Biosorptionofheavymetals
Biosorptionofheavymetals
Biosorptionofheavymetals
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1. Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560 012, INDIA.
2. Department of Zoology, Karnataka University, Dharwad, INDIA.
CONTENTS
Abstract
Introduction
Biosorption
Mechanisms
Conclusion
References
ABSTRACT
The discharge of heavy metals into aquatic ecosystems has become a matter of concern in India
over the last few decades. These pollutants are introduced into the aquatic systems significantly as
a result of various industrial operations. Industrialization in India gained a momentum with
initiation of five year developmental plan in the early 50's. The pollutants of concern include lead,
chromium, mercury, uranium, selenium, zinc, arsenic, cadmium, gold, silver, copper and nickel.
These toxic materials may be derived from mining operations, refining ores, sludge disposal, fly
ash from incinerators, the processing of radioactive materials, metal plating, or the manufacture of
electrical equipment, paints, alloys, batteries, pesticides or preservatives. Heavy metals such as
zinc, lead and chromium have a number of applications in basic engineering works, paper and
pulp industries, leather tanning, organochemicals, petrochemicals fertlisers, etc. Major lead
pollution is through automobiles and battery manufacturers. For zinc and chromium the major
application is in fertliser and leather tanning respectively (Trivedi, 1989). Over the few decades,
several methods have been devised for the treatment and removal of heavy metals.
INTRODUCTION
The commonly used procedures for removing metal ions from aqueous streams include chemical
precipitation, lime coagulation, ion exchange, reverse osmosis and solvent extraction (Rich and
Cherry, 1987). The process description of each method is presented below.
Electrodialysis: In this process, the ionic components (heavy metals) are separated through the use
of semi-permeable ionselective membranes. Application of an electrical potential between the two
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electrodes causes a migration of cations and anions towards respective electrodes. Because of the
alternate spacing of cation and anion permeable membranes, cells of concentrated and dilute salts
are formed. The disadvantage is the formation of metal hydroxides, which clog the membrane.
Ultrafiltration: They are pressure driven membrane operations that use porous membranes for the
removal of heavy metals. The main disadvantage of this process is the generation of sludge.
Ion-exchange: In this process, metal ions from dilute solutions are exchanged with ions held by
electrostatic forces on the exchange resin. The disadvantages include: high cost and partial
removal of certain ions.
Phytoremediation: Phytoremediation is the use of certain plants to clean up soil, sediment, and
water contaminated with metals. The disadvantages include that it takes a long time for removal of
metals and the regeneration of the plant for further biosorption is difficult.
Hence the disadvantages like incomplete metal removal, high reagent and energy requirements,
generation of toxic sludge or other waste products that require careful disposal has made it
imperative for a cost-effective treatment method that is capable of removing heavy metals from
aqueous effluents.
BIOSORPTION
The search for new technologies involving the removal of toxic metals from wastewaters has
directed attention to biosorption, based on metal binding capacities of various biological materials.
Biosorption can be defined as the ability of biological materials to accumulate heavy metals from
wastewater through metabolically mediated or physico-chemical pathways of uptake (Fourest and
Roux, 1992). Algae, bacteria and fungi and yeasts have proved to be potential metal biosorbents
(Volesky, 1986). The major advantages of biosorption over conventional treatment methods
include (Kratochvil and Volesky, 1998 a):
• Low cost;
• High efficiency;
The biosorption process involves a solid phase (sorbent or biosorbent; biological material) and a
liquid phase (solvent, normally water) containing a dissolved species to be sorbed (sorbate, metal
ions). Due to higher affinity of the sorbent for the sorbate species, the latter is attracted and bound
there by different mechanisms. The process continues till equilibrium is established between the
amount of solid-bound sorbate species and its portion remaining in the solution. The degree of
sorbent affinity for the sorbate determines its distribution between the solid and liquid phases.
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Biosorbent material: Strong biosorbent behaviour of certain micro-organisms towards metallic ions
is a function of the chemical make-up of the microbial cells. This type of biosorbent consists of dead
and metabolically inactive cells.
Some types of biosorbents would be broad range, binding and collecting the majority of heavy
metals with no specific activity, while others are specific for certain metals. Some laboratories have
used easily available biomass whereas others have isolated specific strains of microorganisms and
some have also processed the existing raw biomass to a certain degree to improve their biosorption
properties;
Recent biosorption experiments have focused attention on waste materials, which are by-products
or the waste materials from large-scale industrial operations. For e.g. the waste mycelia available
from fermentation processes, olive mill solid residues (Pagnanelli, et al 2002), activated sludge
from sewage treatment plants (Hammaini et aI. 2003), biosolids (Norton et al 2003), aquatic
macrophytes (Keskinkan et aI. 2003), etc.
Norton et aI. 2003, used dewatered waste activated sludge from a sewage treatment plant for the
biosorption of zinc from aqueous solutions. The adsorption capacity was determined to be 0.564
mM/g of biosolids. The use of biosolids for zinc adsorption was favourable compared to the
bioadsorption rate of 0.299 mM/g by the seaweed Durvillea potatorum (Aderhold et aI. 1996).
Keskinkan et al. 2003 studied the adsorption characteristics of copper, zinc and lead on submerged
aquatic plant Myriophyllum spicatum. The adsorption capacities were 46.69 mg/g for lead, 15.59
mg/g for zinc and 10.37 mg/g for copper. Table 1 gives a comparison of heavy metal uptakes of
various macrophytes.
Pagnanelli, et al 2002 have carried out a preliminary study on the 'Use of oli ve mill residues as hea
vy metal sorbent material The results revealed that copper was maximally adsorbed in the range of
5.0 to 13.5 mg/g under different operating conditions.
The simultaneous biosorption capacity of copper, cadmium and zinc on dried activated sludge
(Hammaini et al. 2003) were 0.32 mmoI/g for metal system such as CuCd; 0.29 mmoI/g for Cu-Zn
and 0.32 mmoI/g for Cd-Zn. The results showed that the biomass had a net preference for copper
followed by cadmium and zinc.
Although abundant natural materials of cellulosic nature have been suggested as biosorbents, very
less work has been actually done in that respect.
The mechanism of biosorption is complex, mainly ion exchange, chelation, adsorption by physical
forces, entrapment in inter and intrafibrilliar capillaries and spaces of the structural polysaccharide
network as a result of the concentration gradient and diffusion through cell walls and membranes.
There are several chemical groups that would attract and sequester the metals in biomass:
acetamido groups of chitin, structural polysaccharides of fungi, amino and phosphate groups in
nucleic acids, amido, amino, sulphhydryl and carboxyl groups in proteins, hydroxyls in
polysaccharide and mainly carboxyls and sulphates in polysaccharides of marine algae that belong
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to the divisions Phaeophyta, Rhodophyta and Chlorophyta. However, it does not necessarily mean
that the presence of some functional group guarantees biosorption, perhaps due to steric,
conformational or other barriers.
Choice of metal for biosorption process: The appropriate selection of metals for biosorption
studies is dependent on the angle of interest and the impact of different metals, on the basis of
which they would be divided into four major categories: (i) toxic heavy metals (ii) strategic metals
(iii) precious metals and (iv) radio nuclides. In terms of environmental threats, it is mainly
categories (i) and (iv) that are of interest for removal from the environment and/or from point
source effluent discharges.
Apart from toxicological criteria, the interest in specific metals may also be based on how
representative their behaviour may be in terms of eventual generalization of results of studying
their biosorbent uptake. The toxicity and interesting solution chemistry of elements such as
chromium, arsenic and selenium make them interesting to study. Strategic and precious metals
though not environmentally threatening are important from their recovery point of view.
MECHANISMS
Biosorption Mechanisms: The complex structure of microorganisms implies that there are many
ways for the metal to be taken up by the microbial cell. The biosorption mechanisms are various
and are not fully understood. They may be classified according to various criteria.
According to the dependence on the cell's metabolism, biosorption mechanisms can be divided
into:
According to the location where the metal removed from solution is found, biosorption can be
classified as
Transport of the metal across the cell membrane yields intracellular accumulation, which is
dependent on the cell's metabolism. This means that this kind of biosorption may take place only
with viable cells. It is often associated with an active defense system of the microorganism, which
reacts in the presence of toxic metal.
In the case of precipitation, the metal uptake may take place both in the solution and on the cell
surface (Ercole, et al. 1994). Further, it may be dependent on the cell's' metabolism if, in the
presence of toxic metals, the microorganism produces compounds that favour the precipitation
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process. Precipitation may not be dependent on the cells' metabolism, if it occurs after a chemical
interaction between the metal and cell surface.
Transport across cell membrane: Heavy metal transport across microbial cell membranes may be
mediated by the same mechanism used to convey metabolically important ions such as potassium,
magnesium and sodium. The metal transport systems may become confused by the presence of
heavy metal ions of the same charge and ionic radius associated with essential ions. This kind of
mechanism is not associated with metabolic activity. Basically biosorption by living organisms
comprises of two steps. First, a metabolism independent binding where the metals are bound to the
cell walls and second, metabolism dependent intracellular uptake, whereby metal ions are
transported across the cell membrane. ( Costa, et.al., 1990, Gadd et.al., 1988, Ghourdon et.al., 1990,
Huang et.al., 1990., Nourbaksh et.al., 1994)
Physical adsorption: In this category, physical adsorption takes place with the help of van der
Waals' forces. Kuyucak and Volesky 1988, hypothesized that uranium, cadmium, zinc, copper and
cobalt biosorption by dead biomasses of algae, fungi and yeasts takes place through electrostatic
interactions between the metal ions in solutions and cell walls of microbial cells. Electrostatic
interactions have been demonstrated to be responsible for copper biosorption by bacterium
Zoogloea ramigera and alga Chiarella vulgaris (Aksu et al. 1992), for chromium biosorption by fungi
Ganoderma lucidum and Aspergillus niger .
Ion Exchange: Cell walls of microorganisms contain polysaccharides and bivalent metal ions
exchange with the counter ions of the polysaccharides. For example, the alginates of marine algae
occur as salts of K+, Na+, Ca2+, and Mg2+. These ions can exchange with counter ions such as
CO2+, Cu2+, Cd2+ and Zn2+ resulting in the biosorptive uptake of heavy metals (Kuyucak and
Volesky 1988). The biosorption of copper by fungi Ganoderma lucidium (Muraleedharan and
Venkobachr, 1990) and Aspergillus niger was also up taken by ion exchange mechanism.
Complexation: The metal removal from solution may also take place by complex formation on the
cell surface after the interaction between the metal and the active groups. Aksu et al. 1992
hypothesized that biosorption of copper by C. vulgaris and Z. ramigera takes place through both
adsorption and formation of coordination bonds between metals and amino and carboxyl groups of
cell wall polysaccharides. Complexation was found to be the only mechanism responsible for
calcium, magnesium, cadmium, zinc, copper and mercury accumulation by Pseudomonas syringae.
Microorganisms may also produce organic acids (e.g., citric, oxalic, gluonic, fumaric, lactic and
malic acids), which may chelate toxic metals resulting in the formation of metallo-organic
molecules. These organic acids help in the solubilisation of metal compounds and their leaching
from their surfaces. Metals may be biosorbed or complexed by carboxyl groups found in microbial
polysaccharides and other polymers.
Use of Recombinant bacteria for metal removal: Metal removal by adsorbents from water and
wastewater is strongly influenced by physico-chemical parameters such as ionic strength, pH and
the concentration of competing organic and inorganic compounds. Recombinant bacteria are being
investigated for removing specific metals from contaminated water. For example a genetically
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engineered E.coli, which expresses Hg2+ transport system and metallothionin (a metal binding
protein) was able to selectively accumulate 8 mmole Hg2+/g cell dry weight. The presence of
chelating agents Na+, Mg2+ and Ca2+ did not affect bioaccumulation.
Factors affecting Biosorption : The investigation of the efficacy of the metal uptake by the
microbial biomass is essential for the industrial application of biosorption, as it gives information
about the equilibrium of the process which is necessary for the design of the equipment.
The metal uptake is usually measured by the parameter 'q' which indicates the milligrams of metal
accumulated per gram of biosorbent material and 'qH' is reported as a function of metal
accumulated, sorbent material used and operating conditions.
1. Temperature seems not to influence the biosorption performances in the range of 20-35 0C
(Aksu et al. 1992)
2. pH seems to be the most important parameter in the biosorptive process: it affects the
solution chemistry of the metals, the activity of the functional groups in the biomass and the
competition of metallic ions (Friis and Myers-Keith, 1986, Galun et al. 1987)
3. Biomass concentration in solution seems to influence the specific uptake: for lower values of
biomass concentrations there is an increase in the specific uptake (Fourest and Roux, 1992; Gadd et
al. 1988). Gadd et al. 1988 suggested that an increase in biomass concentration leads to interference
between the binding sites. Fourest and Roux, 1992 invalidated this hypothesis attributing the
responsibility of the specific uptake decrease to metal concentration shortage in solution. Hence
this factor needs to be taken into consideration in any application of microbial biomass as
biosorbent.
4. Biosorption is mainly used to treat wastewater where more than one type of metal ions would
be present; the removal of one metal ion may be influenced by the presence of other metal ions. For
example: Uranium uptake by biomass of bacteria, fungi and yeasts was not affected by the
presence of manganese, cobalt, copper, cadmium, mercury and lead in solution (Sakaguchi and
Nakajima, 1991). In contrast, the presence of Fe2+ and Zn2+ was found to influence uranium uptake
by Rhizopus arrhizus (Tsezos and Volesky, 1982) and cobalt uptake by different microorganisms
seemed to be completely inhibited by the presence of uranium, lead, mercury and copper
(Sakaguchi and Nakajima, 1991).
The equilibrium of the biosorption process is often described by fitting the experimental points
with models (Gadd, et al. 1988) usually used for the representation of isotherm adsorption
equilibrium. The two widely accepted and linearised equilibrium adsorption isotherm models for
single solute system are given by the following:
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where q is milligrams of metal accumulated per gram of the biosorbent material; Ceq is the metal
residual concentration in solution; qmax is the maximum specific uptake corresponding to the site
saturation and b is the ratio of adsorption and desorption rates. This is a theoretical model for
monolayer adsorption.
These models can be applied at a constant pH. These models are used in literature for modeling of
biosorption equilibrium in the presence of one metal. These values are plotted in a 2D line where
the specific uptake q is reported as a function of the metal concentration Ceq
But the above said adsorption isotherms may exhibit an irregular pattern due to the complex
nature of both the sorbent material and its varied multiple active sites, as well as the complex
solution chemistry of some metallic compounds (Volesky and Holan, 1995). Evaluation of
equilibrium sorption performance needs to be supplemented by process-oriented studies of its
kinetics and eventually by dynamic continuous flow tests.
Biosorption by immobilized cells: Microbial biomass consists of small particles with low density,
poor mechanical strength and little rigidity. The immobilization of the biomass in solid structures
Qeates a material with the right size, mechanical strength and rigidity and porosity necessary for
metal accumulation. Immobilisation can also yield beads and granules that can be stripped of
metals, reactivated and reused in a manner similar to ion exchange resins and activated carbon.
Various applications are available for biomass immobilization. The principal techniques that are
available in literature for the application of biosorption are based on adsorption on inert supports,
on entrapment in polymeric matrix, on covalent bonds in vector compounds, or on cell cross-
linking.
Adsorption on inert supports: Support materials are introduced prior to sterilization and
inoculation with starter culture and are left inside the continuous culture for a period oftime, after
which a film of microorganisms is apparent on the support surfaces. This technique has been used
by Zhou and Kiff, 1991 for the immobilization of Rhizopus arrhizus fungal biomass in reticulated
foam biomass support particles; Macaskie et al. 1987, immobilised the bacterium Citrobacter sp. by
this technique. Scott and Karanjakar 1992, used activated carbon as a support for Enterobacter
aerogens biofilm. Bai and Abraham, 2003 immobilized Rhizopus nigricans on polyurethane foam
cubes and coconut fibres.
Entrapment in polymeric matrices: The polymers used are calcium alginate (Babu et al. 1993,
Costa and Leite, 1991, Peng and Koon, 1993, Gulay Bayramoglu et al. 2002), polyacrylamide
(Macaskie et aI., 1987, Michel et al. 1986, Sakaguchi and Nakajima et al. 1991, Wong and Kwok,
1992), polysulfone (Jeffers et al. 1991, Bai and Abraham, 2003) and polyethylenimine (Brierley and
Brierley, 1993). The materials obtained from immobilization in calcium alginate and
polyacrylamide are in the form of gel particles. Those obtained from immobilization in polysulfone
and polyethyleneimine are the strongest.
Covalent bonds to vector compounds: The most common vector compound (carrier) is silica gel.
The material obtained is in the form of gel particles. This technique is mainly used for algal
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Cross-linking: The addition of the cross-linker leads to the formation of stable cellular aggregates.
This technique was found useful for the immobilization of algae (Holan et al. 1993). The most
common cross linkers are: formaldehyde, glutaric dialdehyde, divinylsulfone and formaldehyde -
urea mixtures.
• restore the biosorbent to close to the original condition for effective reuse with undiminished
metal uptake and
While the regeneration of the biosorbent may be accomplished by washing the metal- laden
biosorbent with an appropriate solution, the type and strength of this solution would depend on
the extent of binding of the deposited metal. Dilute solutions of mineral acids like hydrochloric
acid, sulphuric acid, acetic acid and nitric acid can be used for metal desorption from the biomass
(de Rome and Gadd, 1987, Zhou and Kiff, 1991, Luef et.al. 1991, Holan et.al. 1993, Pagnanelli etal.
2002, Baiand Abraham, 2003).
Polysulphone immobilized Rhizopus nigricans were subjected to Cr (VI) recovery experiments using
0.01 N solutions of mineral acids, salt solutions, alkalies, deionised distilled water and buffer
solutions. The percentage desorption by various eluants is given in Table 2.
A few experiments were conducted to desorb the metal ions from the loaded waste fungal biomass
of Aspergillus species (Chandrashekar et al. 1998) as a function of HCl concentration in the case of
iron, calcium and nickel. The results revealed that with increase in HCl concentrations, the
desorption of the metal ions increased and at 5M HCI, complete removal of calcium and iron
would be achieved while about 78% Nickel would be desorbed.
The desorption of the adsorbed Hg (II) from the biosorbent - immobilized and heat inactivated
Trametes versicolor and Pleurotus sajur-caju were studied in a batch system (Arica et al. 2003). Hg (II)
ions adsorbed onto the biosorbents were eluted with 10 mmol dm-3 HCl and the results showed
that more than 97% of the adsorbed Hg (II) ions were desorbed from the biosorbents.
Effect of Pre-treatment on the biosorption of heavy metals: Metal affinity to the biomass can be
manipulated by pretreating the biomass with alkalies, acids, detergents and heat, which may
increase the amount of the metal sorbed. The bioadsorption capacity of autoclaved Mucor rouxii
decreased as compared to the live fungus, attributed to the loss of intracellular uptake (Yan and
Viraraghavan, 2000). Whistler and Daniel (1985) reported that the heat treatment could cause a loss
of amino-functional groups on the fungal surface through the non-enzymic browning reaction.
Aminofunctional groups in the polysaccharides contribute to the binding of heavy metals (Loaec et
al., 1997). However, Galun et al., 1987 reported that Pencillium biomass pretreatment at 100°C for 5
minutes increased the biadsorption of lead, cadmium, nickel and zinc and the increase was
attributed to the exposure of latent binding sites after pre-treatment.
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In the case of alkali pre-treatment, bioadsorption capacity of Mucor rouxii biomass was significantly
enhanced in comparison with autoclaving (Yan and Viraraghavan, 2000). In a study by Galun et al.
(1987), NaOH treated Pencillium digitatum also showed enhancement of cadmium, nickel and zinc
biosorption. Removal of surface impurities, rupture of cell-membrane and exposure of available
binding sites for metal bioadsorption after pre-treatment may be the reason for the increase in
metal bioadsorption. McGahren et al. (1984), Brierly et.al (1985) and Muraleedharan and
Venkobachar (1990) showed that alkali treatment of biomass may destroy autolytic enzymes that
cause putrefaction of biomass and remove lipids and proteins that mask reactive sites. The cell wall
of Mucor rouxii was ruptured by NaOH treatment. Besides, the pre-treatment could release
polymers such as polysaccharides that have a high affinity towards certain metal ions (Mittelman
and Geesey, 1985; Loaec et.al. 1997).
Acid pretreatment of Mucor rouxii significantly decreased the bioadsorption of heavy metals (Yan
and Viraraghavan, 2000), which is in agreement with the observation of Kapoor and Viraraghvan
(1998) in the case of A.niger. This is attributed to the binding ofH+ ions to the biomass after acid
treatment may be responsible for the reduction in adsorption of heavy metals. The polymeric
structure of biomass surface exhibits a negative charge due to the ionization of organic and
inorganic groups (Hughes and Poole, 1989). Bux and Kasan (1994) suggested that the higher the
biomass electronegativity, the greater the attraction and adsorption of heavy metal cations. Thus
the remaining H+ions on the acidic pretreated M.rouxii biomass may change the biomass
electronegativity, resulting in a reduction in bioadsorption capacity.
However, Huang and Huang (1996) reported that acid pretreatment can strongly enhance the
adsorption capacity of Aspergillus.oryzae mycelia. In case of A.oryzae, live biomass after acid
pre-treatment was directly used in bioadsorption of heavy metals instead of being autoc1aved and
dried. The difference in results after a specific pretreatment may be attributed to the different
strains of fungi used and whether the biomass was live or stead when it is used in biosorption of
metal ions. For example; pre-treatment of A.oryzae by HClO4 increased the bioadsorption of lead,
cadmium and nickel, but it was not the case for the species of R.oryzae (Huang and Huang, 1996).
When non-viable biomass is used in the removal of heavy metals, alkali pretreatment is an
effective method to improve the bioadsorption capacity for metal ions (Yan and Viraraghavan,
2000). Hence, the bioadsorption efficiency of dead biomass may be greater, equivalent to, or less
than that of live biomass depending on the pre-treatment method applied. It is necessary to carry
out more detailed studies to understand why enhancement or reduction in adsorption capacity
occurs under specific pre-treatment conditions.
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CONCLUSION
Biosorption is being demonstrated as a useful alternative to conventional systems for the removal
of toxic metals from industrial effluents. The development of the biosorption processes requires
further investigation in the direction of modeling, of regeneration of biosorbent material and of
testing immobilized raw biomasses with industrial effluents. Due to the extensive research and
significant economic benefits of biosorption, some new biosorbent materials are poised for
commercial exploitation.
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