo-first-order kinetic model was applied in biosorption studies carried out by
bacterial biomass for removal of Cr(VI) by Acinetobacter junii (Paul et al., 2012), Ni(II) by
Streptomyces rimosus (Selatnia et al., 2004), Zn(II) by Streptomyces rimosus (Mameri et al.,
1999), Cd(II) by Pseudomonas stutzeri (Hassan et al. 2009) and Pb(II) by Pseudomonas
aeruginosa PU21 (Lin and Lai, 2006).
ii) Pseudo-second-order kinetic model: The pseudo-second-order model is based upon
the sorption capacity of solid/liquid interface in the adsorbent and expressed as:

Where k2 is the equilibrium rate constant of pseudo-second-order kinetics (g mg-1 min-1), qe is

the amount of adsorbate at equilibrium, q is the amount of adsorbed solute, and t is the time at

of
equilibrium.
The linear form of the equation proposed by Ho and McKay (1999) is:

ro
It can also be represented as: -p
re
By rewriting eq(8):
lP

Where
na

The fractional uptake (F) (with respect to equilibrium) at time t can be expressed as:
ur

The pseudo-second-order kinetic model is more applicable for representing kinetic

data in biosorption study than the pseudo-first-order kinetic model (Dadrasnia et al., 2015).
Jo

Several studies have shown that the pseudo-second-order model is more appropriate for the
removal of toxic cations in biosorption processes such as Pb (Colak et al., 2011; Rodríguez-
Tirado et al., 2012; Jin et al., 2017), Cr (Dadrasnia et al., 2015), Cd (Yuan et al., 2009; Rajesh
et al., 2014), Cu (Sag et al., 2002; Jin et al., 2017) and Zn (Li et al., 2010).
To evaluate the efficiency of pseudo-first-order and pseudo-second-order adsorption
kinetics in various biosorption experiments, both the experimental outcomes and calculated
outcomes of the data were taken into consideration. The eq(2) and eq(6) have been followed

25
to represent the pseudo-first-order (k1) and pseudo-second-order (k2) rate constants, and the
linear equations of both rate constants can be expressed in the form of:

Where m and c are the reaction parameters and are utilized for the calculation of the
experimental data. The value of y can be derived at different time intervals tn for n= 1, 2,
3........, N (N is the number of data).
In various biosorption study, the experimental values for and
are expressed at different time intervals (1, 2, 3.........., N). The data are then
analyzed by incorporating the values of y in the linear equations of adsorption kinetics
(for pseudo-first-order kinetic) and (for

of
pseudo-second-order kinetic), which gives the experimental data of the biosorption process.
This can be expressed as qexp. Then the calculated values of the qe are obtained by fitting the

ro
slopes and intercepts with the vertical axis taking t = 0. This can be written as qcal. Further, the
correlation coefficient (r2) of qexp and qcal of both the kinetic models are evaluated, and the
-p
closeness of each calculated values are compared with the experimental data. In general,
pseudo-second-order kinetic showed a greater correlation with the data than the pseudo-first-
re
order kinetic, as reported in Pb(II) sequestration using Bacillus cereus and Bacillus pumilus
(Çolak et al., 2011).
lP

iii) Elovich kinetic model: Elovich kinetic model is generally used to express the
kinetics of chemisorptions. This equation states that the rate of adsorption decreases with an
increase in surface coverage. The equation for the Elovich model is represented as:
na

Where a and b are the parameters of the equation, a is associated with an initial rate
ur

of the reaction, and b is related to the extent of surface coverage and activation energy for
chemisorptions. Following Chien and Clayton (1980), the linearized form of the equation can
be represented as:
Jo

Elovich kinetic model was applied for adsorption of Zn on Mesorhizobium

amorphae biomass with r2 value of more than 0.96 (Hao et al., 2014). This model was also
suitably fitted for the biosorption of Cr(III) by Streptomyces rimosus with an acceptable
regression coefficient (Sahmoune et al., 2009).

26
 iv) Intra-particle diffusion model: This kinetic model is applicable for diffusion
mechanisms, in which multi-step adsorption occurs with the transport of solute from the
liquid phase to the solid phase of the adsorbent surface followed by diffusion into the matrix.
The model was developed by Weber and Morris (1963), and the linearized equation for
expression of the initial rate of intra-particle diffusion is:

Where, kip = Intra-particle diffusion rate constant (mg g-1 min-1/2) and C is the
intercept. According to this model, the graph for uptake (qt) against time (T1/2) must be linear
and is considered as a rate-limiting step when the line passes through the origin (Arami et al.,
2008). However, when the plots do not pass through the origin, it shows a boundary layer in

of
the reaction. The intercept value represents the thickness of the boundary layer. The intra-
particle diffusion model was applied to the adsorption process of Zn(II) by utilizing Zn

ro
sequestering bacterium VMSDCM (Mishra et al., 2013). Podder and Majumder (2015)
showed the multi-linear nature of the intra-particle diffusion model for biosorption of As(III)
-p
and As(V) by immobilized biomass of Corynebacterium glutamicum MTCC 2745.
v) Bangham’s model: The Bangham kinetic model is applicable for the analysis of
re
slow steps that take place at the later stage of the biosorption process. Bangham’s model
equation is generally expressed as:
lP

Where C0= initial concentration of adsorbate (mg L-1), q= amount of uptake at time t,
m= mass of biomass (g), V= volume of solution (L), α >1, and k0 are constants. The
na

application of Bangham’s model in biosorption of As ions by immobilized Corynebacterium

glutamicum MTCC 2745 expressed the role of film diffusion in rate monitoring steps (Podder
ur

and Majumder, 2015). Bangham’s model was applied for Zn(II) biosorption by Zn
sequestering bacterium VMSDCM, but with a lower R2 value than the intra-particle diffusion
Jo

model (Mishra et al., 2013).

6.2. Various isotherm models for characterization of biosorption
The adsorption mechanism becomes more complicated in the multicomponent
systems due to the interaction and competition of the various pollutants. Various components
that influence the adsorption behavior of the pollutants to the adsorbents are their surface
charge, size, structure, and the functional groups present on their surface. The adsorbent
affects the adsorption mechanism by the porosity and the attaching site present on its surface

27
(Baig et al., 2009; Kumar et al., 2011). Therefore, various equilibrium isotherm models are
used to gain information on the metal uptake capacity of the different species (Pagnanelli et
al., 2003). These represent the amount of solute adsorbed per unit mass of the adsorbent, and
as equilibrium concentrations are a function of temperature, their relationship at a specific
temperature is known as adsorption isotherm. There are two types of methods, such as
equilibrium batch-sorption study and dynamic continuous-flow sorption study, to evaluate
the solid-liquid sorption system. Equilibrium adsorption models are best for analyzing
adsorption data in the batch system (Vimala and Das, 2009). It is crucial to find the best-fit
isotherm to assess the efficiency of the used adsorbent to prepare appropriate adsorption
system designs for industrial purposes. For the proper understanding of an adsorption process

of
and effective designing of the adsorption system, adsorption equilibrium information is
crucial (El-Khaiary, 2008). The adsorption equilibrium can be represented as a graphical data

ro
based on calculation; an empirical algebraic form suitably fitted to the data or equation relied
upon molecular statistics of the ongoing process.
i) One-Parameter isotherm
-p
a) Henry’s isotherms: This isotherm model is the simplest among all the adsorption
models. This isotherm suggests that the number of adsorbates in the gas phase is equivalent to
re
the number of adsorbates on the adsorbent (da Rocha et al., 1998). The model is suitable for
the adsorption system, which involves a low concentration of adsorbate. The linear
lP

expression of the equation is:

Where qe= amount of the adsorbate at equilibrium (on the surface) (mg g-1), KHE =
na

Henry’s adsorption constant, Ce= equilibrium concentration of the adsorbate on adsorbent

(pressure).
ii) Two-Parameter isotherm
ur

a) Langmuir isotherm: This adsorption isotherm model is intended to express gas-

solid phase adsorption and also used to evaluate and distinct the adsorptive behavior of
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different adsorbents. Langmuir isotherm balances the dynamic equilibrium by covering the
respective rates of adsorption and desorption. The surface coverage of the adsorbent is
analyzed through Langmuir isotherm, where the surface available for adsorbate binding is
referred to as the rate of adsorption, and the surface covered with adsorbate is referred to as
the rate of desorption. This model assumes the monolayer adsorption of the adsorbate on the
adsorbent surface (Çolak et al., 2011). The linear form of Langmuir Isotherm is as follows:

28
 Ce = concentration of adsorbate at equilibrium (mg g−1), qe = amount of adsorbate at
equilibrium.
Du et al. (2016) investigated the adsorption of Pb(II) and Cd(II) ions from aqueous
solution using Pseudomonas putida X4 associated clay mineral composite. The Langmuir
isotherm model was the best-fitted model of the study representing the correlation coefficient
(R2) of ≥ 0.98 and ≥ 0.97 for single and binary metals. Chakraborty et al. (2018) reported that
the adsorption of Cd(II) by functionalized EPS of Pseudomonas aeruginosa N6P6 was
suitably fitted in the Langmuir isotherm model at all the temperatures, i.e., 25ºC, 35ºC, and
45ºC. Similarly, Hg(II) biosorption employing EPS of Bacillus thuringiensis PW-05 was also

of
fitted well with the Langmuir isotherm model at different temperatures showing R2 value ≥
0.96 (Dash and Das, 2016) (Fig. 7). Langmuir model is based on the fact that the adsorbent

ro
surface is homogenous, and each molecule of adsorbent acquired uniform activation energy.
b) Freundlich isotherm: The adsorption processes that occur on heterogonous
-p
surfaces are assessed through Freundlich isotherm. This isotherm model was used to define
the surface heterogeneity, active sites, and their energies. The Freundlich isotherm can be
re
written in the following linear form (Öztürk, 2007):
lP

Kf = adsorption capacity (L/mg), 1/n = adsorption intensity, which also gives the idea
about relative dissemination of energy and the diversity of the adsorbate sites.
na

Ansari and Malik (2007) studied the adsorption of Ni and Cd by Escherichia coli
WS11 isolated from the industrial wastewater. The Freundlich isotherm model described the
equilibrium data of both the metals in single and combined metal ion solutions. The
ur

adsorption of multiple metal ions by Exiguobacterium sp. ZM-2 was also fitted to the
Freundlich isotherm model (Alam and Ahmed, 2013). The Freundlich isotherm model can be
Jo

applied for the multilayer adsorption process. However, this model is suitable for explaining
adsorption within low intermediate concentration ranges.
c) Dubinin-Radushkevich isotherm: Dubinin-Radushkevich isotherm model is a
practical adsorption model applicable to measure the adsorption system with Gaussian energy
distribution onto heterogeneous surfaces. The differentiation among physical and chemical
adsorption of metal ions is the typical feature of this isotherm model. Instead of surface
coverage, this model is based upon filling pores for gas adsorption (Dubinin, 1960). A unique

29
aspect of this isotherm is that it depends upon temperature. So, by plotting of adsorption data
at various temperatures as a function of the logarithm of the amount adsorbed versus the
square of potential energy, all the appropriate data can be obtained. The equation of Dubinin-
Radushkevich isotherm can be represented as follows:

As the adsorption model is based upon Polanyi adsorption potential theory and the
concentration of solute is dimensionless, the equation for the calculation of Polanyial energy

of
is substituted with the standard concentration value of the solution (Zhou, 2020). The
modified equation can be represented as,

ro
-p
Where, lnqe = logarithm of amount adsorbed at equilibrium, lnqm= logarithm of the
amount absorbed at saturation, E = potential energy, ϵ = Polanyi potential (Jmol-1), β=
re
Dubinin-Radushkevich constant for the mean free energy of sorption per mole of the
adsorbate (mol2J-2), R= gas constant (8.31 Jmol−1 k−1), T = Absolute temperature (K), E =
lP

mean adsorption energy, Ce is the solute concentration in equilibrium solution (mol dm-3) and
C0 = concentration of the solution in the standard state (1 mol dm-3).
Cayllahua et al. (2009) applied the Dubinin-Radushkevich isotherm model to
na

investigate the adsorption of Ni(II) ions with the Rhodococcus opacus biomass. In the
adsorption process of Cd(II), Cu(II), Ni(II), Zn(II), and Mn(II) by using Geobacillus
toebii sub.sp. decanicus and Geobacillus thermoleovorans sub.sp. stromboliensis cells, the
ur

Dubinin-Radushkevich model was applied for estimating the mean free energy values of the
adsorption process. The result indicated that the physical interaction between the metals and
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adsorbent is solely responsible for the biosorption of metal ions (Özdemir et al., 2009). The
Dubinin-Radushkevich isotherm model depends on the porous nature of the adsorbent and
accurate determination of Polanyi potential is essential for applying this model in adsorption
processes.
d) Temkin isotherm: Temkin isotherm model is applied for the indirect interaction
between the adsorbent and adsorbate on the adsorption process. According to this isotherm,
the increase in surface coverage results in a decrease in the heat of adsorption (ΔHads) of all

30
the molecules in the layer. The Temkin isotherm is applicable only for an intermediate
concentration range. The linear form of Temkin isotherm can be expressed as:

Where b = Temkin constant related to the heat of sorption (J mol−1), KT = Temkin isotherm
constant (Lg−1).
Temkin model explained the adsorption of Zn(II) ions on to the Zinc sequestering
bacterium VMSDCM and at temperature 25ºC and 30ºC, it was appropriately fitted into the
experimental data (Mishra et al., 2014). According to Chen et al. (2009), the Temkin
isotherm model assumes that the heat of adsorption of all the molecules or ions in the layer
decreases linearly with the thickness of the coverage due to the interaction of Cu(II) and

of
Pb(II) ions with the amidoximated bacterial cellulose. Temkin isotherm model is not valid for
complex adsorption reactions with a very low and high range of concentrations.

ro
e) Flory-Huggins isotherm: This isotherm model can evaluate the degree of surface
coverage ability of the adsorbate on the adsorbent. This isotherm model can explain the
-p
feasibility and spontaneity of an adsorption mechanism. The linear equation of Flory-Huggins
isotherm can be written as:
re
lP

Where θ= degree of surface coverage, n= number of adsorbates occupying adsorption sites,

KFH = Flory-Huggins equilibrium constant (L mol−1). The Flory-Huggins isotherm model was
applied in Ni(II) biosorption carried out by immobilized biomass of Bacillus cereus and
na

suggested that the process is exothermic (Naskar and Bera, 2018). Generally, this isotherm
model gives an idea about the spontaneity of the reaction medium.
iii) Three-Parameter isotherm
ur

a) Redlich-Peterson isotherm: This isotherm is a combination of the Langmuir and

Freundlich isotherms. The numerator is from Langmuir isotherm and has the advantage of
Jo

approaching the Henry region at infinite dilution (Redlich and Peterson, 1959). This is a
three-parameters containing empirical model and incorporates both the Langmuir and
Freundlich equations. This model applies to a wide concentration range in various adsorption
equilibrium studies. The linear equation of Redlich-Peterson isotherm can be expressed as:

31
 Where KRP = Redlich-Peterson equilibrium constant, Ce = concentration of adsorbate
at equilibrium. It reduces to Langmuir isotherm when β = 1. The isotherm model was best
fitted to Pb(II) adsorption data by utilizing Bacillus cereus cells as adsorbent (Pan et al.,
2006). Due to the flexibility of the Redlich-Peterson isotherm, it can be applied to both
homogenous and heterogeneous adsorption systems. However, it does not follow the ideal
monolayer adsorption.
The accuracy in the result achieved from the adsorption practices predominantly
depends upon the appropriate modeling and interpretation of adsorption isotherms.
Equilibrium concentrations are a function of temperature, but analysis of a single temperature
value may not exhibit a realistic result. Hence, different isotherm models with varying values

of
of temperature are required for getting the most relevant result. The isotherm model with the
best R2 value under the test temperatures should be taken as the best isotherm model to

ro
describe the adsorption behavior of an adsorbate on a given adsorbent.
7. Desorption and recovery of heavy metal ions
The desorption phenomenon can be defined as removing the adsorbate, i.e., heavy
-p
metal ions from the biosorbent surface. The desorption mechanism can be expressed by solid
to liquid ratio; in which the adsorbent that allows heavy metal adsorption is referred to as
re
solid phase, and the desorption medium that helps in the recovery of metal ions is referred to
as liquid phase (Vijayaraghavan and Yun, 2008; Wang and Chen, 2009). The desorption
lP

mechanism should be aimed to recover maximum metal ions from the biosorption medium,
and the recovered biomass should have the metal-binding affinity (Alluri et al., 2007). The
desorption mechanism of metal ions from loaded biomass followed by the recovery of heavy
na

metals and the regeneration of bacteria biosorbent has been depicted in Fig. 8.
Desorption of heavy metal from the biosorbent requires a suitable eluant, which
should be metal selective, cost-effective, eco-friendly, and should not adversely affect the
ur

biosorbent. The desorption process is crucial for the economic feasibility and the continuous
supply of adsorbent to the biosorption reaction.
Jo

7.1. Eluents used for desorption of heavy metals

Various eluents have been applied to restore the bacterial biosorbents, such as mineral
acids/bench acids (H2SO4, HNO3, and HCl), organic acids (acetic acids, citric acid, and lactic
acid), alkalis (NaOH, KOH, etc.), and chelating agents (EDTA and thiosulphate), etc. (Sar et
al., 1999; Leung et al., 2000; Goyal et al., 2003; Sar et al., 2004; Ahemad and Kibret, 2013;
Vishan et al., 2019). The desorption of metal ions by the eluents may be due to the
dissolution and/or chelation of metals in the liquid desorption medium. For most metals, acid

32
eluents are used to dissolve metal ions that are adsorbed on the biosorbent surface. Similarly,
alkaline eluents are used for the extraction of some anionic metal species. However, the use
of chemicals (EDTA) also solubilizes the adsorbed metals within the liquid medium.
i) Mineral acids as desorbent: The acid solution acts as a suitable medium for the
desorption of metal ions. A lower pH value of the solution promotes metal desorption.
Competition between metal ions and protons for binding sites on the biosorbents gives rise to
the delocalization of metal ions in the acid eluent. The common inorganic acids, such as
H2SO4, HNO3, and HCl, are applied as eluents for the desorption of heavy metals from the
biosorbent. H2SO4 is a strong mineral acid that dissociates the adsorbed metals by protonation
of the biosorbent surface. H2SO4 has been utilized for the desorption of various heavy metals

of
like Cu, Cd, Zn, Ni, etc. This desorbent recovered more than 90% of Cu(II) ions from the
bacterial biomass (Loutseti et al., 2009; Tassist et al., 2009). However, recovery of Pb was

ro
not suitable by utilizing H2SO4, as Pb precipitates by reacting with the acid and forms the lead
sulfate (Puranik and Paknikar 1999). A very dilute solution of H2SO4 can efficiently recover
various metal ions.
-p
HNO3 is one of the most effective eluents for heavy metal recovery and has shown
better desorption efficiency than other mineral acids. HNO3 can efficiently recover Pb, Ni,
re
Cu, and Cd from the biosorbent surface (Teemu et al., 2008; Kumari et al., 2017). The
advantage associated with HNO3 is that it does not affect the quality of biosorbent. Besides,
lP

HNO3 contains many proton ions that interact with the sorbents and break the bond between
sorbent and heavy metals (Davila-Guzman et al., 2016). A dilute solution of HNO3 is
efficient in removing metal from the biosorbent. As HCl is a strong mineral acid, a very high
na

concentration can damage the binding efficiency of bacterial biosorbent. HCl is also an
efficient desorbing agent for recovery of Pb, Cd, and Cu from Enterobacter sp. biomass (at
pH 2) with high biomass regeneration and metal recovery efficiency. 0.1M HCl could recover
ur

more than 90% of all the metals even after four cycles of the adsorption-desorption process.
Besides, HCl was able to restore the regeneration efficiency of the biomass above 75% for all
Jo

the metals in successive biosorption study (Lu et al., 2006). The desorption efficiency of 0.1N
HCl along with 0.01M thiourea was ~97% for Pd(II) from polyethylenimine coated
Escherichia coli biomass composite fiber (Cho et al., 2016). Both HCl and H2SO4 were able
to recover 95% of trivalent Cr from Streptomyces rimosus biomass (Sahmoune et al., 2009).
The desorption of Cd(II) from Brevundimonas sp. ZF12 biomass was done using HCl at
different pH, i.e., from 2 to 5, where >90% of metal ions were recovered in all pH conditions
except pH 5 (Masoudzadeh et al., 2011). However, a very dilute solution of HCl is not

33
suitable for metal desorption, as the pH of the dilute HCl is not sufficient to dissociate the
bond between adsorbate and adsorbent. Recovery of metal ions by applying acid solution is
relatively faster than other eluents and is chiefly utilized for desorption of metal ions.
ii) Alkalis as desorbent: A few metals such as Cr and As may be present in the
anionic form. Hence, the application of alkalis as a desorbing solution can effectively recover
those metals from the biosorbents. Some of the dilute alkaline solutions are NaOH, Na2CO3,
and NaHCO3, predominantly employed as desorbents and were reported to effectively
recover Cr ions from bacterial biomass (Mishra, 2014). NaOH is a strong base; hence, it plays
a vital role in recovering some anionic metal ions. NaOH was proved to be a suitable eluent
for Cr(III) desorption (81%) from Bacillus subtilis biomass. The increase in recovery of

of
trivalent Cr was observed with an increase in the concentration of NaOH solution, and the
maximum recovery was obtained at 5M solution of the same eluent (Aravindhan et al., 2012).

ro
Further, NaOH has been predominantly utilized as a desorbing eluent for Cr recovery from
various other bacterial adsorbents (Sivaprakash et al., 2009; Zhang et al., 2015; Sathvika et
al., 2019). The application of 0.1M NaOH to the desorbed biomass of Brevibacterium sp.
-p
strain HZM-1 fully restored the Zn(II) adsorption ability of the adsorbent (Taniguchi et al.,
2000). Alam and Ahmad (2011) reported more than 90% desorption of both Cr(III) and
re
Cr(VI) ion from the dead biomass of Stenotrophomonas maltophilia ZA-6, Exiguobacterium
sp. ZM-2, Pantoea sp. KS-2 and Aeromonas sp. KS-14. NaOH is more effective than other
lP

alkalis for desorption process.

Due to the stable alkaline property of Na2CO3, it can be effectively used as an eluent
for heavy metal recovery. Na2CO3 (0.01-0.1M) was found to preserve the regeneration
na

efficiency of the Pseudomonas aeruginosa Strain CSU biomass for U removal and
successfully recovered 95% of the adsorbed U (Hu and Reeves, 1997). 0.1M sodium
carbonate efficiently recovered Cr(VI) ions from the different bacterial biomass, and these
ur

were successfully reused for further removal of Cr with 83.4–91.7% adsorption efficiency
(Oyetibo et al., 2013). Na2CO3 has been applied for the desorption of various metals and
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exhibited more regeneration efficiency than other eluents.

iii) Chelating agents as desorbent: Chelating agents have the efficiency to release
the metal ions from the biosorbent surface by complex formation with the metal ions.
Chelating agents generate water dissolved compounds by desorbing the heavy metals from
the bonded surface (Akhtar and Iram, 2014). The desorption percentage of Cu(II) and Pb(II)
ions from diethylenetriamine-bacterial cellulose were higher by applying EDTA as a
desorbing agent than the HCl solution (Shen et al., 2009). Along with HNO3, the dilute

34
solution of EDTA was able to recover 100% adsorbed Pb(II) and Cd(II) from Lactobacillus
fermentum and Bifidobacterium longum (Teemu et al., 2008). EDTA is a potent desorbing
agent that retained the adsorption efficiency of the biosorbent in several cycles of the
adsorption process with little or no damage to the binding sites. Chelating agents in the
desorption process can eliminate the exhaustion of huge amounts of acid or alkalis.
There are extensive screening experiments conducted to select suitable eluents for
maximum recovery of metal ions (Table 3). Apart from the appropriate eluent, the desorption
process also depends on various factors for better yield, such as the pH of the desorbing
agent, reaction temperature, shaking condition, and the contact time of the eluent with the
adsorbent. However, it was observed that a single eluent that was ideal for the recovery of

of
metal from one adsorbent was not essentially suitable for another adsorbent. Hence, further
studies are needed to develop a noble desorbent that can be effective for multiple adsorbents.

ro
7.2. Estimation of heavy metals after desorption
Once the desorption process is over, most of the desorbents contain a high
concentration of heavy metals. Several spectroscopic techniques analyze the heavy metal
-p
content in the eluent. The metal loaded biomass regains their adsorption ability after
treatments with desorbents.
re
The percentage of heavy metal desorption can be calculated from the following
equation:
lP

Where C0 represents the amount of heavy metals (mg L-1) bonded to the adsorbent
na

before the desorption process, and Ci represents the amount of heavy metals (mg L-1) bonded
to the adsorbent after the desorption process.
ur

Where q = the amount of heavy metal per unit of adsorbent after desorption
Jo

experiment (mg g-1), V = volume of desorbent or eluant solution, Cf = heavy metal

concentration in the eluant solution after desorption experiment (mg L-1), and M = mass of
the adsorbent taken (g) (Bhuvaneshwari et al., 2012).
The combined process of biosorption, desorption, and reprocessing of biosorbent
makes the adsorption experiment successful. Regeneration of biosorbents for their
reprocessing for the maximum number of cycles needs suitable desorbents with non-
detrimental effects. The regeneration efficiency of Bacillus sphaericus biomass was retained

35
up to five cycles of Ni(II) adsorption (Aryal, 2015). However, the regeneration efficiency of
Staphylococcus equisimilis for Cr(VI) was decreased after the first cycle of biosorption
(Goyal et al., 2003). Hence, the utilization of an appropriate eluent is vital for maximum
regeneration efficiency. Regeneration efficiency of biosorbent can be calculated by using the
following formula:

In which, A(r) refers to the regenerated adsorption capacity of the adsorbent (mg g-1),
and A(o) refers to the original adsorption capacity of the adsorbent (mg g-1) (Lu et al., 2006).
Metal recovery efficiency can be calculated as:

of
ro
After completion of the desorption process, the eluents contain a significant amount
of metal ions. Therefore, it is necessary to extract heavy metals from these desorbents
-p
employing various electrochemical techniques, and further use them for other purposes (Yasri
and Gunasekaran, 2017). Besides, the exhausted biomasses should also be discarded safely
without causing harm to the environment. As the dumping and burning of exhausted
re
adsorbents are associated with soil, air, and groundwater contamination, suitable alternative
methods such as thermal desorption and maximum possible recycling of biosorbents are
lP

necessary for sustainable utilization.

8. Conclusion
In the advancement of better cleanup technology, such as removing heavy metals for
na

the purification of wastewater, biosorption can play an imperative role. Biosorption is an

advantageous process which is associated with the removal of heavy metal pollutants.
ur

Microbial biomass is one of the cheapest adsorbents and can efficiently remove heavy metals
from the polluted environment. Bacterial biomass that acts as a sorbent for metal removal is
environmentally and economically feasible, but studies are needed to face challenges in
Jo

treating large-scale industrial effluents. Applications of these biosorbents in pilot-scale are

vital for assessing their efficiency in dynamic environmental conditions at a full-scale
approach. Various parameters that positively regulate the adsorption process in the laboratory
studies may adversely affect full-scale practices. The controlled pH of the reaction medium
enhances the metal removal efficiency of the biosorbent. However, varying pH of the
industrial discharge may affect the potency of the adsorbent as it is not feasible to maintain

36
the pH of the real wastewater. In the laboratory experiments, an increase in adsorption of
heavy metals is observed with an increase in the contact time of the adsorbent with the metal
ions. In industrial or pilot-scale applications, the contact time is shorter (as industrial
discharge releases tonnes of effluents), resulting in reduced metal removal. Agitation is an
important parameter that enhances the adsorption rate of metals by increasing the diffusion
rate of metal ions all over the biosorbent surface. In the case of full-scale application, the
rapid process saturates the biosorbent surface within very less time. Most of the laboratory-
based techniques need to be established, commercialized, and scale-up. Other concerns
include optimal dosages of biosorbents and optimization of process parameters for the
industrial application of biosorbents. As the industrial effluents carry several metal ions with

of
varying concentrations, more vigorous screening procedures should be implemented and
advanced statistical analysis to deal with the wastewater. The execution of various kinetic and

ro
isotherm models will provide information about environmental conditions, which will further
help optimize the biosorption process. In addition to biosorption, several associated methods
like desorption, regeneration, and reuse of biosorbents should be estimated at a large scale.
-p
The development of more selective biosorbents and the selection of suitable biosorption
models is crucial for applying the biosorption process.
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Declaration of interest statement
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The authors declare no conflict of interest.

Declaration of interest statement

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I, on behalf of all the authors, declare that we do not have any conflict of interest.
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Acknowledgements
The authors acknowledge the authorities of NIT Rourkela for providing the research
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facility. MP acknowledges the Department of Science & Technology, Govt of India, for the
INSPIRE Award (IF170195) for doctoral study.

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Table 1. Different functional groups of bacteria that interact with heavy metals
Heavy Functional
Biomass Mechanism References
metals groups
Electrostatic
Amine,
reaction and Chakravarty and
Acidophillic bacteria Cd Carboxyl and
complex Banerjee, 2012.
hydroxyl group
formation

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Golab and
Electrostatic
Streptomyces pilosus Cu Carboxyl Breitenbach,
binding

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1995.
Ionic
Pseudomonas -NH, -OH, -CH exchange and
Cd Guo et al., 2012
plecoglossicida and –CONH -pelectrostatic
interaction
Carboxyl,
Cyanobacteria
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Sb hydroxyl and Complexation Wu et al., 2012
mycrocystis
Amino group
Carboxyl,
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Cyanobacteria Sb hydroxyl and Complexation Sun et al., 2011

mycrocystis
Amino group
Amino and Complexation,
carboxyl group adsorption and Ahalya et al.,
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Zoogloea ramigera Cu
electrostatic 2003
interaction
-CH Ion exchange Quintelas et al.,
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Escherichia coli Ni
2009)
Bacillus cereus Amino, Physical-
Pseudomonas carboxyl, chemical
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Zn Joo et al., 2010

aeruginosa hydroxyl and adsorption and
carbonyl ion exchange
Ochrobactrum carboxylic,
intermedium LBr Cu & Surface
hydroxyl and Fan et al., 2014
Cupriavidus Cr complexation
metallidurans CH34 amino groups
carboxylic, Surface
Hasan et al.,
Aeromonas hydrophilla Pb hydroxyl and complexation
2009
amine groups and ion

64
 exchange
carboxylic,
Pb, Cr Bueno et al.,
Rhodococcus opacus hydroxyl and Complexation
& Cu 2008
amine groups
Cd,
Bacillus Ion exchange,
Cr, amino, carboxyl,
thuringiensis strain complexation
Pb, hydroxyl, and Oves et al., 2013
OSM29 and surface
Cu, carbonyl groups
adsorption
and Ni
carboxyl,
surface
hydroxyl, and Selatnia et al.,
Streptomyces rimosus Pb adsorption,
carbonyl, amide 2004
complexation
groups

of
Bacillus sp. MC3B-22 Carbonyl, amide Camacho-Chab
and Microbacterium sp. Cd Complexation
groups et al., 2018
MC3B-10
Hydroxyl,

ro
carboxyl, amine Complexation, Lameiras et al.,
Arthrobacter viscosus Cr
and sulphate ion exchange 2008

Pseudomonas sp. Lk9

Cd,
Cu
groups

Amino
-p
Ion exchange,
Luo et al., 2014
complexation
re
and Pb
Pseudoalteromonas sp. Pb, CdCarboxyl, amide,
Surface
SCSE709-6 and acyl and Jiang et al., 2017
adsorption
lP

Zn hydroxyl
Pseudomonas Carboxyl and
Cr Complexation Kang et al., 2007
aeruginosa amine groups
Cr(III) Amine, carboxyl Ion exchange Aryal and
na

Mycobacterium sp. and and phosphate and Liakopoulou-

strain Spyr1
Cr(VI) groups complexation Kyriakides, 2014
Amine, carboxyl Ion exchange
Arthrobacter sp. Sphe3
ur

Cu(II) and phosphate and Aryal et al., 2012

Bacillus sphaericus
groups complexation
Cd(II),
Hydroxyl,
Jo

Pseudomonas Cu(II) Surface Choińska-Pulit et

carboxyl and
azotoformans JAW1 & adsorption al., 2018
amine groups
Pb(II)
Complexation,
Pb(II) Carboxyl, amide electrostatic
Bacillus sp. strain
& and phosphate interaction Shao et al., 2019
MRP-3
Cr(VI groups and ion
exchange
Cronobacter muytjensii Cd(II), Hydroxyl, Precipitation Saranya et al.,

65
KSCAS2 Cu(II), amine, 2018
Cr(VI) phosphate,
& carbonyl,
Zn(II)
Carboxyl,
hydroxyl,
Cu(II)
phosphate,
Bacillus cereus & Complexation Pan et al., 2007
amino, and
Pb(II)
amide functional
groups
Carboxyl, Complexation
Bacillus cereus Pb(II) hydroxyl and and Pan et al., 2006
amino groups precipitation

of
Caboxyl, amide,
Complexation Oh et al., 2009
Pseudomonas stutzeri Pb(II), hydroxyl,
and surface
Cd(II) phosphate,
adsorption

ro
& sulphate
Cu(II) Hydroxyl,
Surface Rajesh et al.,
Halomonas BVR 1 Cd(II) carboxyl and
amino groups -p adsorption 2014
re
lP
na
ur
Jo

66
Table 2. Heavy metal removal by bacteria in single and multimetal solution
Uptake Uptake
Heavy capacity in capacity in
Bacteria Observation Reference
metals single metal multimetal
ion solution solution
Pseudomonas Cr & 278 mg.gˉ1 150 mg.gˉ1 Uptake of Cd(II)
sp. Cd (Cd) (Cd) & Cr(VI) by
95 mg.gˉ 1
25 mg.gˉ 1

of
both bacterial
(Cr) (Cr) biomass in single
metal ion Ziagova et

ro
Cr & 250 mg.gˉ 1
130 mg.gˉ 1
solution is higher al., 2007
Staphylococcus Cd (Cd) (Cd) in comparison to
xylosus
143 mg.gˉ 1
12.5mg.gˉ 1
combined
(Cr) (Cr)
-p mixture of Cd(II)
and Cr(VI) ion
solution
re
Uptake of
Cr(VI), Cu(II) &
Ni(II) from their
lP

61 mg.gˉ 1
individual metal
100 mg.gˉ1
(Cr) ion solutions are
(Cr) Masood
Acenetobacter Cr, Cu 53 mg.gˉ 1
greater than their
83.8 mg.gˉ1 and Malik,
na

sp. FM4 & Ni (Cu) uptake from

(Cu) 2015
25 mg. gˉ 1
ternary metal ion
47mg. gˉ1 (Ni)
(Ni) solution with a
more affinity of
ur

the bacterium
towards Cr(VI)
Jo

67
 Removal of
metal ions from
each metal salt
60 mg.gˉ1 43.48 solution is higher
(Pb) mg.gˉ1 than the removal
Bacillus Pb, Cr 40.32 mg.gˉ1 25.67 of respective Sati et al.,
megaterium & Cd (Cd) mg.gˉ1 ions in ternary 2014
18.92 mg.gˉ1 13.56 metal ion
(Cr) mg.gˉ1 solution with a
greater affinity
of the bacterium
towards Pb(II)
The uptake of
192 μmol g 105.5 μmol g
-1 -1
ions from each

of
(Co) (Co) metal solutions
Pseudomonas Co, Ni 172 μmol g-1
105.4 μmol g-1 is greater than Kang et
aeruginosa & Cr (Ni) (Ni) the removal of al., 2005

ro
154 μmol g-1
90.9 μmol g-1 respective ions
(Cr) (Cr) in multimetal
-p condition
Biosorption of
all the three
16% (Cu),
re
22 % (Cu), heavy metals is Parungao
Stenotrophomo Cu, Cd 8% (Cd),
24% (Cd), & higher in et al.,
nas maltophilia & Pb and
42.75% (Pb) 35% (Pb) primary solution 2007
lP

than in ternary
solution
The rate of
removal
na

bimetallic
solution is higher
55.31 μg.ml-1 26.89 μg.ml-1 due to
Ansari
Ni & (Ni) (Ni) competition of
ur

Escherichia and Malik,

coli WS11 Cd 45.37 μg.ml-1 26.42 μg.ml-1 both the metals
2007
(Cd) (Cd) for binding sites
Jo

or due to the
inhibition of
effective binding
site
208.08 mg.gˉ1 58.93 mg.gˉ1 The metal with
Bacillus (Cd) (Cd) high efficiency
Cd, Hg Wang and
subtilis 382.43 mg.gˉ1 377.84 mg.gˉ1 in metal removal
& Pb Sun, 2013
(Hg) (Hg) in the mono-
400.75 mg.gˉ1 288.95 mg.gˉ1 component

68
 (Pb) (Pb) system showed a
higher inhibitory
effect on the
biosorption of
other metals in
the ternary metal
solution

of
ro
-p
re
lP
na
ur
Jo

69
Table 3. Desorption medium and recovery of heavy metal ions by various bacterial species.

f
Operating conditions

oo
Biomass Maximum
Initial metal Desorption No of Agitation References
Biomass conc. (g L- recovery of metal T
1) ion conc. medium cycles pH Speed
ions (In %) (°C)
(RPM)
Cu(II)

pr
Leung et al.,
Micrococcus luteus 1.5 0.787mM L- 0.05M H2SO4 90% (5 min) 5 6 25 250
1
2000

e-
For U(VI)- 70%,
1M HCL, 1M
72%, 70%, 5%, 92%

Pr
HNO3, 1M
U(VI) & & 20% respectively.
H2SO4, 1M
Pseudomonas sp. 0.5 Th(IV) For Th(IV)- 80%, 4 4 30 150 Sar et al., 2004
CaCO3, 0.01M
100mgL-1 80%, 91%, 93%,
CaCO3, &
l 83% & 36%
na
0.01M EDTA
respectively (24 h)
Cu(II) Wong et al.,
Micrococcus sp. 1.5 0.05M H2SO4 >90% (45 min) 5 6 25 250
ur

50 mgL-1 2001
Cu(II)
Jo

Bacillus sp. 1 to 2 2 and 0.1M HNO3 >90% (5 min) 5 5 25 Lo et al., 2003

50mgL-1
Goethite–Bacillus Cu(II) and 38.3%(Cu) & 22.7%
0.0436 0.1M Ca(NO3)2 1 4.5 25 Zhu et al., 2012
thuringiensis complex Cr(VI) (Cu+Cr) and 29%

70
 4µM (Cr) & 18.2%

f
(Cr+Cu) (8 h)

oo
For Ni(II)- 71% &
Ni(II) and
21% respectively.
Pseudomonas Cu(II) CaCO3 and

pr
0.15 For Cu(II)- 57% & 1 37 150 Sar et al., 1999
aeruginosa 0-1000 mgL- Na2CO3 10mM
1
88% respectively.

e-
(24 h)
Arthrobacter strain 1M HCL and
Cu(II) 62.36mgg-1 and Aryal et al.,
Sphe3 and Bacillus 1 0.8M HCL 5 5 180

Pr
300mgL-1 51.96mgg-1 (30 min) 2012
sphaericus respectively
Staphylococcus Cr(VI) 25- 42% Goyal et al.,
0.1M NaOH 1 25
equisimilis 150 mgL-1 (20 min) 2003
l
na
Pseudomonas
77.6% (EPS + Ca-
aeruginosa N6P6 Per unit of
Pb(II) 15.4 Alginate), >45% Kumari et al.,
(EPS + Ca-Alginate), alginate 10mM HNO3 3 25 400
mgL-1
ur

(Biomass + Ca- 2017

(Biomass + Ca- beads
Alginate) (30 min)
Alginate)
Jo

Ni(II)
Bacillus sphaericus 1 0.1M HNO3 97.76% (60 min) 5 Aryal, 2015
50mgL-1
Citrobacter strain 1g per Pb(II), 0.1M HCL and For Pb(II)- 92.4% Puranik and
3
MCM B-181 25ml Cd(II) & 0.1M EDTA and 78.26% Pakniker, 1999

71
 Zn(II) 1mM respectively. For

f
Cd(II) – 96% and

oo
84.55%
respectively. For

pr
Zn(II)- 93.88% and
80.63%

e-
respectively.
0.11gg-1
Sphaerotilus natans
inside Beolchini et al.,

Pr
(immobilized in Cu(II) 0.05M CaCl2 >80% 10 4.4 30
beads 2003
polysulfone beads)
(4.6gL-1)
Aryal and
l Cr(III) &
na
0.2molL-1 Liakopoulou-
Mycobacterium sp. 1.0 Cr(VI) 100% (60 min) 5
HNO3 Kyriakides,
100mgL-1
2014
ur

36% Fowle and

Bacillus subtilis 10 Cd(II) 10-4M 0.1M NaClO4 24 4 20
(1 h) Fein, 2000
Jo

Cu(II) Tassist et al.,

Streptomyces rimosus 10 H2SO4 99.77% 1.5 500
250mg L-1 2009

72
Figure legends
Fig. 1. Mechanisms involved in heavy metal biosorption by bacterial biomass. (a)
Precipitation involves chemical interaction between the cell surface and metal species, (b)
Complexation of heavy metals occurs by interaction of the metal ions with the surface-
active groups of bacterial biomass, (c) Exchange of bivalent metal ions occurs with the
counter ions present on the bacterial cell in the ion exchange mechanism, (d) Diffusion is
the simple process of biosorption without the involvement of any rate-limiting step, (e)
Surface adsorption is a rapid and reversible process in which metal cations bind to the
bacterial surface anions via non-specific attraction forces, and (f) Intracellular
accumulation of heavy metals takes place by the transport of metal ions across the cell

of
membrane. IM- Inner Membrane, OM- Outer Membrane, EI- Exchangeable Ions, TP-
Transport Proteins.

ro
Fig. 2. Schematic diagram of bacterial biofilm mediated adsorption of heavy metals from
metal-contaminated water.
Fig. 3. Mechanisms of interactions between EPS and heavy metal ions. Bacterial EPS is
-p
synthesized metabolically using glucose molecules. Glucose is converted to glucose 6-
phosphate by hexokinase, which either initiates glycolysis pathway or forms glucose 1-
re
phosphate and further converted to UDP glucose. UDP-glucose then enters into the
anabolic pathway for synthesis of EPS. The complex composition of bacterial EPS exhibits
lP

several mechanisms for metal binding, (a) Ion exchange between the metal cations and
negatively charged functional groups of polysaccharides in EPS, (b) Surface precipitation
of metal is induced by pH of the medium as well as the anionic charge of surface
na

active sites, (c) Complexation involves metal binding by the reaction between heavy metals
and the proteins and polysaccharides of EPS, and (d) Native adsorption of the metal ions
occurs at their native state on to the porous surface of the EPS.
ur

Fig. 4. EDX analysis elucidates the metal accumulation in bacteria and EPS. (a) EPS of P.
pseudoalcaligenes NP103 entrapped 6.4% Pb(II) (Kumari et al., 2017), (b) Bacillus
Jo

pumilus showed adsorption of Cr(III) on the cell surface (Kanagaraj et al., 2014), and (c)
Halomonas sp. showed adsorption of Cd(II) on the cell surface (Rajesh et al., 2014).
Fig. 5. FTIR analysis showing the interaction of biofilm-EPS of various bacteria with
different concentrations of the toxic metals. (a) P. aeruginosa JP-11 with Cd (Chakraborty
and Das, 2014), (b) B. cereus BW-201B with Hg (Dash et al., 2017), and (c) P.
pseudoalcaligenes NP103 with Pb (Kumari et al., 2017).
Fig. 6. Preparation and modification of bacterial biosorbent for increasing the sorption
ability of bacterial biomass. The biosorbents are pre-treated by various methods such as (a)
Physical method for pre-treatment of bacterial biomass, (b) Chemical method for pre-
treatment of bacterial biomass, and (c) Biological method for modification of bacterial cell.
Fig. 7. Langmuir plot showing the adsorption of the toxic metals at different temperatures.
(a) Cd adsorption with the functionalized EPS of P. aeruginosa N6P6 (Chakraborty et al.,
2018), and (b) Hg(II) adsorption by EPS of Bacillus thuringiensis PW-05 (Dash and Das,
2016).
Fig. 8. Strategy for desorption and recovery of heavy metal ions and regeneration of
bacterial biosorbents.

of
ro
-p
re
lP
na
ur
Jo

74
 Fig. 1
Jo
ur
na
lP
re
-p
ro
of

75
 Fig. 2
Jo
ur
na
lP
re
-p
ro
of

76
 Fig. 3
Jo
ur
na
lP
re
-p
ro
of

77
(a)

of
ro
-p
re
lP

(b)
na
ur
Jo

(c)
Fig. 4

78
(a)

of
ro
-p
re
lP

(b)
na
ur
Jo

(c)
Fig. 5

79
 Fig. 6
Jo
ur
na
lP
re
-p
ro
of

80
 (a)

(b)
Fig. 7
Jo
ur
na
lP
re
-p
ro
of

81
 Fig. 8
Jo
ur
na
lP
re
-p
ro
of

82