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1. Biosorbents
Biosorbents for the removal of metals/dyesmainly come under the following categories:
bacteria, fungi, algae, industrial wastes, agricultural wastes and other polysaccharide materials.
In general, all types of biomaterials have shown good biosorption capacities towards all types of
metal ions. Potent metal biosorbents under the class of bacteria include genre of Bacillus,
Pseudomonas and Streptomyces, etc. Important fungal biosorbents include Aspergillus, Rhizopus
and Penicillium etc. Since these microorganisms are used widely in different food/
pharmaceutical industries, they are generated as waste, which can be attained free or at low cost
from these industries. Another important biosorbent, which has gained momentum in recent
years, is seaweed. Marine algae, popularly known as seaweeds, are biological resources, which
are available in many parts of the world. Algal divisions include red, green and brown seaweed;
of which brown seaweeds are found to be excellent biosorbents. This is due to the presence of
alginate, which is present in gel form in their cell walls. Also, their macroscopic structure offers
a convenient basis for the production of biosorbent particles that are suitable for sorption process
applications. However, it should be noted that seaweeds are not regarded as wastes; in fact they
are the only source for the production of agar, alginate and carrageenan. Therefore, utmost care
should be taken while selecting seaweeds for a biosorption process. Metal sorbing properties of
one of the best metal sorbent Sargassum seaweed and brown seaweeds. k
Recently, numerous approaches have been made for the development of low-cost sorbents from
industrial and agricultural wastes. Of these, crab shells, activated sludge, rice husks, egg shell
and peat moss deserve particular attention. Crab shells possess excellent arsenic, chromium,
copper, cobalt and nickel sorbent.
With respect to dye biosorption, microbial biomass (bacteria, fungi, microalgae, etc.)
outperformed macroscopic materials (sea- weeds, crab shell, etc.). The reason for this
discrepancy is due to the nature of the cell wall constituents and functional groups involved in
dye binding. Many bacteria, fungi and microalgae have been found to bind a variety of dye
classes. Corynebacterium glutamicum as a potent biosorbent of Reactive red4,which can bind
104.6 mg/g at pH 1. Rhizopus arrhizus was capable of binding 773 mg/g of Gemazol Turquise
blue-G at 45 °C and pH 2. the biosorption capacity of Chlorella vulgaris, using several reactive
dyes, and identified that the microalga was capable of binding 419.5 mg/g of Remazol black B.
Very little effort has been made to utilize seaweeds for the biosorption of dyes, Sargassum
muticum for the removal of methylene blue and utilized Laminaria sp. for the removal Reactive
black 5. Hundreds of biosorbents have been proposed for the removal of metals and dyes;
therefore, their consolidation in a single review would be impossible. Therefore, in this study,
bacterial biosorbents have been taken in general, with other biosorbents considered only in
special instances. Hence, the important aspects of biosorption will be discussed, but will not be
limited to bacteria. Fungal and other low-cost biosorbents

2. History of bacterial biosorption
Early 1980 witnessed the capability of some microorganisms to accumulate metallic elements.
Numerous research reports have been published from toxicological points of view, but these
were concerned with the accumulation due to the active metabolism of living cells, the effects of
metal on the metabolic activities of the microbial cell and the consequences of accumulation on
the food chain. However, further research has revealed that inactive/dead microbial biomass can
passively bind metal ions via various physicochemical mechanisms. With this new finding,
research on biosorption became active,with numerous biosorbents of different origins being
proposed for the removal of metals/dyes. Researchers have understood and explained that
biosorption depends not only on the type or chemical composition of the biomass, but also on the
external physicochemical factors and solution chemistry. Many investigators have been able to
explain the mechanisms responsible for biosorption, which may be one or combination of ion
exchange, complexation, coordination, adsorption, electrostatic interaction, chelation and micro-
precipitation.
A direct comparison of experimental data is not possible, due to different systematic
experimental conditions employed (pH, pH control, temperature equilibrium time and biomass
dosage). However, Also for most metal ions, weak acidic pH resulted in maximum biosorption.
This is because of the involvement of carboxyl group and other acidic functional groups, which
are responsible for binding metal cations through vari ous mechanisms.
In addition, the formation of metal hydroxide and other metal-ligand complexes significantly
reduce the amount of metal ions sorbed at high pH. However, the mechanisms for the
biosorption have not always been confirmed or discussed in most studies; therefore,
generalizations are not possible in these cases. The extent of biosorption not only depends on the
type of metal ions, but also on the bacterial genus, due to variations in the cellular constituents.
Very short contact times were generally sufficient to attain metal-bacterial biomass steady state.
This is because biomass was either used in the form of fine powder or wet cells; where mass
transfer resistances are usually negligible. The rapid kinetics observed with bacterial biomasses
represents an advantageous aspect for the design of waste water treatment systems.
It was surprising to see that much less attention has been paid on employing dead bacterial
biomass for the sorption of dyes. Most of the earlier works on dye biosorption have focused on
utilization of fungal biomasses and other low-cost adsorbents. Of the limited results on bacterial
biosorption, C. glutamicum have been shown to perform well in the biosorption of reactive dyes,
with dye uptakes in the range of 0.1– 0.4 times that of its dry weight.
Bacterial structure and mechanism of bacterial biosorption
1. Bacterial structure
Bacteria are a major group of unicellular living organisms belonging to the prokaryotes, which
are ubiquitous in soil and water, and as symbionts of other organisms. Bacteria can be found in a
wide variety of shapes, which include cocci (such as Streptococcus), rods (such as Bacillus),
spiral (such as Rhodospirillum) and filamentous (such as Sphaerotilus). Eubacteria have a

3. relatively simple cell structure, which lack cell nuclei, but possess cell walls. The bacterial cell
wall provides structural integrity to the cell, but differs from that of all other organisms due to
the presence of peptidoglycan (poly-N-acetylglucosamine and N-acetylmuramic acid), which is
located immediately outside of the cytoplasmic membrane. Peptidoglycan is responsible for the
rigidity of the bacterial cell wall, and determines the cell shape. It is also relatively porous and
considered as an impermeability barrier to small substrates. The cell walls of all bacteria are not
identical. In fact, the cell wall composition is one of the most important factors in the analysis
and differentiation of bacterial species. Accordingly, two general types of bacteria exist, of
which Gram-positive bacteria (Fig. 1) are comprised of a thick peptidoglycan layer connected by
amino acid bridges. Imbedded in the Gram-positive cell wall are polyalcohols, known as teichoic
acids, some of which are lipid-linked to form lipoteichoic acids. Because lipoteichoic acids are
covalently linked to lipids within the cytoplasmic membrane, they are responsible for linking
peptidoglycan to the cytoplasmic membrane. The cross-linked peptidoglycan molecules form a
network, which covers the cell like a grid. Teichoic acids give the Gram-positive cell wall an
overall negative charge, due to the presence of phosphodiester bonds between the teichoic acid
monomers. In general, 90% of the Gram-positive cell wall is comprised of peptidoglycan. On the
contrary, the cell wall of Gram-negative bacteria (Fig. 1) is much thinner, and composed of only
10–20% peptidoglycan. In addition, the cell wall contains an additional outer membrane
composed of phospholipids and lipopolysaccharides. The highly charged nature of
lipopolysaccharides confers an overall negative charge on the Gram-negative cell wall. the
anionic functional groups present in the peptidoglycan, teichoic acids and teichuronic acids of
Gram-positive bacteria, and the peptidoglycan, phospholipids, and lipopolysaccharides of Gram-
negative bacteria were the components primarily responsible for the anionic character and metal-
binding capability of the cell wall. Extracellular polysaccharides are also capable of binding
metals. However, their availability depends on the bacterial species and growth conditions; and
they can easily be removed by simple mechanical disruption or chemical washing.
2. Mechanism of bacterial biosorption

4. The bacterial cell wall is the first component that comes into contact with metal ions/dyes, where
the solutes can be deposited on the surface or within the cell wall structure. Since the mode of
solute uptake by dead/inactive cells is extracellular, the chemical functional groups of the cell
wall play vital roles in biosorption. Due to the nature of the cellular components, several
functional groups are present on the bacterial cell wall, including carboxyl, phosphonate, amine
and hydroxyl group.
As they are negatively charged and abundantly available, carboxyl groups actively participate in
the binding of metal cations. Several dye molecules, which exist as dye cations in solutions, are
also attracted towards carboxyl and other negatively charged groups. Carboxyl groups of the cell
wall peptidoglycan of Streptomyces pilosus were responsible for the binding of copper. Also,
amine groups are very effective at removing metal ions, as it not only chelates cationic metal
ions, but also adsorbs anionic metal species or dyes via electrostatic interaction or hydrogen
bonding. amine groups protonated at pH 3 and attracted negatively charged chromate ions via
electrostatic interaction. amine groups of C. glutamicum were responsible for the binding of
reactive dye anions via electrostatic attraction. In general, increasing the pH increases the overall
negative charge on the surface of cells until all the relevant functional groups are deprotonated,
which favors the electrochemical attraction and adsorption of cations. Anions would be expected
to interact more strongly with cells with increasing concentration of positive charges, due to the
protonation of functional groups at lower pH values. The solution chemistry affects not only the
bacterial surface chemistry, but the metal/dye speciation as well. Metal ions in solution undergo
hydrolysis as the pH increases. The extent of which differs at different pH values and with each
metal, but the usual sequence of hydrolysis is the formation of hydroxylated monomeric species,
followed by the formation of polymeric species, and then the formation of crystalline oxide
precipitates after aging. For example, in the case of nickel solution, within the pH range from 1
to 7, nickel existed in solution as Ni 2+ions (90%); whereas at pH 9, Ni 2+ (68%), Ni 4 OH 4
4+
(10%) and Ni (OH)+ (8.6%) co-existed. The different chemical species of a metal occurring with
pH changes will have variable charges and adsorbability at solid– liquid interfaces. In many
instances, biosorption experiments conducted at high alkaline pH values have been reported to
complicate evaluation of the biosorbent potential as a result of metal precipitation.

5. 3. Characterization of bacterial surface
Characterization of bacterial biomass and the biosorption mechanisms can be elucidated using
different methods, including potentiometric titrations, Fourier transform infrared spectroscopy,
X-ray diffraction, scanning electron microscopy, transmission electron microscopy and energy
dispersive X-ray microanalysis. Potentiometric titrations have aided several researchers in the
determination of the type and number of binding sites.
In recent years, interest has been focused on increasing the sorption capacity of the biomass.
Several biomasses, regarded as industrial wastes following certain processes, possess low
biosorption capacities. As sorption mainly takes place on the biomass surface,
increasing/activating the binding sites on the surface would be an effective approach for
enhancing the biosorption capacity.
Chemically modified biosorbents
Chemical modification procedures include pretreatment, binding site enhancement, binding site
modification and polymerization. Common chemical pretreatments include acid, alkaline,
ethanol and acetone treatments of the biomass. The success of a chemical pretreatment strongly
depends on the cellular components of the biomass itself. In many instances, acidic pretreatment
has proved successful; this is because some of the impurities and ions blocking the binding sites
can easily be eliminated. Metal (Cu2+ and Ni2+) uptake capacity of lyophilized Pseudomonas
auruginosa cells was enhanced when pretreated with NaOH, NH4OH or toluene; whereas, oven
heating (80 °C), autoclaving, acid, detergent and acetone treatments were inhibitory. Even
though these chemical pretreatments are almost essential for most of the biosorbents, especially
industrial wastes, vast improvements in their biosorption capacities cannot always be expected.
Conversely, enhancement or modification of the binding sites on a biomass seems to enhance the
biosorption capacities by multiple folds. Carboxyl, amine, phosphonate, sulfonate and hydroxyl
groups have become well established as being responsible for metal/dye binding. As the density
of these groups is low, most biosorbents show low sorption capacities. Various procedures are
available for the enhancement of these functional groups on the biomass. In general, futile/less
important functional groups can be converted into active binding groups via several chemical
treatment methods. Carboxylated biomass was treated with ethylenediamine followed by
carbodiimide to form aminated biomass and increase in amine groups increased mercury uptake
by 47% compared to that of control. Citric acid also modify an alkali-saponified biomass, which
increased the total acidic sites, but a decrease of basic sites. In particular, they reported that
biomass modified using 0.6 mol/L citric acid at 80 °C for 2 h exhibited cadmium uptake capacity
twice than that of the raw biomass. Biosorption of 111.8 mg Reactive black 5/g for virgin C.
glutamicum, but when the carboxyl groups were masked from participation, the biomass
exhibited biosorption of 257.3 mg Reactive black 5/g.
Another efficient way for the introduction of functional groups onto the biomass surface is the
grafting of long polymer chains onto the biomass surface via direct grafting or polymerization of
a monomer. However, very little research has focused specifically on this aspect.

6. Genetically modified biosorbents
Genetic engineering has the potential to improve or redesign microorganisms, where biological
metal-sequestering systems will have a higher intrinsic capability as well as specificity and
greater resistance to ambient conditions. It is well known that virgin biosorbents usually lack
specificity in metal-binding, which may cause difficulties in the recovery and recycling of the
desired metal(s). Genetic modification is a potential solution to enhance the selectivity as well as
the accumulating properties of the cells. Genetic modification would be feasible especially when
the microbial biomass is produced from fermentation processes where genetically engineered
microorganisms are used. Nowa-days, many kinds of amino acids and nucleic acids are being
produced in an industrial scale by using genetically engineered microbial cells.
Higher organisms respond to the presence of metals, with the production of cysteine-rich
peptides, such as glutathione (GSH), phytochelatins (PCs) and metallothioneins (MTs), which
can bind and sequester metal ions in biologically inactive forms. The over expression of MTs in
bacterial cells will result in an enhanced metal accumulation and; thus, offers a promising
strategy for the development of microbial-based biosorbents for the remediation of metal
contamination. Neurospora crassa metal-lothionein gene within the periplasmic space) was
rapid. Greater than 75% Cd uptake occurred in the first 20 min, with maximum uptake achieved
in less than 1 h. However, the expression of such cysteine-rich proteins is not devoid of
problems, due to the predicted interference with redox pathways in the cytosol. More
importantly, the intracellular expression of MTs may prevent the recycling of the biosorbents, as
the accumulated metals cannot be easily released. However, the development of organisms
overexpressing PCs requires a thorough knowledge of the mechanisms involved in the synthesis
and chain elongation of these peptides. Several biosorbents, displaying metal-binding peptides
on the cell surface, have been successfully engineered. A typical example includes creating a
repetitive metal-binding motif, consisting of (Glu-Cys)n Gly. These peptides emulate structure of
PCs; however, they differ in the fact that the peptide bond between the glutamic acid and
cysteine is a standard α peptide bond. Phytochelatin analogs were found to be present on the
bacterial surface, which enhanced the ccumulation of Cd2+ and Hg2+ by 12 and 20-fold,
respectively. Attempts to create recombinant bacteria with improved metal-binding capacity
have so far been restricted to mostly Escherichia coli. This is because E. coli greatly facilitates
genetic engineering experiments and it is found to have more surface area per unit of cell mass,
which potentially should give higher rates of metal removal from solution. Nevertheless, a
Gram-positive surface display system also possesses its own merits compared to Gram-negative
bacteria:
(a) translocation through only one membrane is required, and (b) Gram-positive bacteria have
been shown to be more rigid and; therefore, less sensitive to shear forces due to the thick cell
wall surrounding the cells, which potentially make them more suitable for field applications,
such as bioadsorption. Recombinant Staphylococcus xylosus and Staphylococcus carnosus
strains, with surface-exposed chimeric proteins containing polyhistidyl peptides most widely

7. used. Both strains of staphylococci gained improved nickel-binding capacities due to the
introduction of the H1or H2 peptide into their surface proteins. Owing to their high selectivity,
genetically engineered biosorbents may prove very competitive for the separation of toxins and
other pollutants from dilute contaminated solutions.
Immobilized biosorbents
Microbial biosorbents are basically small particles, with low density, poor mechanical strength
and little rigidity. Even though they have merits, such as high biosorption capacity, rapid steady
state attainment, less process cost and good particle mass transfer, they often suffer several
drawbacks. The most important include solid–liquid separation problems, possible biomass
swelling, inability to regenerate/reuse and development of high pressure drop in the column
mode.
Several established techniques are available to make biosorbents suitable for process
applications. Among these, immobilization techniques such as entrapment and cross linking have
been found to be practical for biosorption.
Immobilization of microorganisms within a polymeric matrix has exhibited greater potential,
especially in packed or fluidized bed reactors, with benefits including the control of particle size,
regeneration and reuse of the biomass, easy separation of biomass and effluent, high biomass
loading and minimal clogging under continuous-flow conditions.The choice of immobilization
matrix is a key factor in the environmental application of immobilized biomass. The polymeric
matrix determines the mechanical strength and chemical resistance of the final biosorbent
particle to be utilized for successive sorption–desorption cycles.
However, care must be taken to avoid the practical problems generated during the
immobilization process; in particular, the mass transfer limitations and additional process costs.
After immobilization, the biomass will usually be retained within the interior of the matrix used
for the immobilization; hence, mass transfer resistance will play a vital role in deciding the rate
of biosorption. The presence of mass transfer resistance usually slows the attainment of
equilibrium; however, a successful immobilization matrix should allow all the active binding
sites to have access to the solute, even at a slower rate. immobilization of C. glutamicum within a
polysulfone matrix has delayed the attainment of equilibrium; however, the dye uptake was
almost comparable to that of the free biomass. Next, immobilizing the biomass usually enhances
the process costs. Biosorption is usually portrayed as a cost effective process, which is often
highlighted as attractive option compared to that of other proven technologies. Although
immobilizing the biomass for the sole purpose of biosorption will enhance the process costs, it is
often necessary for practical implementation of biosorption in real applications. The need for
microbial biomass in biosorption applications is arguable especially when there is availability of
highly rigid and efficient biosorbents such as seaweeds. Raw/unprocessed seaweeds have been
shown to be good biosorbents for metal ions, and are also highly stable under acidic conditions.
These have successfully regenerated and reused seaweeds over a number of cycles for the
removal of metal ions. However, the stability of seaweeds under alkaline conditions is of
concern, as they tend to swell under high pH conditions, mainly due to their cellular constituents.

8. In general, seaweeds are not very efficient in the biosorption of dyes, due to the nature of the
binding sites. Conversely, microbial biomaterials, such as bacteria and fungi, exhibit high metal
and dye uptakes. Also, the microbial wastes generated by many fermentation/food industries
cause a nuisance, and their disposal is of great concern. For instance, C. glutamicum, a Gram-
positive bacterium, is widely used for the biotechnological production of amino acids. Currently,
the production of amino acids from fermentative processes using C. glutamicum amounts to
1,500,000 and 550,000 t per year of L-glutamate and L-lysine, respectively. Hence the waste C.
glutamicum generated after fermentation is usually high and the potential utilization of this waste
is of interest. Yun and co-workers examined the biosorption potential of C. glutamicum and
identified its excellent reactive dye binding capacity. However, this biomass is associated with
problems during desorption, as it tends to swell under alkaline environments. Furthermore, the
continuous supply of biomass cannot be assured, which will have a huge impact on its successful
application in industrial biosorption applications.
Desorption and regeneration
Biosorption is a process of treating pollutant-bearing solutions to make it contaminant free.
However, it is also necessary to be able to regenerate the biosorbent. This is possible only with
the aid of appropriate elutants, which usually results in a concentrated pollutant solution.
Therefore, the overall achievement of a biosorption process is to concentrate the solute, i.e.,
sorption followed by desorption. Desorption is of utmost importance when the biomass
preparation/generation is costly, as it is possible to decrease the process cost and also the
dependency of the process on a continuous supply of biosorbent. A successful desorption process
requires the proper selection of elutants, which strongly depends on the type of biosorbent and
the mechanism of biosorption. Also, the elutant must be (i) nondamaging to the biomass, (ii) less
costly, (iii) environmental friendly and (iv) effective. Therefore, it would be logical to make the
cell surface negative using alkaline solutions to repel the negatively charged reactive dyes .
At the same time, the volume of the solution should be sufficient to provide maximum solubility
for the desorbed solute. Desorbed sorbate stays in the solution and a new equilibrium is
established between that and the one (remaining) still fixed on the biosorbent. This leads to the
concept of a “desorption isotherm” where the equilibrium is strongly shifted towards the sorbate
dissolved in the solution. Thus, it is necessary to evaluate the suitable elutant volume, which can
be performed using experiments with different solid-to-liquid ratios. The solid-to-liquid ratio is
defined as the mass of solute-laden biosorbent to the volume of elutant.
Continuous biosorption
Continuous biosorption studies are of utmost importance to evaluate the technical feasibility of a
process for real applications. Among the different column configurations, packed bed columns
have been established as an effective, economical and most convenient for biosorption processes.
They make best use of the concentration difference, which is known to be the driving force for
sorption, and allow more efficient utilization of the sorbent capacity, resulting in better effluent

9. quality. Also, packed bed sorption has a number of process engineering merits, including a high
operational yield and the relative ease of scaling up procedures. Other column contactors, such as
fluidized and continuous stirred tank reactors, are very rarely used for the purpose of biosorption.
Continuous stirred tank reactors are useful when the biosorbent is in the form of a powder;
however, they suffer from high capital and operating costs. Fluidized bed systems, which operate
application of biosorption to real industrial effluents, biosorption is a proven technique
potentially for the removal of metals/dyes from aqueous solutions. However, its performance
under real industrial conditions is of concern. There have been few investigations examining the
compatibility of the biosorbent for real industrial effluents. This case would not be feasible if the
biomass was immobilized in a polymeric matrix. If the biomass is free of cost, or the
transportation and processing costs are minimal, the metal or dye loaded biosorbents can be used
to sorb other solutes. For example, with molybdate loaded chitosan beads, the chelating affinity
of molybdate for arsenic has been used for the recovery of As(V) from dilute solutions. In
another instance, Cibacron blue F3GA-attached poly vinyl butyral microbeads have been shown
to be effective in the removal of Cu(II), Cd(II) and Pb(II) ions. However, one should understand
that waste microbial biomasses originating from their respective industries are already creating a
disposal nuisance. The biosorbents developed from these waste microbial biomasses are;
thereby, solving their own disposal problems as well as adding value to their waste. The
developed biosorbent, after serving multiple times in the remediation of metal/dye polluted
effluents, should be regarded as having served its purpose.
Scope and future directions
Bacterial biomass represents an efficient and potential class of biosorbents for the removal of
both dyes and metal ions. Unfortunately, the difficulties in reusing the microbial biomass, as well
as the poor selectivity, hinder their applications under real conditions. Although some attempts
have been made at the commercialization of biosorption for wastewater treatment, the progress is
very modest considering that there has been more than a decade of fundamental research. The
important features required for the successful application of biosorption technology to real
situations include, but are not limited to:
 Screening and selection of the most promising biomass, with sufficiently high biosorption
capacity and selectivity.
 Optimizing the conditions for maximum biosorption, including optimization of pH,
temperature, ionic strength and co-ion effects, etc
 Improving the selectivity and uptake via chemical and/or genetic modification methods.
 Examining the mechanical strength of biomass and if insufficient for reuse, improving
rigidity by proper immobilization or other chemical methods.
 Testing the performance of biosorbents under different modes of operation.
 Analyzing the behavior of biosorbent for use with real industrial effluents and,
simultaneously analyzing the impact of water quality on the biosorption uptake of the
specific pollutant of interest.

10. Conversely, it is no small feat to replace well established conventional techniques. However, in
addition to being cost effective, biosorption has huge potential, as many biosorbents are known
to perform well, if not better than most conventional methods. Also being aware of the hundreds
of biosorbents able to bind various pollutants, sufficient research has been performed on various
biomaterials to understand the mechanism responsible for biosorption. Therefore, through
continued research, especially on pilot and full-scale biosorption process, the situation is likely to
change in the near future, with biosorption technology becoming more beneficial and attractive
than currently used technologies.