Bacterial Bios or Bents and Ion

Research review paper
Bacterial biosorbents and biosorption

K. Vijayaraghavan
, Yeoung-Sang Yun
Division of Environmental and Chemical Engineering, Research Institute of Industrial Technology, Chonbuk National University, Chonju 561-756, South Korea
Received 31 July 2007; received in revised form 5 February 2008; accepted 7 February 2008
Available online 15 February 2008
Abstract
Biosorption is a technique that can be used for the removal of pollutants from waters, especially those that are not easily biodegradable such as
metals and dyes. A variety of biomaterials are known to bind these pollutants, including bacteria, fungi, algae, and industrial and agricultural
wastes. In this review, the biosorption abilities of bacterial biomass towards dyes and metal ions are emphasized. The properties of the cell wall
constituents, such as peptidoglycan, and the role of functional groups, such as carboxyl, amine and phosphonate, are discussed on the basis of their
biosorption potentials. The binding mechanisms, as well as the parameters influencing the passive uptake of pollutants, are analyzed. A detailed
description of isotherm and kinetic models and the importance of mechanistic modeling are presented. A systematic comparison of literature,
based on the metal/dye binding capacity of bacterial biomass under different conditions, is also provided. To enhance biosorption capacity,
biomass modifications through chemical methods and genetic engineering are discussed. The problems associated with microbial biosorption are
analyzed, and suitable remedies discussed. For the continuous treatment of effluents, an up-flow packed column configuration is suggested and the
factors influencing its performance are discussed. The present review also highlights the necessity for the examination of biosorbents within real
situations, as competition between solutes and water quality may affect the biosorption performance. Thus, this article reviews the achievements
and current status of biosorption technology, and hopes to provide insights into this research frontier.
2008 Elsevier Inc. All rights reserved.
Keywords: Biosorption; Bacteria; Metals; Dyes; Wastewater treatment; Packed column; Isotherm model; Kinetic model; Biomass reuse; Multicomponent biosorption
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
2. Overview of treatment methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
3. Biosorbents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
4. History of bacterial biosorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
5. Bacterial structure and mechanism of bacterial biosorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
5.1. Bacterial structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
5.2. Mechanism of bacterial biosorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
5.3. Characterization of bacterial surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
6. Preparation of bacterial biosorbents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
6.1. Chemically modified biosorbents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
6.2. Genetically modified biosorbents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
6.3. Immobilized biosorbents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
7. Biosorption experimental procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
7.1. Factors influencing bacterial batch biosorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
7.2. Biosorption isotherms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
7.3. Batch experimental data modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
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Biotechnology Advances 26 (2008) 266291
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Corresponding authors. Tel.: +82 63 270 2308; fax: +82 63 270 2306.
E-mail addresses: drkvijy@chonbuk.ac.kr (K. Vijayaraghavan), ysyun@chonbuk.ac.kr (Y.-S. Yun).
0734-9750/$ - see front matter 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2008.02.002
7.3.1. Empirical modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
7.3.2. Mechanistic modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
7.4. Batch kinetic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
8. Desorption and regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
9. Continuous biosorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
9.1. Column biosorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
9.2. Column regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
9.3. Modeling of column data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
10. Multicomponent systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
11. Application of biosorption to real industrial effluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
12. Fate of exhausted biosorbent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
13. Scope and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
1. Introduction
Comprising over 70% of the Earth's surface, water is un-
deniably the most valuable natural resource existing on our planet.
Without this invaluable compound, the life on the Earth would be
non-existent. Although this fact is widely recognized, pollution of
water resources is a common occurrence. In particular, potable
water has become greatly affected, and in many instances has lost
its original purpose.
There are many sources of water pollution, but two main
general categories exist: direct and indirect contaminant sources.
Direct sources include effluent outfalls from industries, refineries
and waste treatment plants; whereas, indirect sources include
contaminants that enter the water supply from soils/ground water
systems and from the atmosphere via rain water. In general, con-
taminants come under two broad classes, viz. organic and inor-
ganic. Some organic water pollutants include industrial solvents,
volatile organic compounds, insecticides, pesticides and food
processing wastes, etc. Inorganic water pollutants include metals,
fertilizers and acidity caused by industrial discharges, etc. To limit
our scope, this review takes into consideration only dyes, which
come under organic, and metals, which come under inorganic
pollutants. They are common contaminants in industrial waste-
waters and many of themare known to be toxic and carcinogenic.
A dye can generally be described as a colored substance with
an affinity to the substrate to which it is applied. Dyes are widely
used in industries, such as textiles, paper, plastics and leather,
etc., for the coloration of products. The effluents emanating from
these industries often contain high concentrations of dye wastes.
Two percent of the dyes produced are discharged directly in
aqueous effluent, with a further 10% subsequently lost during
the textile coloration process (Easton, 1995). It has been reported
that over 100,000 dyes are commercially available, with a pro-
duction of over 710
5
tonnes per year (Zollinger, 1987; Aksu,
2005). Dyes are generally believed to be toxic and carcinogenic
or prepared from other known carcinogens (Banat et al., 1996).
The discharge of these dye stuffs from industries into rivers and
lakes results in a reduced dissolved oxygen concentration caus-
ing anoxic conditions, which subsequently affect aerobic organ-
isms (Chander and Arora, 2007). Apart from the toxicological
properties of dyes, their color is one of the first signs of
contamination recognized in a wastewater. Since a very small
quantity of dyes in water is highly visible, it often affects the
aesthetic merit and water transparency (Banat et al., 1996).
Another group of contaminants of concern, which comes
under the inorganic division, are metals. Metals are extensively
used in several industries, including mining, metallurgical,
electronic, electroplating and metal finishing. The presence of
metal ions in final industrial effluents is extremely undesirable, as
they are toxic to both lower and higher organisms. Under certain
environmental conditions, metals may accumulate to toxic levels
and cause ecological damage (Jefferies and Firestone, 1984). Of
the important metals, mercury, lead, cadmium and chromium(VI)
are regarded as toxic; whereas, others, such as copper, nickel,
cobalt and zinc are not as toxic, but their extensive usage and
increasing levels in the environment are of serious concerns
(Brown and Absanullah, 1971; Moore, 1990; Volesky, 1990).
Radionuclides, such as uranium, possess high toxicity and radio-
activity, and exhibit a serious threat, even at small concentrations.
In most developed and developing countries, stricter envi-
ronmental regulations, with regard to contaminants discharged
fromindustrial operations, are being introduced. This means that
industries need to develop on-site or in-plant facilities to their
own effluents and minimize the contaminant concentrations to
acceptable limits prior to their discharge (Banat et al., 1996).
However, before selecting a wastewater treatment facility, a
considerable amount of laboratory and engineering work must
be completed prior to system design (Atkinson et al., 1998).
2. Overview of treatment methods
Various techniques have been employed for the treatment of
dye/metal bearing industrial effluents, which usually come
under two broad divisions: abiotic and biotic methods. Abiotic
methods include precipitation, adsorption, ion exchange,
membrane and electrochemical technologies. Much has been
discussed about their downside aspects in recent years
(Atkinson et al., 1998; Crini, 2006), which can be summarized
as expensive, not environment friendly and usually dependent
on the concentration of the waste. Therefore, the search for
efficient, eco-friendly and cost effective remedies for waste-
water treatment has been initiated.
In recent years, research attention has been focused on
biological methods for the treatment of effluents, some of which
267 K. Vijayaraghavan, Y.-S. Yun / Biotechnology Advances 26 (2008) 266291
are in the process of commercialization (Prasad and Freitas, 2003).
There are three principle advantages of biological technologies for
the removal of pollutants; first, biological processes can be carried
out in situ at the contaminated site; Second, bioprocess tech-
nologies are usually environmentally benign (no secondary pollu-
tion) and third, they are cost effective.
Of the different biological methods, bioaccumulation and
biosorption have been demonstrated to possess good potential to
replace conventional methods for the removal of dyes/metals
(Volesky and Holan, 1995; Malik, 2004). Some confusion has
prevailed in the literature regarding the use of the terms bio-
accumulation and biosorption based onthe state of the biomass.
Herein, therefore, bioaccumulation is defined as the phenomenon
of living cells; whereas, biosorption mechanisms are based on the
use of dead biomass. To be precise, bioaccumulation can be
defined as the uptake of toxicants by living cells. The toxicant can
transport into the cell, accumulate intracellularly, across the cell
membrane and through the cell metabolic cycle (Malik, 2004).
Conversely, biosorption can be defined as the passive uptake of
toxicants by dead/inactive biological materials or by materials
derived frombiological sources. Biosorption is due to a number of
metabolism-independent processes that essentially take place in
the cell wall, where the mechanisms responsible for the pollutant
uptake will differ according to the biomass type.
Biosorption possesses certain inherent advantages over bio-
accumulation processes, which are listed in Table 1. In general, the
use of living organisms may not be an option for the continuous
treatment of highly toxic organic/inorganic contaminants. Once the
toxicant concentration becomes too high or the process operated for
a long time, the amount of toxicant accumulated will reach
saturation (Eccles, 1995). Beyond this point, an organism's
metabolism may be interrupted, resulting in death of the organism.
This scenario can be avoided in the case of dead biomass, which is
flexible to environmental conditions and toxicant concentrations.
Thus, owing to its favorable characteristics, biosorption has, not
surprisingly, received much attention in recent years.
3. Biosorbents
Biosorbents for the removal of metals/dyes mainly 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 (Nakajima and Tsuruta, 2004; Tunali et al., 2006),
Pseudomonas (Chang et al., 1997; Uslu and Tanyol, 2006) and
Streptomyces (Mameri et al., 1999; Selatnia et al., 2004a), etc.
Important fungal biosorbents include Aspergillus (Kapoor and
Viraraghavan, 1997; Jianlong et al., 2001; Binupriya et al., 2006),
Rhizopus (Bai and Abraham, 2002; Park et al., 2005) and Peni-
cillium (Niu et al., 1993; Tan and Cheng, 2003), 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 (Davis et al., 2003). This is
due to the presence of alginate, which is present in gel formin their
cell walls. Also, their macroscopic structure offers a convenient
basis for the production of biosorbent particles that are suitable for
sorption process applications (Vieira and Volesky, 2000). 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
Table 1
Comparison of the features of biosorption and bioaccumulation
Features Biosorption Bioaccumulation
Cost Usually low. Most biosorbents used were industrial, agricultural and other
type of waste biomass. Cost involves mainly transportation and other
simple processing charges.
Usually high. The process involves living cells
and; hence, cell maintenance is cost prone.
pH The solution pH strongly influences the uptake capacity of biomass. However,
the process can be operated under a wide range of pH conditions.
In addition to uptake, the living cells themselves
are strongly affected under extreme pH conditions.
Temperature Since the biomass is inactive, temperature does not influence the process.
In fact, several investigators reported uptake enhancement with temperature rise.
Temperature severely affects the process.
Maintenance/storage Easy to store and use External metabolic energy is needed for
maintenance of the culture.
Selectivity Poor. However, selectivity can be improved by modification/processing of
biomass
Better than biosorption
Versatility Reasonably good. The binding sites can accommodate a variety of ions. Not very flexible. Prone to be affected by high
metal/salt conditions.
Degree of uptake Very high. Some biomasses are reported to accommodate an amount of toxicant
nearly as high as their dry weight.
Because living cells are sensitive to high
toxicant concentration, uptake is usually low.
Rate of uptake Usually rapid. Most biosorption mechanisms are rapid. Usually slower than biosorption. Since
intracellular accumulation is time consuming.
Toxicant affinity High under favorable conditions. Depends on the toxicity of the pollutant.
Regeneration and reuse High possibility of biosorbent regeneration, with possible reuse over a
number of cycles.
Since most toxicants are intracellularly accumulated,
the chances are very limited.
Toxicant recovery With proper selection of elutant, toxicant recovery is possible. In many instances,
acidic or alkaline solutions proved an efficient medium to recover toxicants.
Even if possible, the biomass cannot be utilized
for next cycle.
Table 2
Important results from the literature on metal biosorption by various bacterial species
Metal Organism Operating conditions Uptake (mg/g) Reference
pH Temp (C) Other information
Aluminum Chryseomonas luteola 5.0 NA M=1 g/l, t
eq
=1 h 55.2 (L) Ozdemir and Baysal, 2004
Chromium (VI) Aeromonas caviae 2.5 20 M=0.5 g/l; t
eq
=2 h 284.4 (L) Loukidou et al., 2004a,b
Bacillus coagulans 2.5 283 M=2 g/l, t
eq
=1 h 39.9 (E) Srinath et al., 2002
Bacillus licheniformis 2.5 50 M=1 g/l, t
eq
=2 h 69.4 (L) Zhou et al., 2007
Bacillus megaterium 2.5 283 M=2 g/l, t
eq
=1 h 30.7 (E) Srinath et al., 2002
Bacillus thuringiensis 2.0 25 M=1 g/l 83.3 (L) ahin and ztrk, 2005
Chryseomonas luteola 4.0 NA M=1 g/l, t
eq
=1 h 3.0 (L) Ozdemir and Baysal, 2004
Pseudomonas sp. 4.0 NA M=1 g/l, t
eq
=1.5 h 95.0 (L) Ziagova et al., 2007
Staphylococcus xylosus 1.0 NA M=1 g/l, t
eq
=1.5 h 143.0 (L) Ziagova et al., 2007
Zoogloea ramigera 2.0 25 NA 27.5 (L) Sa and Kutsal, 1989
Copper Bacillus sp. (ATS-1) 5.0 25 M=2 g/l, t
eq
=2 h 16.3 (E) Tunali et al., 2006
Bacillus subtilis IAM 1026 5 25 M=0.5 g/l, t
eq
=1 h 20.8 (L) Nakajima et al., 2001
Enterobacter sp. J1 5.0 25 t
eq
=24 h 32.5 (L) Lu et al., 2006
Micrococcus luteus IAM 1056 5 25 M=0.5 g/l, t
eq
Pseudomonas aeruginosa PU21 5.0 NA M=12 g/l; t
eq
=24 h 23.1 (L) Chang et al., 1997
Pseudomonas cepacia 7 30 NA 65.3 (L) Savvaidis et al., 2003
Pseudomonas putida 6.0 NA NA 6.6 (L) Pardo et al., 2003
Pseudomonas putida 5.5 30 M=1 g/l, t
eq
=24 h 96.9 (L) Uslu and Tanyol, 2006
Pseudomonas putida CZ1 4.5 30 M=1 g/l; t
eq
=24 h 15.8 (L) Chen et al., 2005
Pseudomonas stutzeri IAM 12097 5 25 M=0.5 g/l, t
eq
Sphaerotilus natans 6 NA M=3 g/l; t
eq
=0.5 h 60 (E) Beolchini et al., 2006
Sphaerotilus natans
b
5.5 30 NA 5.4 (L) Beolchini et al., 2006
Streptomyces coelicolor 5.0 25 M=1 g/l; t
eq
=8 h 66.7 (L) ztrk et al., 2004
Thiobacillus ferrooxidans
a
6.0 37 M=0.2 g/l; t
eq
=2 h 198.5 (L) Ruiz-Manriquez et al., 1997
a
5.0 40 M=300 g/l; t
eq
=2 h 39.84 (L) Liu et al., 2004
Cadmium Aeromonas caviae 7.0 20 M=1 g/l; t
eq
=2 h 155.3 (L) Loukidou et al., 2004a,b
Bacillus circulans 7.0 30 M=0.5 g/l; t
eq
=2 h 26.5 (E) Yilmaz and Ensari, 2005
eq
=24 h 46.2 (L) Lu et al., 2006
eq
=24 h 42.4 (L) Chang et al., 1997
Pseudomonas sp. 7.0 NA M=1 g/l, t
eq
=1.5 h 278.0 (L) Ziagova et al., 2007
Staphylococcus xylosus 6.0 NA M=1 g/l, t
eq
=1.5 h 250.0 (L) Ziagova et al., 2007
Streptomyces pimprina
a
5.0 NA t
eq
=1 h 30.4 (L) Puranik et al., 1995
Streptomyces rimosus
a
8.0 NA M=3 g/l 64.9 (L) Selatnia et al., 2004a
Iron (III) Streptomyces rimosus
a
NA NA M=3 g/l; t
eq
=4 h 122.0 (L) Selatnia et al., 2004c
Lead Bacillus sp. (ATS-1) 3.0 25 M=2 g/l, t
eq
=2 h 92.3 (E) Tunali et al., 2006
Corynebacterium glutamicum 5.0 202 M=5 g/l, t
eq
=2 h 567.7 (E) Choi and Yun, 2004
eq
=24 h 50.9 (L) Lu et al., 2006
eq
=24 h 79.5 (L) Chang et al., 1997
Pseudomonas aeruginosa PU21
b
5 50 M=200 g/l 0.7 (L) Lin and Lai, 2006
Pseudomonas putida 5.5 25 M=1 g/l, t
eq
=24 h 270.4 (L) Uslu and Tanyol, 2006
a
NA NA M=3 g/l; t
eq
=3 h 135.0 (L) Selatnia et al., 2004b
Streptoverticillium cinnamoneum
a
4.0 283 M=2 g/l, t
eq
=0.5 h 57.7 (E) Puranik and Paknikar, 1997
Mercury Bacillus sp. 6 25 M=2 g/l, t
eq
=2 h 7.9 (L) Green-Ruiz, 2006
Nickel Bacillus thuringiensis 6 35 M=1 g/l, t
eq
=8 h 45.9 (L) ztrk, 2007
a
6.5 NA M=3 g/l, t
eq
=2 h 32.6 (L) Selatnia et al., 2004d
Palladium Desulfovibrio desulfuricans 2.0 30 M=0.15 g/l, t
eq
=4 d 128.2 (L) de Vargas et al., 2004
Desulfovibrio fructosivorans 2.0 30 M=0.15 g/l, t
eq
=4 d 119.8 (L) de Vargas et al., 2004
Desulfovibrio vulgaris 2.0 30 M=0.15 g/l, t
eq
=4 d 106.3 (L) de Vargas et al., 2004
Platinum Desulfovibrio desulfuricans 2.0 30 M=0.15 g/l, t
eq
=4 d 62.5 (L) de Vargas et al., 2004
Desulfovibrio fructosivorans 2.0 30 M=0.15 g/l, t
eq
=4 d 32.3 (L) de Vargas et al., 2004
Desulfovibrio vulgaris 2.0 30 M=0.15 g/l, t
eq
=4 d 40.1 (L) de Vargas et al., 2004
Thorium Arthrobacter nicotianae IAM 12342 3.5 30 M=0.15 g/l, t
eq
=1 h 75.9 (E) Nakajima and Tsuruta, 2004
Bacillus licheniformis IAM 111054 3.5 30 M=0.15 g/l, t
eq
Bacillus megaterium IAM 1166 3.5 30 M=0.15 g/l, t
eq
Bacillus subtilis IAM 1026 3.5 30 M=0.15 g/l, t
eq
Corynebacterium equi IAM 1038 3.5 30 M=0.15 g/l, t
eq
Corynebacterium glutamicum IAM 12435 3.5 30 M=0.15 g/l, t
eq
Micrococcus luteus IAM 1056 3.5 30 M=0.15 g/l, t
eq
Nocardia erythropolis IAM 1399 3.5 30 M=0.15 g/l, t
eq
Zoogloea ramigera IAM 12136 3.5 30 M=0.15 g/l, t
eq
(continued on next page)
carrageenan. Therefore, utmost care should be taken while select-
ing seaweeds for a biosorption process. Many investigators have
worked on brown seaweed and in particular Volesky and his co-
workers (Davis et al., 2000; Yang and Volesky, 1999a; Volesky
et al., 2003) investigated the metal sorbing properties of one of the
best metal sorbent Sargassum seaweed. Davis et al. (2003)
reviewed the metal sorbing properties of brown seaweeds, and
highlighted their biosorption mechanisms.
Recently, numerous approaches have been made for the
development of low-cost sorbents fromindustrial and agricultural
wastes. Of these, crab shells (Lee et al., 1997), activated sludge
(Al-Qodah, 2006), rice husks (Chuah et al., 2005), egg shell
(Vijayaraghavan et al., 2005b) and peat moss (Sharma and
Forster, 1993) deserve particular attention. Recent publications
indicate that crab shells possess excellent arsenic (Niu et al.,
2007), chromium (Kim, 2003), copper (Vijayaraghavan et al.,
2006b), cobalt (Vijayaraghavan et al., 2006b) and nickel sorbent
abilities (Vijayaraghavan et al., 2004).
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. Won et al. (2005) identified
Corynebacterium glutamicum as a potent biosorbent of Reactive
red 4, which canbind104.6mg/gat pH1. Aksuandaatay (2006)
indicated that Rhizopus arrhizus was capable of binding 773 mg/g
of Gemazol Turquise blue-G at 45 C and pH 2. Aksu and Tezer
(2005) explored 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. Verylittle effort
has been made to utilize seaweeds for the biosorption of dyes, but of
note, Rubin et al. (2004) employed Sargassum muticum for the
removal of methylene blue and Vijayaraghavan and Yun (2008)
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. Readers are encouraged to refer to other reviews for
information on fungal (Kapoor and Viraraghavan, 1995), algal
(Davis et al., 2003) and other low-cost biosorbents (Crini, 2006).
4. 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 (Volesky, 1987). However, further research has revealed that
inactive/dead microbial biomass can passively bind metal ions via
various physicochemical mechanisms. With this new finding,
research onbiosorptionbecame 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 microprecipita-
tion (Vegli and Beolchini, 1997; Volesky and Schiewer, 1999).
Table 2 summarizes some of the important results of metal
biosorption using bacterial biomasses. A direct comparison of
experimental data is not possible, due to different systematic
experimental conditions employed (pH, pH control, temperature,
Table 2 (continued)
Metal Organism Operating conditions Uptake (mg/g) Reference
Uranium Arthrobacter nicotianae IAM 12342 3.5 30 M=0.15 g/l, t
eq
Bacillus licheniformis IAM 111054 3.5 30 M=0.15 g/l, t
eq
Bacillus megaterium IAM 1166 3.5 30 M=0.15 g/l, t
eq
Bacillus subtilis IAM 1026 3.5 30 M=0.15 g/l, t
eq
Corynebacterium equi IAM 1038 3.5 30 M=0.15 g/l, t
eq
Corynebacterium glutamicum IAM 12435 3.5 30 M=0.15 g/l, t
eq
Micrococcus luteus IAM 1056 3.5 30 M=0.15 g/l, t
eq
Nocardia erythropolis IAM 1399 3.5 30 M=0.15 g/l, t
eq
Zoogloea ramigera IAM 12136 3.5 30 M=0.15 g/l, t
eq
Zinc Aphanothece halophytica 6.5 30 M=0.2 g/l, t
eq
=1 h 133.0 (L) Incharoensakdi and Kitjaharn, 2002
Pseudomonas putida CZ1 5.0 30 M=1 g/l; t
eq
=24 h 17.7 (L) Chen et al., 2005
Streptomyces rimosus 7.5 20 M=3 g/l 30.0 (L) Mameri et al., 1999
a
7.5 20 M=3 g/l 80.0 (L) Mameri et al., 1999
Streptoverticillium cinnamoneum
a
5.5 283 M=2 g/l, t
eq
=0.5 h 21.3 (E) Puranik and Paknikar, 1997
a
6.0 25 M=0.2 g/l, t
eq
=2 h 82.6 (L) Celaya et al., 2000
a
6.0 40 M=300 g/l; t
eq
=2 h 172.4 (L) Liu et al., 2004
(E) =experimental uptake, (L) =uptake predicted by the Langmuir model; M=biomass dosage, t
eq
=equilibrium time, NA=not available.
a
Chemically modified.
b
Immobilized.
equilibrium time and biomass dosage). However, Table 2
provides basic information to evaluate the possibility of using
bacterial biomass for the uptake of metal ions. Also, it should be
noted that Table 2 is only comprised of biosorption studies that
employed either inactive or dead bacterial biomasses.
Some variability in the results has been observed when the same
bacterium was employed for the same metal, but under different
instances. Apart from the different experimental conditions, this is
due to the fact that the biomass was pretreated or immobilized to
improve the biosorbent characteristics, as highlighted in Table 2.
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 various 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 formof
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.
Table 3 summarizes some of the important results of dye
biosorption using bacterial biomass. 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 (Fuand Viraraghavan, 2001; Crini, 2006).
Also, most research focused on studying the biodegradation/deco-
lorization potential of bacteria (Forgacs et al., 2004; Pandey et al.,
2007). Of the limited results on bacterial biosorption, C. glutamicum
have been shown to performwell in the biosorption of reactive dyes,
with dye uptakes in the range of 0.10.4 times that of its dry weight.
5. Bacterial structure and mechanism of bacterial biosorption
5.1. 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 Rho-
dospirillum) and filamentous (such as Sphaerotilus). Eubacteria
have a relatively simple cell structure, which lack cell nuclei,
but possess cell walls (Salton, 1964). 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
(Rogers et al., 1980). Peptidoglycan is responsible for the
rigidity of the bacterial cell wall, and determines the cell shape
(Kolenbrander and Ensign, 1968). 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
(Beveridge, 1981; Dijkstra and Keck, 1996) 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 cytoplas-
mic 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 (Sonnenfeld et al., 1985). 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 1020% pepti-
doglycan (Kolenbrander and Ensign, 1968; Beveridge, 1999). In
addition, the cell wall contains an additional outer membrane
composed of phospholipids and lipopolysaccharides (Sheu and
Freese, 1973). The highly charged nature of lipopolysaccharides
confers an overall negative charge on the Gram-negative cell wall.
Sherbert (1978) showed that the anionic functional groups
present in the peptidoglycan, teichoic acids and teichuronic acids
of Gram-positive bacteria, and the peptidoglycan, phospholi-
pids, 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 (McLean
Table 3
Important results from the literature on dye biosorption by various bacterial species
Organism Dye Operating conditions Uptake (mg/g) Ref.
Corynebacterium glutamicum Reactive black 5 1.0 35 M=2.5 g/l; t
eq
=12 h 419.0 (L) Vijayaraghavan and Yun, 2007b
Reactive red 4 1.0 202 M=10 g/l; t
eq
=24 h 104.6 (L) Won et al., 2005
Reactive orange 16 1.0 202 M=10 g/l; t
eq
=24 h 186.6 (L) Won et al., 2004
Reactive blue 4 4.0 252 M=10 g/l; t
eq
=24 h 173.1 (L) Han and Yun, 2007
Reactive yellow 2 1.0 252 M=10 g/l; t
eq
=24 h 178.5 (L) Won and Yun, 2008
Streptomyces rimosus Methylene blue NA 20 M=4.5 g/l, 34.3 (L) Nacra and Aicha, 2006
(L) =uptake predicted by the Langmuir model; M=biomass dosage, t
eq
=equilibrium time, NA=not available.
et al., 1992). However, their availability depends on the bacterial
species and growth conditions; and they can easily be removed
by simple mechanical disruption or chemical washing (Yee and
Fein, 2001).
5.2. Mechanism of bacterial biosorption
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 (Beveridge and
Murray, 1976; Doyle et al., 1980). 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, phospho-
nate, amine and hydroxyl groups (Doyle et al., 1980; van der Wal
et al., 1997).
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. Golab and Breitenbach (1995) indicated that the 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. Kang et al. (2007)
observed that amine groups protonated at pH 3 and attracted
negatively charged chromate ions via electrostatic interaction.
Vijayaraghavan and Yun (2007b) confirmed that the 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 (Baes and Mesmer, 1976). For example, in the case
of nickel solution, Lpez et al. (2000) indicated that 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 solidliquid 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 (Selatnia et al., 2004b;
Iqbal and Saeed, 2007).
5.3. Characterization of bacterial surface
Characterization of bacterial biomass and the biosorption
mechanisms can be elucidated using different methods, includ-
ing potentiometric titrations (Texier et al., 2000; Yee and Fein,
2001; Phoenix et al., 2002), Fourier transform infrared spectro-
scopy (Beveridge and Murray, 1980; Jiang et al., 2004; Vannela
and Verma, 2006), X-ray diffraction (Carito et al., 1967; Lpez
et al., 2000; Kelly et al., 2002; Kazy et al., 2006), scanning
electron microscopy (Tunali et al., 2006; Lu et al., 2006;
Vijayaraghavan et al., 2007), transmission electron microscopy
Fig. 1. Structure of Gram-positive and negative bacteria.
(Mullen et al., 1989; Kazy et al., 2006; Vannela and Verma,
2006) and energy dispersive X-ray microanalysis (Small et al.,
1999; Kazy et al., 2006).
Potentiometric titrations have aided several researchers in the
determination of the type and number of binding sites. Yee and
Fein (2001) titrated two Gram-negative and seven Gram-positive
bacteria, and determined the pK
a
values and number of available
binding sites. Davis et al. (2000) successfully correlated the
amount of acidic groups, determined via potentiometric titrations,
with the metal uptake capacity. The nature of the binding sites and
their involvement during biosorption can be approximately eval-
uated using FT-IR. Vannela and Verma (2006) analyzed the FT-IR
spectra of virgin and Cu
2+
exposed Spirulina platensis. Several
band transformations allowed the authors to predict the possible
involvement of amide, amino and carboxyl groups in the bio-
sorption of Cu
2+
. Won et al. (2005) used FT-IRspectra to confirm
the presence of carboxyl, amine and phosphonate groups in
C. glutamicum biomass.
EDX can provide information regarding the chemical and
elemental characteristics of a biomass. Tunali et al. (2006)
analyzed both Pb(II) and Cu(II) loaded Bacillus sp. using EDX,
and confirmed the involvement of an ion exchange mechanism
during their biosorption. In order to elucidate the chemical nature
of bacterial cell-bound lanthanum, Kazy et al. (2006) employed
XRD analysis, and confirmed the involvement of cellular
carboxyl and phosphate groups in the binding of lanthanum by
Pseudomonas biomass. To analyze the morphology of the cell
surface before and after biosorption, SEM micrographs are often
used. With the aid of SEM photographs, Lu et al. (2006)
visualized the surface of metal-loaded Enterobacter sp., which
appeared to be vague and damaged by the heavy-metal ions.
Vijayaraghavan et al. (2007) used SEM photographs to show the
pattern of C. glutamicum immobilization within a polysulfone
matrix. Methods for analyzing the biomass surface and possible
biosorption mechanism; therefore, are well established.
6. Preparation of bacterial biosorbents
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 bio-
sorption 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.
6.1. Chemically modified biosorbents
Chemical modification procedures include pretreatment, bind-
ing site enhancement, binding site modification and polymeriza-
tion. Common chemical pretreatments include acid, alkaline,
ethanol and acetone treatments of the biomass (Vijayaraghavan
and Yun, 2007b; Selatnia et al., 2004a; Gksungur et al., 2005; Bai
and Abraham, 2002). 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. Vijayaraghavan and Yun (2007b)
employed several chemical agents (mineral acids, NaOH,
Na
2
CO
3
, CaCl
2
and NaCl) for the pretreatments of C. glutamicum
in the biosorption of Reactive black 5. The authors identified0.1 M
HNO
3
as being most suitable for opening newbinding sites, which
enhanced Reactive black 5 uptake capacity by 1.3 times as that of
the raw biomass. However, utmost care and careful screening
methods must be employed for selecting appropriate chemical
agents for pretreatment. Sar et al. (1999) observed that the metal
(Cu
2+
and Ni
2+
) uptake capacity of lyophilized Pseudomonas
auruginosa cells was enhanced when pretreated with NaOH,
NH
4
OH 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 res-
ponsible 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. Jeon and Hll (2003) used
chloroacetic acid to introduce carboxyl in the place of hydroxyl
groups. Then the carboxylated biomass was treated with ethyl-
enediamine followed by carbodiimide to formaminated biomass.
The authors observed that increase in amine groups increased
mercury uptake by 47% compared to that of control. Li et al.
(2007) employed citric acid to 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.
Many studies have focused on enhancing the active binding
sites to improve the biosorption; however, less attention has been
paid to the inhibition sites. For instance, amine groups are
responsible for the binding of dye anions via electrostatic
interaction; whereas the presence of negatively charged groups,
such as carboxyl, may repel dye anions. Vijayaraghavan and Yun
(2007a) observed 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. Deng and Ting (2005a,b,c, 2007) worked
extensively with polyethylenimine, composed of a large number
of primary and secondary amine groups, which when cross-linked
with biomass exhibited good biosorption abilities towards
chromium (VI), copper, lead, nickel and arsenic. In 2005b,
Deng and Ting copolymerized acrylic acid onto the biomass
surface to enhance the carboxyl groups, which resulted in five and
seven fold enhancements for the uptakes of copper and cadmium,
respectively, compared with the pristine biomass. Poly(amic
acid), from reaction of pyromellitic dianhydride and thiourea,
which comprises a large number of carboxyl and secondary amine
groups in a molecule, when grafted on the biosorbent surface,
exhibited 15- and 11-fold increases in the uptakes of cadmiumand
lead, respectively, compared to the pristine biomass. (Yu et al.,
2007).
6.2. 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 (Bae et al., 2000; Majare
and Bulow, 2001). 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
(Pazirandeh et al., 1995).
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)
(Singhal et al., 1997), phytochelatins (PCs) and metallothioneins
(MTs) (Mehra and Winge, 1991), which can bind and sequester
metal ions in biologically inactive forms (Hamer, 1986; Bae et al.,
2000). The overexpression 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 (Pazirandeh et al., 1995).
In addition to the high selectivity and accumulation capacity,
Pazirandeh et al. (1995) demonstrated that the uptake by re-
combinant E. coli (expressing the 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 (Gadd and White, 1993). Chen and Georgiou
(2002) suggested a solution to bypass this transport problem by
expressing MTs on the cell surface. Sousa et al. (1996)
demonstrated the possibility of inserting MTs into the permissive
site 153 of the LamB sequence. The expression of the hybrid
proteins on the cell surface dramatically increased the whole-cell
accumulation of cadmium. Also, the expression of proteins on the
surface offers an inexpensive alternative for the preparation of
affinity adsorbents (Georgiou et al., 1993).
The use of PCs in a similar manner to MTs has also been
suggested (Bae et al., 2000). PCs are short, cysteine-rich
peptides, with the general structure (Glu-Cys)
n
Gly (n=211)
(Zenk 1996). PCs offer many advantages over MTs, due to their
unique structural characteristics, particularly the continuously
repeating Glu-Cys units. Also, PCs have been found to exhibit
higher metal-binding capacity (on a per cysteine basis) than
MTs (Mehra and Mulchandani, 1995). However, the develop-
ment 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 exam-
ple includes creating a repetitive metal-binding motif, consisting
of (Glu-Cys)
n
Gly (Bae et al., 2000). These peptides emulate the
structure of PCs; however, they differ in the fact that the peptide
bond between the glutamic acid and cysteine is a standard
peptide bond. Phytochelatin analogs were found to be present on
the bacterial surface, which enhanced the accumulation of Cd
2+
and Hg
2+
by 12- (Bae et al., 2000) and 20-fold (Bae et al., 2001),
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 (Chen and Wilson, 1997). Nevertheless, a
Gram-positive surface display system also possesses its own
merits compared to Gram-negative bacteria (Malik et al., 1998,
Samuelson et al., 2000): (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 (Kelemen and Sharpe, 1979) due to the thick cell wall
surrounding the cells, which potentially make themmore suitable
for field applications, such as bioadsorption. Samuelson et al.
(2000) generated recombinant Staphylococcus xylosus and Sta-
phylococcus carnosus strains, with surface-exposed chimeric
proteins containing polyhistidyl peptides. 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 bio-
sorbents may prove very competitive for the separation of toxins
and other pollutants from dilute contaminated solutions.
6.3. 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 trans-
fer, they often suffer several drawbacks. The most important
include solidliquid separation problems, possible biomass
swelling, inability to regenerate/reuse and development of high
pressure drop in the column mode (Vegli and Beolchini, 1997;
Vijayaraghavan and Yun 2007a).
Several established techniques are available to make biosor-
bents suitable for process applications. Among these, immobili-
zation techniques such as entrapment and cross linking have been
found to be practical for biosorption (Vegli and Beolchini, 1997;
Volesky, 2001). 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 (Hu and
Reeves, 1997). Very few efforts have been made to utilize the
immobilization concept for dye (Fu and Viraraghavan, 2003;
Vijayaraghavan et al., 2007) compared to metal biosorption (Hu
and Reeves, 1997; Prakashamet al., 1999; Yan and Viraraghavan,
2001; Khoo and Ting, 2001; Beolchini et al., 2003; Bai and
Abraham, 2003). Important immobilization matrices used in
biosorbent immobilization include sodium alginate (Bai and
Abraham, 2003; Xiangliang et al., 2005), polysulfone (Beolchini
et al., 2003; Vijayaraghavan et al., 2007), polyacrylamide (Bai
and Abraham, 2003) and polyurethane (Hu and Reeves, 1997).
The choice of immobilization matrix is a key factor in the
environmental application of immobilized biomass. The poly-
meric matrix determines the mechanical strength and chemical
resistance of the final biosorbent particle to be utilized for
successive sorptiondesorption cycles (Bai and Abraham, 2003).
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. Vijayaraghavan
et al. (2007) reported the immobilization of C. glutamicumwithin
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. Several investigators have successfully regen-
erated and reused seaweeds over a number of cycles for the
removal of metal ions (Vijayaraghavan et al., 2005a; Senthilkumar
et al., 2006). Volesky et al. (2003) regenerated and reused virgin
Sargassum filipendula loaded flow-through packed column over
ten cycles during the biosorption of copper. 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. 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 usedfor 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 (Hermann, 2003). 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 exam-
ined 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. Vijayaraghavan et al. (2007)
immobilized C. glutamicum into several polymeric matrices,
and identified polysulfone as a favorable and practical immobiliz-
ing agent, which showed the ability for the biosorption of reactive
dyes over twenty successive sorptiondesorption cycles.
A review of literature relating to biosorption revealed that
several microbial biomasses have been cultivated and explored
for their biosorption potential. The cost for producing biomass
for the sole purpose of its transformation into biosorbents has
been shown to be too expensive (Tsezos, 2001). Furthermore,
the continuous supply of biomass cannot be assured, which will
have a huge impact on its successful application in industrial
biosorption applications.
7. Biosorption experimental procedures
Abiosorption process can be performed via several modes; of
which, batch and continuous modes of operation are frequently
employed to conduct laboratory scale biosorption processes.
Although most industrial applications prefer a continuous mode
of operation, batch experiments have to be used to evaluate the
required fundamental information, such as biosorbent efficiency,
optimum experimental conditions, biosorption rate and possibi-
lity of biomass regeneration.
7.1. Factors influencing bacterial batch biosorption
A schematic representation of the batch biosorption process is
illustrated in Fig. 2. Batch experiments usually focus on the study
of factors influencing biosorption, which are important in the
evaluation of the full biosorption potential of any biomaterial. The
important factors include:
# Solution pH
# Temperature
# Ionic strength
# Biosorbent dosage
# Biosorbent size
# Initial solute concentration
# Agitation rate
Of these, the solution pH usually plays a major role in bio-
sorption, and seems to affect the solution chemistry of metals/
dyes and the activity of the functional groups of the biomass.
For metals, the pH strongly influences the speciation and
biosorption availability of the metal ions (Yang and Volesky,
1999b; Esposito et al., 2002). At higher solution pH, the
solubility of metal complexes decreases sufficiently allowing
precipitation, which may complicate the sorption process. The
activity of binding sites can also be altered by adjustment of the
pH. For instance, during the biosorption of metal ions by
bacterial biomass, pH 3 to 6 has been found favorable for
biosorption (Table 2), due to the negatively charged carboxyl
groups (pK
a
=35), which are responsible for the binding metal
cations via ion exchange mechanism. In the case of the
biosorption of dyes, different dye classes require different pH
ranges. For instance, basic dyes require alkaline or neutral
conditions (Farah et al., 2007); whereas, reactive dyes demand
strong acidic conditions (O'Mahony et al., 2002) for their
optimum biosorption.
Temperature seems to affect biosorption only to a lesser
extent within the range from 20 to 35 C (Vegli and Beolchini,
1997). Higher temperatures usually enhance sorption due to the
increased surface activity and kinetic energy of the solute (Sa
and Kutsal, 2000; Vijayaraghavan and Yun, 2007b); however,
physical damage to the biosorbent can be expected at higher
temperatures. Due to the exothermic nature of some adsorption
processes, an increase in temperature has been found to reduce
the biosorption capacity of the biomass (Mameri et al., 1999;
Suhasini et al., 1999). It is always desirable to conduct/evaluate
biosorption at room temperature, as this condition is easy to
replicate.
Another important parameter in biosorption is the ionic
strength, which influences the adsorption of solute to the
biomass surface (Daughney and Fein, 1998; Borrok and Fein,
2005). The effect of ionic strength may be ascribed to the
competition between ions, changes in the metal activity, or in the
properties of the electrical double layer. When two phases, e.g.
biomass surface and solute in aqueous solution are in contact,
they are bound to be surrounded by an electrical double layer
owing to electrostatic interaction. Thus, adsorption decreases
with increase in ionic strength (Dnmez and Aksu, 2002). Some
inorganic ions, such as chloride, may formcomplexes with some
metal ions and therefore, affect the sorption process (Boro-
witzka, 1988).
The dosage of a biosorbent strongly influences the extent of
biosorption. In many instances, lower biosorbent dosages yield
higher uptakes and lower percentage removal efficiencies (Aksu
and aatay, 2006; Vijayaraghavan et al., 2006b). An increase
in the biomass concentration generally increases the amount of
solute biosorbed, due to the increased surface area of the
biosorbent, which in turn increases the number of binding sites
(Esposito et al., 2001). Conversely, the quantity of biosorbed
solute per unit weight of biosorbent decrease with increasing
biosorbent dosage, which may be due to the complex interaction
of several factors. An important factor at high sorbent dosages is
that the available solute is insufficient to completely cover the
available exchangeable sites on the biosorbent, usually resulting
in low solute uptake (Tangaromsuk et al., 2002). Also, as
suggested by Gadd et al. (1988), the interference between
binding sites due to increased biosorbent dosages can not be
overruled, as this will result in a low specific uptake.
The size of the biosorbent also plays a vital role in biosorption.
Smaller sized particles have a higher surface area, which in turn
favors biosorption and results in a shorter equilibration time.
Simultaneously, a particle for biosorption should be sufficiently
resilient to withstand the applicable pressures and extreme
conditions applied during regeneration cycles (Volesky, 2001).
Therefore, preliminary experiments are mandatory to decide the
suitable size of a biosorbent. If a biosorbent is available in
powdered form, such as industrial waste, efforts should be made
to improve the mechanical strength, such as granulation, for its
effective use in biosorption columns.
The initial solute concentration seems to have impact on
biosorption, with a higher concentration resulting in a high
Fig. 2. Schematic diagram of batch biosorption equilibrium experimental procedure.
solute uptake (Ho and McKay, 1999b; Ho and McKay, 2000;
Binupriya et al., 2007a). This is because at lower initial solute
concentrations, the ratio of the initial moles of solute to the
available surface area is low; subsequently, the fractional
sorption becomes independent of the initial concentration.
However, at higher concentrations, the sites available for
sorption become fewer compared to the moles of solute present
and; hence, the removal of solute is strongly dependent upon the
initial solute concentration. It is always necessary to identify the
maximum saturation potential of a biosorbent, for which
experiments should be conducted at the highest possible initial
solute concentration.
In some instances, external filmdiffusion can influence the rate
of a biosorption process. With appropriate agitation, this mass
transfer resistance can be minimized. When increasing the agi-
tation rate, the diffusion rate of a solute fromthe bulk liquid to the
liquid boundary layer surrounding particles becomes higher due
to the enhanced turbulence and the decrease in the thickness of the
liquid boundary layer (Evans et al., 2002). Under these condi-
tions, the value of the external diffusion coefficient becomes
larger (Shen and Duvnjak, 2005). Finally, at higher agitation rates,
the boundary layer becomes very thin, which usually enhances the
rate at which a solute diffuse through the boundary layer.
7.2. Biosorption isotherms
The quality of a biosorbent is judged by how much sorbate it
can attract and retain in an immobilized form. The solute uptake
by a biosorbent can be calculated from the differences between
the initial quantities of solute added to that contained in the
supernatant, which is achieved using the following equation:
Q V
0
C
0
V
f
C
f
=M 1
where Q is the solute uptake (mg/g); C
0
and C
f
the initial and
equilibriumsolute concentrations in solution (mg/l), respectively;
V
0
and V
f
the initial and final solution volumes (l), respectively;
and M the mass of biosorbent (g). The sorption uptake can be
expressed in different units depending on the purpose of the
exercise: for example, milligrams of solute sorbed per gramof the
(dry) biosorbent material (the basis for engineering processmass
balance calculations), or mmol/g (when the stoichiometry and/or
mechanism are to be considered).
A biosorption isotherm, the plot of uptake (Q) versus the
equilibriumsolute concentration in the solution (C
f
), is often used
to evaluate the sorption performance. Isotherm curves can be
evaluated by varying the initial solute concentrations, while fixing
the environmental parameters, such as pH, temperature and ionic
strength. In general, the uptake increases with increase in concen-
tration, and will reach saturation at higher concentrations. In most
biosorption studies, pHseems to be an important parameter for the
evaluation of an isotherm. However, confusion prevails in report-
ing isotherms based on the pH. In the literature pertaining to
biosorption, isotherms have been reported on the basis of the
initial (Aksu et al., 2002; Fu and Viraraghavan, 2002), final
(Reddad et al., 2002) or controlled (Davis et al., 2000; Esposito
et al., 2001) pH conditions. This is because; during biosorption,
the pHof the reaction mixture tends to change due to the chemical
interaction between the biomass and sorbent. The chemical
constituents of a biosorbent and the nature of the biosorption
mechanismseemto be mainly responsible for any pHchange. For
instance, a protonated bacterial biomass releases H
+
ions during
the biosorption of metals/dyes, which in turn decreases the solu-
tion pH. These changes in pHare rapid during the initial period, as
most of the reaction tends to occur during the initial stage, fol-
lowed by slow attainment of equilibrium. Several reports have
stressed that the pH should be controlled over the entire contact
period until equilibrium is reached (Kratochvil and Volesky,
1998; Vieira and Volesky, 2000). However, this is a fairly
complex task, which necessitates sophisticated instrumentation.
Also, the addition of chemical agents to maintain the pH at the
optimumlevel should be incorporated when calculating a specific
uptake (Eq. (1)). Biosorption has also been reported on the basis
of the final pH, with the claim that final equilibrium pH
determines the performance of a sorption system. Since batch
experiments are designed to evaluate the fundamental information
regarding a biosorbent, cases where the pHis controlled will give
a real picture of the potential of a biosorbent and usage in chemical
equilibrium modeling.
7.3. Batch experimental data modeling
Models have an important role in technology transfer from a
laboratory- to industrial-scale. Appropriate models can help in
understanding process mechanisms, analyze experimental data,
predict answers to operational conditions and optimize processes.
As an effective quantitative means to compare binding strengths
and design biosorption processes, employing mathematical
models for the prediction of binding capacities can be useful
(Volesky and Holan, 1995; Limousin et al., 2007).
Biosorption modeling can be performed in two general ways:
empirical or mechanistic equations, which are able to explain,
represent and predict the experimental behavior.
7.3.1. Empirical modeling
Empirical models are simple mathematical relationships,
characterized by a limited number of adjustable parameters,
which give a good description of the experimental behavior
over a large range of operating conditions (Esposito et al.,
2002). Some frequently employed and well established
empirical models involve two, three or even four parameters
to model the isotherm data (Vijayaraghavan et al., 2006a).
Although these conventional empirical models do not reflect
the mechanisms of sorbate uptake, they are capable of
reflecting the experimental curves (Kratochvil and Volesky,
1998). Also, in most cases, the assumptions from which these
models were derived are not valid for biosorption. Despite this,
conventional adsorption isotherm models are used with a high
rate of success for replicating biosorption isotherm curves.
Within the literature, the Langmuir (Langmuir, 1918) and
Freundlich (Freundlich, 1907) models (two-parameter models)
have been used to describe biosorption isotherm. The models
are simple, well-established and have physical meaning and
are easily interpretable, which are some of the important rea-
sons for their frequent and extensive use.
The Langmuir model can be represented as:
Q
Q
max
b
L
C
f
1 b
L
C
f
2
This classical model incorporates two easily interpretable
constants: Q
max
, which corresponds to the maximum achievable
uptake by a system; and b
L
, which is related to the affinity
between the sorbate and sorbent. The Langmuir constant Q
max
is often used to compare the performance of biosorbents; while

the other constant b
L
characterizes the initial slope of the iso-
therm. Thus, for a good biosorbent, a high Q
max
and a steep
initial isotherm slope (i.e., high b
L
) are generally desirable
(Kratochvil and Volesky, 1998).
The Freundlich isotherm can be represented as:
Q K
F
C
1=n
F
f
3
The Freundlich isotherm was originally empirical in nature,
but was later interpreted as the sorption to heterogeneous surfaces
or surfaces supporting sites with various affinities. It is assumed
that the stronger binding sites are initially occupied, with the
binding strength decreasing with increasing degree of site
occupation. It incorporates two constants: K
F
, which corresponds
to the binding capacity; and n
F
, which characterize the affinity
between the sorbent and sorbate.
Some other two-parameter models widely used for describ-
ing biosorption isotherms include,
Temkin (Temkin, 1934):
Q
RT
b
Te
ln a
Te
C
f
4
DubninRadushkevich (Dubinin, 1960):
Q Q
D
exp B
D
RTln 1
1
C
f

2

5
where b
Te
is the Temkin constant related to the heat of sorption; a
Te
the Temkin isotherm constant; R the gas constant (8.314 J/mol K);
T the absolute temperature; Q
D
the DubininRadushkevich mod-
el uptake capacity and B
D
the DubininRadushkevich model
constant.
Despite the simplicity of these two-parameter models, some
three-parameter models have also been widely used by investiga-
tors, including:
RedlichPeterson (Redlich and Peterson, 1959):
Q
K
RP
C
f
1 a
RP
C
b
RP
f
6
Sips (Sips, 1948):
Q
K
S
C
b
S
f
1 a
S
C
b
S
f
7
Khan (Khan et al., 1997b):
Q
Q
max
b
K
C
f
1 b
K
C
f

a
K
8
RadkePrausnitz (Radke and Prausnitz, 1972a):
Q
a
R
r
R
C
b
R
f
a
R
r
R
C
b
R
1
f
9
Toth (Toth, 1971):
Q
Q
max
b
T
C
f
1 b
T
C
f

1=n
T

n
T
10
where K
RP
is the RedlichPeterson model isotherm constant,
a
RP
the RedlichPeterson model constant;
RP
the Redlich
Peterson model exponent; K
S
the Sips model isotherm constant;
a
S
the Sips model constant;
S
the Sips model exponent; b
K
the
Khan model constant; a
K
the Khan model exponent; a
R
and r
R
are RadkePrausnitz model constants;
R
the RadkePrausnitz
model exponent; b
T
the Toth model constant and n
T
the Toth
model exponent.
Of these three-parameter models, the RedlichPeterson and
Sips models have been used with most success (Ho et al., 2002;
Vijayaraghavan et al., 2005a). The RedlichPeterson model is
comprised of an exponent (
RP
), which lies between 0 and 1.
There are two limiting behaviors: the Langmuir form for
RP
=1
and Henry's law form for
RP
=0. In the case of the Sips model,
under low sorbate concentrations, it effectively reduces to the
Freundlich isotherm and; thus, does not obey Henry's law. For
high sorbate concentrations, it predicts a monolayer sorption
capacity, characteristic of the Langmuir isotherm (Ho et al.,
2002). The descriptions and assumptions of these two- and
three-parameter models have been explained elsewhere (Aksu,
2005; Vijayaraghavan et al., 2006a; Limousin et al., 2007).
The most common method found in the literature for the
determination of the isotherm constants from the two-parameter
models involves the transformation of the isotherm variables so
the equation can be converted into a linear form, with linear
regression analysis then applied. Although a linear regression
analysis is not possible for three- or four-parameter models, a trial
and error procedure can be employed (Ho et al., 2002). Recently,
there has been concern over the use of the linearization procedure.
Linearization of non-linear models distorts the fit, resulting in
prediction errors. Also, the use of linearization has become
obsolete with recent advancement in computers and software. The
successful predication of isotherm constants should make use of
these extremely powerful tools for the acquisition of more
accurate results. Non-linear optimization provides a more
complex, yet mathematically rigorous, method for determining
isotherm parameter values (Khan et al., 1997a; Ho et al., 2002).
7.3.2. Mechanistic modeling
Mechanistic models have been proposed to describe solute
adsorption onto the surfaces of biomass (Plette et al., 1995; Fein
et al., 1997, 2001; Cox et al., 1999; Haas et al., 2001). The
development of a mechanistic model is usually based on
preliminary biomass characterization, with the formulation of a
set of hypothesized reactions between the sorbent sites and
solutes, which also considers the particular solution chemistry
of the solutes. Mechanistic models can often be characterized by
the different degrees of complexity or accuracy in a system
description to account for the surface heterogeneity and other
factors that contribute to non-ideal adsorption phenomena
(Pagnanelli et al., 2005). Mechanistic modeling of biosorption
has been attempted in several investigations, with significant
success (Yang and Volesky 1999b; Pagnanelli et al., 2000; Yun
et al., 2001).
Yang and Volesky (1999b) used the ion exchange relationship
between protons in the biomass and hydrolyzed uranium ion
species to develop a biosorption model, which was able to predict
the biosorption isotherms at different pH values as well as the
equilibrium uranium desorption concentrations. With respect to
the chromium speciation, Yun et al. (2001) developed a model to
predict the equilibrium sorption experimental data at different pH
values and chromiumconcentrations. Metal hydrolysis was found
to play an important part in the biosorption of chromium, with the
developed model able to describe the experimental data with high
accuracy. Yee and Fein (2001) developed an adsorption model to
correlate the cadmium adsorption behavior over a wide range of
different bacterial species. For this purpose, they employed
several Gram-positive and -negative bacteria, and postulated that
metalbacteria adsorption was not dependent on the bacterial
species involved. Pagnanelli et al. (2003) formulated mechanistic
models that accounted for the acidic properties of the cell wall
constituents derived from the characterization of Arthrobacter
biomass. These models revealed the complexity of metal bio-
sorption phenomenon and also showed a good agreement with the
experimental data.
From literature accounts, it must be emphasized that empirical
models have often been employed to describe experimental data.
Greater complexity, the requirement of titration or other biomass
characterization data and the solution chemistry often limit the
application of these mechanistic models. However, this approach
would be useful in the understanding and isolation of the
operating binding mechanisms as well as the proper and true
representation of experimental sets. Also, it must be stressed that
only metals, humic (Fein et al., 1999; Borrok and Fein, 2004) and
fulvic (Borrok and Fein, 2004) substances had previously been
used as model solutes in mechanistic modeling, with no attempts
made to consider dyes as the model solute, the reason for which is
unknown, but is probably associated with the complication in
understanding the mechanism of dye sorption by biomaterials.
7.4. Batch kinetic studies
For any practical applications, the process design, operation
control and sorption kinetics are very important (Azizian, 2004).
The sorption kinetics in a wastewater treatment is significant, as
it provides valuable insights into the reaction pathways and the
mechanism of a sorption reaction (Ho and McKay 1999a). Also,
the kinetics describes the solute uptake, which in turn controls
the residence time of a sorbate at the solid-solution interface (Ho
et al., 2000).
Since biosorption is metabolism-independent, it would be
expected to be a rapid process. Usually, free cell microbial bio-
sorption is comprised of two phases: a very fast initial uptake for
3060 min, followed by slow attainment of equilibrium within 2
to 3 h (Celaya et al., 2000; Choi and Yun, 2004; Lu et al., 2006).
However, when the bacterial biomass is immobilized, a delay in
the attainment of equilibrium would be expected as a result of
mass transfer resistances (Vegli and Beolchini, 1997; Wu and
Yu, 2007).
Sorption is a multi-step process, comprising of four con-
secutive elementary steps in the case of immobilized beads (Guo
et al., 2003): (1) transfer of solute fromthe bulk of solution to the
liquid filmsurrounding the beads, (2) transport of the solute from
the boundary liquid film to the surface of the bead (external
diffusion), (3) transfer of solute from the surface to the internal
active binding sites (intraparticle diffusion), and (4) interaction
of the solute with the active binding sites. In general, the first two
steps (external diffusion) are usually fast; as long as sufficient
agitation is provided to avoid the formation of a concentration
gradient within solution. If the fourth step is assumed to be rapid,
the subsequent intraparticle diffusion becomes the rate-limiting
step. Intraparticle diffusion has often been shown to be an
important factor in deciding the attainment of equilibrium with
the use of immobilized beads (Vijayaraghavan et al., 2007;
Vijayaraghavan and Yun, 2007a).
The most commonly used technique for identifying the
involvement of intraparticle diffusion during sorption is fitting
of the kinetic data to an intraparticle diffusion plot, as previously
suggested by Weber and Morris (1963) which is as follows,
q
t
k
i
t
1=2
11
This involves plotting the uptake at a given time versus the
square root of that time. If this plot passes through the origin,
then intraparticle diffusion is the rate determining step.
Over 25 models have been reported in the literature, all of which
have attempted to quantitatively describe the kinetic behavior
during the adsorption process (Khraisheh et al., 2002; Ho, 2006).
Each adsorption kinetic model has its own limitations, which are
derived according to specific experimental and theoretical
assumptions. Even though they violate the fundamental assump-
tions, many adsorption models have been used to successfully test
experimental biosorption data. Of these, pseudo-first and -second
order models have often been used to describe biosorption kinetic
data.
Pseudo-first order model
q
t
q
e
1 exp k
1
t 12
Pseudo-second order model
q
t
q
e
1
1
1 q
e
kt

13
where q
e
is the amount of solute sorbed at equilibrium (mg/g); q
t
the amount of solute sorbed at time t (mg/g); k
1
the first order
equilibrium rate constant (min
1
) and k
2
the second order equi-
librium rate constant (g/mg min). In most published cases
involving biosorption, the pseudo-first order equation was found
to not fit well over the entire contact time range, but was generally
applicable over the initial periods of the sorption process. This is
mainly due to use of linearized form of Eq. (12), which requires
previous knowledge of the equilibrium sorption capacity (q
e
).
Therefore, a means of extrapolating the experimental data to t =,
or treat q
e
as an adjustable parameter, has to be employed for a
trial and error determination (McKay et al., 1999). Conversely,
there is no prior need to know q
e
for solving the linear form of a
pseudo-second order equation. It is also based on the sorption
capacity of the solid phase, which predicts the behavior over the
entire study range, with a chemisorption mechanismbeing the rate
controlling step (McKay et al., 1999). Due to above merits, the
pseudo-second order equation is able to describe almost all kinetic
data originating from metal/dye interactions with biomaterials
(Ho and McKay, 1999a; Reddad et al., 2002; Deng and Ting,
2005b). Also, it should again be stressed that the use of the non-
linear form of equations may avoid this error in kinetic modeling.
8. Desorption and regeneration
Biosorption is a process of treating pollutant-bearing solu-
tions 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 achieve-
ment 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 de-
pendency 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) non-
damaging to the biomass, (ii) less costly, (iii) environmental
friendly and (iv) effective. Several investigators have conducted
exhaustive screening experiments to identify appropriate
elutants for this process. Of these, the work of Kuyucak and
Volesky (1989) is noteworthy; they examined several chemical
agents to desorb Co
2+
from cobalt-laden Ascophyllum nodosum,
and identified CaCl
2
in the presence of HCl as a suitable elutant.
Even though some chemical agents perform well in deso-
rption, they may be detrimental to the biosorbent. As discussed in
Section 7, bacterial biomasses pose problems during desorption
due to their microscopic structure. They tend to be affected by the
presence of both strong acidic and alkaline conditions, which are
often used during desorption processes. Vijayaraghavan et al.
(2007) observed that C. glutamicum performed well in reactive
dye biosorption; however, the biomass was severely damaged
when desorption was attempted using 0.1 M NaOH.
The performance of an elutant also strongly depends on the
type of mechanism responsible for the biosorption. For instance,
electrostatic attraction was found to be the main mechanism
responsible for the biosorption of negatively charged dye anions
to a positively charged cell surface (O'Mahony et al., 2002).
Therefore, it would be logical to make the cell surface negative
using alkaline solutions to repel the negatively charged reactive
dyes (Won and Yun, 2008).
Elution is also often influenced by the volume of the elutant,
which should be as low as practically possible to obtain the
maximum solute concentration in the smallest possible volume
(Volesky, 2001). At the same time, the volume of the solution
should be sufficient to provide maximum solubility for the
desorbed solute. Also, one has to realize that the desorbed sorbate
stays in the solution and a newequilibriumis established between
that and the one (remaining) still fixed on the biosorbent. This
leads to the concept of a desorption isotherm where the equilib-
rium is strongly shifted towards the sorbate dissolved in the
solution (Yang and Volesky, 1996). Thus, it is necessary to eval-
uate 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. Davis et al. (2000) observed that the solid-to-
liquid ratio affected the copper elution efficiency of CaCl
2
solutions, while it was nearly independent in the case of 0.1 M
HCl. The purpose of desorption is to unbind a contaminant froma
biosorbent, so both the recovered solute and biosorbent can be
reused. After desorption, the biosorbent should be close to its
original form, both morphologically and effectually. Also, during
the desorption process; removal of all the bound sorbate from the
biosorbent should be assured. If this does not happen, an un-
diminished uptake can not be expected in the next cycle. In the
field of bacterial biosorption, Puranik and Paknikar (1999) re-
generated and reused polysulfone-immobilized Citrobacter strain
over three cycles for the biosorption of lead, cadmium and zinc,
using 0.1 M HCl and 0.1 M EDTA as elutants; but only with
limited success, and emphasized the need for further screening
work. Beolchini et al. (2003) immobilized Sphaerotilus natans
into a polysulfone matrix for the biosorption of copper, and with
the aid of 0.05 M CaCl
2
, regenerated and reused the beads over
10 cycles, with satisfactory results.
One of the main attractions of biosorption is the potential
ability to regenerate the biomass. However, most of the published
work has aimed to evaluate the binding ability of biomass and the
parameters affecting the process. Less attention has been paid to
the regeneration ability of the biosorbent, which often decides the
industrial applicability of a process. Thus, biosorption studies
should emphasize the possibility of biomass regeneration to
improve the process viability.
9. 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 (Zhao et al., 1999; Saeed
and Iqbal., 2003; Volesky et al., 2003; Chu, 2004). 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 quality (Aksu and
Gnen, 2004). Also, packed bed sorption has a number of process
engineering merits, including a high operational yield and the
relative ease of scaling up procedures (Aksu, 2005). Other column
contactors, such as fluidized and continuous stirred tank reactors,
are very rarely used for the purpose of biosorption (Prakasham
et al., 1999; Solisio et al., 2000). Continuous stirred tank reactors
are useful when the biosorbent is in the formof a powder (Cossich
et al., 2004); however, they suffer fromhigh capital and operating
costs (Volesky, 1987). Fluidized bed systems, which operate
continuously, require high flow rates to keep the biosorbent parti-
cles in suspension (Muraleedharan et al., 1991).
9.1. Column biosorption
A schematic representation of a packed bed configuration is
shown in Fig. 3, which basically comprises a cylindrical column
packed firmly with sorbent, through which wastewater is allowed
to flow. Initially, most of the solute will be sorbed as it is exposed
to the fresh biosorbent bed and; thus, almost zero concentration
would be expected at the column outlet. Theoretically, this is
where the highest mass transfer occurs. However, as time is
required (and column length) for stabilized performance, the
initial column behavior can not be considered as this will only
represent a transient and unsteady state regime (Naja and Volesky,
2006a). With increasing time, the biosorbent bed will become
saturated with solute, the concentration of which will gradually
increase at the column outlet. Here, the breakthrough/service
concentration can be fixed, which depends on the toxicity of the
solute. For most solutes, 0.01 to 1 mg/l is considered the break-
through concentration. When the solute concentration exceeds
this limit in real industrial applications, the column has to be
removed from active operation, with the column regenerated or
the flow switched to another column. However, for laboratory
trails, the operation of the column should be terminated only when
inlet solute concentration approximately equals that at the outlet.
This is because complete column saturation, which results in S-
shaped breakthrough curve, is important to evaluate the
characteristics and dynamic response of a biosorption column
(Aksu, 2005). A typical breakthrough curve is shown in Fig. 3.
Recording the concentration profile at the column exit usually
results in a typical S-shaped curve, whose shape and slope are the
result of equilibrium sorption isotherm relationships, mass trans-
fer to and throughout the sorbent in the column, and operational
macroscopic fluid-flow parameters (da Silva et al., 2002).
Various parameters can be used to characterize the perfor-
mance of a packed bed biosorption, including the length of the
sorption zone, uptake, removal efficiency and slope of the
breakthrough curve (Volesky et al., 2003; Vijayaraghavan et al.,
2004). A mass transfer zone will develop between the gradually
saturated section of the column and the fresh biosorbent section
(Naja and Volesky, 2006a). The length of this zone is important
practically, which can be calculated from:
Z
m
Z 1
t
b
t
e

14
where Z denotes the bed depth (cm), and t
b
and t
e
the column
breakthrough and exhaustion times (h), respectively.
The uptake is an important parameter often used to charac-
terize the performance of a biosorbent in a packed column. The
column uptake (Q
col
) can be calculated by dividing the total mass
of biosorbed sorbate (m
ad
) by that of the biosorbent (M). The mass
of biosorbed sorbate is calculated from the area above the
breakthrough curve (C vs. t) multiplied by the flow rate.
The removal efficiency (%) can be calculated from the ratio
of the sorbate mass biosorbed to the total mass of sorbate sent to
the column, as follows:
Removal efficiency k
m
ad
C
0
Ft
e
100 15
where C
0
and F are the inlet solute concentration (mg/l) and
flow rate (l/h), respectively. It is important to note that the
removal efficiency is independent of the biosorbent mass, but
solely dependent on the flow volume. Therefore, it is necessary
Fig. 3. Schematic diagram of packed column arrangement with biosorption breakthrough and elution curves.
to consider both the uptake and removal efficiency when eval-
uating the biosorbent potential.
The slope of the breakthrough curve from t
b
to t
e
(dc / dt) is
often used to characterize the shape of the curve (Volesky et al.,
2003). It is always preferential to have an extended breakthrough
curve with a steep slope, as a steep slope is usually the result of a
shorter mass transfer zone, which implies a longer column ser-
vice time and greater utilization of the sorbent portion inside the
column (Kratochvil and Volesky, 1998).
Thus, for good biosorbents, a delayed breakthrough, earlier
exhaustion, shortened mass transfer zone, high uptake, steep
breakthrough curve and high removal efficiency would be
expected.
Some microorganisms show high biosorption capacities in
batch tests, but fail when applied to continuous-flow processes.
This is because the performance in a column mode strongly
depends on the mechanical strength of biosorbent and the kinetics
of the process. This is the reason for non-applicability of free
microbial biomasses in column mode. Vijayaraghavan and Yun
(2007a) indicated that it was not possible to use the free biomass
of C. glutamicum in a packed column, as it tended to swell and
form dense slurry that blocked the liquid flow, but suggested
immobilization as a potential remedy to this limitation. However,
a column operation provides only a short contact time for solute
and sorbent, and the mass transfer resistances prevailing in im-
mobilized beads strongly affect the column biosorption perfor-
mance. Therefore, utmost care must be taken in specifically
preparing a biosorbent for use in the column mode. In addition,
systematic evaluation of the biosorbent and parameters affecting
the biosorption should be evaluated. Some important parameters
affecting the biosorption in a packed column include, bed depth,
flow rate and initial solute concentration.
The accumulation of a solute in a fixed column is largely
dependent on the amount of biosorbent loaded into the column.
Zulfadhly et al. (2001) reported that the metal uptake increased on
increasing the bed height in a Pycnoporus sanguineus loaded-
fixed column. Similarly, Vijayaraghavan et al. (2004) observed an
increase in the nickel biosorptioncapacity when the bed height was
increased from 15 to 25 cm in a crab shell loaded packed column.
The increase in the uptake capacity with increasing bed depth was
due to the increased surface area of the sorbent, providing a greater
amount of available binding sites for biosorption.
The flow rate is a crucial characteristic in the evaluation of
sorbents for the continuous treatment of effluents in an industrial
scale. In general, a low flow rate favors biosorption, which can
be explained as follows: (1) when the flow rate increases, the
residence time of the solute in the column decreases, which
causes the effluent to leave the column prior to the attainment of
equilibrium; (2) when the process is controlled by intraparticle
mass transfer, a slower flowrate favors sorption, but if controlled
by external mass transfer, a higher flow rate will decrease the
film resistance (Ko et al., 2000).
The driving force for a sorption process is the concentration
difference between the solute on the sorbent and that in the
solution. Thus, an increased inlet solute concentration increases
the concentration difference, which favors biosorption. Padmesh
et al. (2006) indicated during Acid blue 15 biosorption, the
highest initial dye concentration resulted in a favorable break-
through curve, as well as high uptake and percentage removal.
9.2. Column regeneration
Regeneration of a biosorbent is relatively easier in a packed
column arrangement, with the aid of an appropriate elutant. When
the column becomes saturated, the contaminant solution flow
should be switched to the elutant flow. In general, an elution
process is usually fast compared to that of sorption. Thus, a high
contaminant concentration in a small elutant volume would be
expected under optimized process conditions. Also, it is always
desirable to limit the contact of the elutant with the sorbent. This is
because, extreme process conditions such as highly alkaline or
acidic solutions are often employed for elution; and thus morpho-
logical damage to the biosorbent can be expected. Therefore, the
optimal flow rate for the elution should be identified for
successful reuse of the biosorbent over multiple cycles. A typical
elution curve is shown in Fig. 3. Usually, a sharp concentration
increase would be expected at the beginning, followed by a grad-
ual decrease (Volesky et al., 2003; Vijayaraghavan et al., 2005a).
Even with the successful optimization of an elution process,
several investigators have observed a decrease in the biosorp-
tion performance over subsequent cycles (Saeed and Iqbal,
2003; Volesky et al., 2003; Vijayaraghavan et al., 2004). A loss
of sorption performance during long-term use may occur for a
variety of reasons; changes in the chemistry and structure of the
biosorbent, as well as the flow and mass transport conditions
within the column.
Fromthe literature, it is evident that very fewattempts have been
made to examine bacterial biomass in a continuous mode of oper-
ation. Many investigators have proposed the suitability of bacterial
biomass for industrial applications, based on batch experimental
results. As discussed earlier, a continuous mode of operation is
preferred for most industrial applications and; thus, only careful and
systematic investigation of biosorbent performance in a column
mode will ensure its application in real situations.
9.3. Modeling of column data
Mathematical models for flow-through fixed bed columns
have mainly originated from research on activated carbon sorp-
tion and ion exchange or chromatographic applications. Numer-
ous models have been tested for fixed bed biosorption columns,
including:
BohartAdams model (Bohart and Adams, 1920):
C
C
0
exp k
AB
C
0
t k
AB
N
0
Z
U
0

16
Thomas model (Thomas, 1944):
C
0
C
1 exp
k
TH
F
Q
0
M C
0
V
eff

17
YoonNelson model (Yoon and Nelson, 1984):
C
C
0
exp k
YN
t sk
YN

1 exp k
YN
t sk
YN

18
Modified doseresponse model (Yan et al., 1999):
C
C
0
1
1
1
V
eff
b
mdr

a
mdr
19
Clark model (Clark, 1987):
C
C
0
1
1
C
n1
0
C
n1
break
1

e
rt
break
e
rt
1=n1
20
where k
AB
is the BohartAdams rate constant (ml/min mg); N
0
the
bed saturation capacity (mg/l); U
0
the superficial velocity (cm/
min); k
TH
the Thomas model rate constant (ml/min mg); Q
0
the
maximum solid-phase concentration of the solute (mg/g); V
eff
the
volume of solute passed into the column (l); k
YN
the YoonNelson
model rate constant (1/min); the time required for 50% sorbate
breakthrough (min); a
mdr
and b
mdr
the modified doseresponse
model constants; n the Freundlich constant; r the adsorption rate
(mg/l min); and C
break
and t
break
the breakthrough concentration
(mg/l) and time (min), respectively.
The successful design of a column sorption process requires
the concentration-time profile or effluent breakthrough curve to
be predicted (Volesky and Prasetyo, 1994); the maximum sorp-
tion capacity of a sorbent is also required in the design. Both the
BohartAdams and Thomas models have been found to fulfill
this purpose and thus widely applied to many investigations.
Texier et al. (2002) successfully employed the BohartAdams
model to determine the characteristic parameters during the
biosorption of lanthanide ions onto Pseudomonas aeruginosa.
Addour et al. (1999) used a linearized Thomas equation to
describe the biosorption of zinc by Streptomyces rimosus bio-
mass, and successfully calculated the maximum biosorption
capacity. Despite its wide usage, the Thomas model suffers a
major disadvantage i.e., it has a fixed value when the experi-
mental time is zero, which will not be the case in reality. Yan
et al. (1999) highlighted this aspect and proposed a new model
(modified-dose response), which minimized the error resulting
from the use of the Thomas model.
Apart these models, several investigators have used mass
transfer and mechanistic models to describe column biosorption
(Kratochvil and Volesky, 2000; Hatzikioseyian et al., 2001;
Zulfadhly et al., 2001; Naja and Volesky, 2006a). Since the
mechanism responsible for dye/metal biosorption onto biologi-
cal materials is different from that of activated carbon, it is not
appropriate to employ conventional column adsorption models
with these types of biosorption data (Naja and Volesky, 2006a).
However, careful understanding of the mechanism and its
incorporation into the model might be an appropriate approach
towards column modeling.
10. Multicomponent systems
Considerable amount of information is available on the bio-
sorption of single-component systems, but many industries dis-
charge effluents that contain several components. Correspondingly,
knowledge of how one solute may influence the uptake of another
is desirable. Many studies have analyzed biosorbents using pure
metal/dye solutions, with their use recommended for real effluents
which is usually misleading as the behavior of a biosorbent tends to
differ in multicomponent systems. In these systems, the biosorption
of the solute of interest not only depends on the biomass surface
properties and physicalchemical parameters of a solution such as
pH and temperature, but also on the number of solutes and their
concentrations. In such cases, the biosorption will become com-
petitive, with one solute competing with another to occupy the
binding sites.
Multicomponent biosorption has been the subject of limited
studies (Kratochvil and Volesky, 2000; Sa et al., 2000, 2001;
Pagnanelli et al., 2002; Borrok and Fein, 2004; Borrok et al.,
2004; Naja and Volesky, 2006b; Park et al., 2006). The extent of
competition and nature of the mechanism in multicomponent
systems should be clearly understood. However, with the
exception of a few cases, the mechanism and competition effect
have been inadequately understood. Biosorption depends on the
size and relative concentration of each solute, which usually play
vital roles in deciding the nature of competition between solutes.
Maurya et al. (2006) reported that during the biosorption of
Methylene blue and Rhodamine B, the ionic radii played a vital
role and; owing to its greater ionic radius, Methylene blue was
biosorbed to a greater extent than Rhodamine B. However, some
studies revealed that the ionic radii cannot be used as sole
deciding factor when interpreting high solute biosorption during
multicomponent systems. For instance, Texier et al. (1999) ob-
served no correlation with the ionic radius, as the smaller sized
ytterbium ions were found to be significantly affected in the pre-
sence of the larger lanthanum ions. Metal biosorption by bacteria
can also be influenced by metal speciation in the aqueous phase as
well as by the surface properties, such as charge and orientation of
the functional groups on the cell surface. Premuzic et al. (1991)
showed that in addition to metal selectivity, there was also a
species-dependent differentiation in the uptake capacity. These
authors also found significant differences in the uptake of uranium
and thorium by two strains of P. aeruginosa.
Comparatively, multi-metal biosorption has been more ex-
tensively studied than multi-dye systems. The reason for this is
unclear, but it is our belief that it is not associated with the analysis
of multi-dye solutions. The spectrophotometric procedure for
determining the concentration of each dye in a binary system is
well established, and has been successfully employed in some
biosorption studies; however, its validity strongly depends on the
solution chemistry of each dye (Al-Duri and McKay, 1991).
The prediction of multicomponent biosorption data has always
been complicated due to the interactive and competitive effects
involved. Consequently, the appropriate selection of a model,
where the competitive mechanismis taken into account is crucial.
However, an analysis of the literature revealed that single-compo-
nent isotherm data have frequently been used to predict multi-
component data. The extended Langmuir equation is one such
model, which has been widely employed for multicomponent
adsorption data (Sa et al., 1998; Choy et al., 2000). This model
assumes a constant energy of sorption, no interaction between
components and equal competition between species for the sorp-
tion sites (Choy et al., 2000). These assumptions are not valid
under real conditions, since sorbate interactions tend to occur in
multicomponent systems. To incorporate sorbatesorbate inter-
action and competition, an interaction factor () has been intro-
duced in an extended Langmuir equation (McKay and Al-Duri,
1987), which for binary mixtures has the following forms:
Q
1

Q
max1
b
1
C
1
=g
1

1 b
1
C
1
=g
1
b
2
C
2
=g
2

21
Q
2

Q
max2
b
2
C
2
=g
2

1 b
1
C
1
=g
1
b
2
C
2
=g
2

22
These equations have been used with some success (Choy
et al., 2000; Aksu and Isoglu, 2007); however, the use of single-
component data to describe a multicomponent isotherm often
limits its applicability. Similarly, Sheindrof et al. (1981)
proposed a multicomponent Freundlich type equation, which
included a competitive coefficient (
ij
), based on the assumption
that there is an exponential distribution of adsorption energies
available for each solute. This can be represented as follows:
Q
j
i
K
F
i
C
f
i
C
f
i
h
ij
C
f
j

1=n
i
1
23
where (Q)
i
j
is the amount of solute, i, sorbed per unit weight of
sorbent in the presence of solute j, K
Fi
the single-component
Freundlich constant for solute i and n
i
the Freundlich exponent
for solute i. Eq. (23) also involves single-component equilibrium
data and thus, often fail to accurately calculate experimental
mixture equilibria. In addition to the two equations above, several
equations; for example, those of Fritz and Schluender (1974),
Crittenden and Weber (1978) and Khan et al. (1996, 1997a), with
additional adjustable parameters have been proposed. However,
as the number of solutes in a mixture increases, the number of
additional parameters to be determined from the experimental
mixture equilibria becomes higher, where the use of these equa-
tions becomes impractical.
The Ideal Adsorbed Solution (IAS) theory has been successfully
used to characterize the competitive adsorption for activated
carbons. Myers and Prausnitz (1965) presented the IAS theory for
gaseous phase multicomponent adsorption. Radke and Prausnitz
(1972b) later extended the IAS theory to dilute liquid solutions.
Suzuki and Misic (1973) applied the Freundlich isotherm to the
IAS theory, and developed easy-to-use equations for the estimation
of multicomponent equilibria. Finally, Furuya et al. (1986) exten-
ded Suzuki's model, and developed a technique to determine the
isotherm parameters from multicomponent aqueous samples.
Apart from being widely used for gas-phase sorption of
hydrocarbons (Li and Orhan, 1994), the IAS theory has become
the focus to study the sorption of dyes/metals onto adsorbents
(Ko et al., 2004; Choy et al., 2004). The major advantages of the
IAS theory are that it has a sound thermodynamic basis and
requires only single-component isotherm parameters to predict
the sorption equilibrium for a multicomponent system. The IAS
theory is based on the assumption that the adsorbed phase can be
treated as an ideal solution to that of the adsorbed components.
The theory has proved successful in several cases; for instance,
Choy et al. (2004) tested four isotherms using the IAS theory,
namely the Langmuir, Freundlich, RedlichPeterson and Sips
isotherms. Of the isotherms examined, the authors identified the
IAST-RedlichPeterson model provides the best prediction of
the binary data for the sorption of acidic dyes onto activated
carbon. However, the IAS theory requires time-consuming
graphical solutions or extensive computational methods.
Few investigators have attempted to perform mechanistic
modeling of multicomponent biosorption data. Chong and
Volesky (1995) proposed a mechanistic model that considered
not only a Langmuir type interaction, but also the simultaneous
presence of ion exchange reactions, which took into account the
possible effect of the reverse reactions of the displaced ions
actually present at the beginning on the active sites. Fowle and
Fein (1999) proposed a mechanistic model for both binary and
ternary metal biosorption onto two Gram-positive bacteria. This
model considered the metal speciation, and hypothesized a set
of reactions, combining constants and site balances.
As a concluding remark, it should be highlighted that very
less work has been performed on the study of the competition
effects during biosorption especially in the case of dyes. The
proper understanding and evaluation of competition mechan-
isms are mandatory for the successful application of biosorption
to real effluents.
11. Application of biosorption to real industrial effluents
As highlighted in the previous sections, biosorption is a proven
technique potentially for the removal of metals/dyes fromaqueous
solutions. However, its performance under real industrial condi-
tions is of concern. There have been fewinvestigations examining
the compatibility of the biosorbent for real industrial effluents.
Kratochvil and Volesky (1998) explained the necessity of
extended testing of a biosorption process before its commercial
application. In general, industrial effluents can be classified into
two broad categories: those bearing low contaminant concentra-
tions in large volumes, i.e. mining wastewaters, and those charac-
terized by high TDS values in small volumes, i.e. electroplating
and textile dye bath effluents. For the first case, a biosorbent with
strong affinity towards the contaminant of interest is mandatory;
whereas, the latter case requires a biosorbent with high uptake
capacity (Atkinson et al., 1998). However, irrespective of the
effluent, interference by counter ions, such as light metal ions,
should be considered. Vijayaraghavan et al. (2006c) characterized
two effluents originating from nickel electroplating industries,
which contained high amounts of light metal ions and anions,
coupled with high nickel concentrations. They observed strong
interference from co-ions during the removal of nickel(II) in both
batch and column modes of operation. The presence of anions can
lead to the following: 1) Formation of complexes with higher
affinity for the sorbent than the free metal ions (i.e., an enhance-
ment of sorption); 2) Formation of complexes with lower affinity
for the sorbent than free metal ions (i.e., a reduction of sorption)
(Volesky and Schiewer, 1999). There may be instances, owing to
the occurrence of competitive ion exchange in the column, where
one or more of the contaminants present at trace levels may exceed
the acceptable column effluent limit well before the targeted
contaminant, thereby reducing the column service time (Kratoch-
vil and Volesky, 1998).
Since mixtures are comprised of organic as well as inorganic
constituents, it is often mandatory to study the solution chemistry
of the effluents for the proper implementation of biosorption
technology. If necessary, an appropriate effluent pretreatment
should be performed prior to applying the biosorption process. It
has been reported that extreme characteristics (pH, conductivity
and total hardness) may affect the binding abilities of a biosorbent
(Vijayaraghavan et al., 2006c). As indicated previously, there
have been very few reports on employing biosorbents for real
effluents; of which Tobin and Roux (1998) used Mucor meihi
biomass to remove chromium from tanning industry effluents.
The authors thoroughly characterized the effluent and success-
fully removed chromium from the effluent; with performances
corresponding closely to those of commercial ion exchange res-
ins. In another study, Aravindhan et al. (2004) used Turbinaria
spp. to treat a tanning industry effluent comprising of 750 mg/l
chromium. As the Cr(III) concentration was high, the authors
employed a five stage biosorption process, which successfully
reduced the Cr(III) concentration to 2 mg/l.
Considering the number of biosorbents proposed for metal/
dye removal, it would be expected that more attempts be made to
commercialize biosorption in the fields of wastewater treatment.
However, there have only been few instances where biosorption
processes have managed to reach commercialization. One such
example involved the process developed by AMT-Bioclaim,
which comprises of Bacillus subtilis, treated using strong caustic
solution, washed with water, and immobilized as porous balls
onto polyethyleneimine and glutaraldehyde (Brierley, 1990).
This commercial biosorbent is capable of accumulating gold,
cadmium and zinc from cyanide solutions and; therefore, would
be suitable for many metal-finishing operations (Atkinson et al.,
1998). The biosorbent BIO-FIX is made up of a variety of
biomasses, including bacteria, fungi, sphagnum peat moss and
algae, immobilized onto high density polysulfone. This
biosorbent has been found to be selective for toxic heavy metals
over that of alkaline earth metals. The biosorbent AlgaSORB
consists of C. vulgaris and other non-living algae immobilized
in a silica gel matrix (Brierley, 1990). The material has shown
remarkable heavy-metal ion affinities; for instance when applied
at US nuclear sites to clean up ground water contaminated with
uranium and mercury, at concentrations of 200 and 30 g/l,
respectively, it exhibited process efficiencies of about 95% for
both metals (Eccles, 1995). However, other attempts have failed
to obtain successful commercial application in the market
(Tsezos, 2001). To the best of our knowledge, no biosorbent
capable of accumulating dye has reached the commercial level.
Atkinson et al. (1998) highlighted questions that should be
considered relating to the feasibility of a potential biosorbent for the
removal of metals/dyes from industrial effluents. These includes
the effluent characteristics, such as volume, type of contaminant
and competitive ions, solution chemistry, pH and temperature
adjustment; biomass characteristics, such as availability, mechan-
ical stability, regeneration ability, contaminant specificity and
reaction kinetics; and process characteristics, such as capital and
operating costs, batch/continuous and land space requirements.
The design and type of process to be employed (batch/con-
tinuous) is entirely dictated by the choice of biomass and its
method of immobilization. If it is feasible to operate a biomass in a
batch contactor configuration, the initial capital expenditure for
the process development and setup can be estimated as being
similar to that of chemical precipitation methods. Both systems
require the same basic equipments, such as a contact vessel, some
mode of agitation, piping and other peripheral equipment, includ-
ing pH probes and level controllers (Atkinson et al., 1998). If
proper and cheap immobilization techniques are available, a
biosorbent can be used in a packed or fluidized column mode of
operation. The choice can be made on whether to use an up-flow
or down-flow packed column, but the latter is the most cost
effective to operate; however, there is no control over the effluent
retention times, which may affect the biosorbent capacity. The
waste stream may also allow for passing through the columns/
reactors in series, where more effective results will be obtained.
However, care must be taken that the automation and complexity
of a treatment facility may substantially increase the costs.
Other important aspects requiring consideration are the cost
and availability of the biomass. Even if the biomass can be
acquired free of charge, the processing and transportation costs
should be considered. Almost all biomasses require drying and
chemical pretreatment, at least with acid or alkali pretreatment,
for their effective performance. As with any industrial process,
the nearer the source of raw material to the point of application,
the more feasible will be the process. Tsezos (2001) clearly
pointed out that successful biosorption technology not only
depends on the biosorption potential, but also on the continuous
supply of biomass for the process. The source for raw material
must be enthusiastic to secure a steady supply of waste biomass.
12. Fate of exhausted biosorbent
One of the more common questions aroused by biosorption
processes involves the fate of the biosorbent after the process.
Also, the fate of the concentrated metal/dye solutions obtained
after the elution process remains relatively unanswered. Care
must be taken that solving one problem should not create
another. Since the ultimate purpose of a biosorption process is to
concentrate a solute, very high concentrations, in the order of 10
times higher than that of the initial solute, can commonly be
expected by the end of elution process (Vijayaraghavan et al.,
2006c). The recovery of a solute from these high concentrated
solutions can be accomplished using another process, such as
precipitation or electrowinning. Volesky and Schiewer (1999)
suggested it is often feasible to use electrowinning procedures to
recover metals from concentrated solutions. Binupriya et al.
(2007b) desorbed Reactive blue MR from dye-loaded Trametes
versicolor using ethanol and; thereby suggested that Reactive
blue MR-rich ethanol medium can be distilled to remove dyes
and the recovered dye can be used as low-grade dyes in colored
glass, plastic and ceramic industries.
Even if the biosorbent can be efficiently reused over several
cycles, the final disposal of the material should be addressed.
The common answer to the disposal of the final material is via
landfill or incineration. However, due to the increasing levels of
landfill tax, and the potential restrictions due to contamination
of ground waters, the landfill option has become less attractive.
Alternatively, when recycling is not considered worthwhile,
biomass combustion would yield ash with a high concentration
of the desired metal (Volesky, 1987). 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 pro-
cessing 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 (Dambies et al., 2000). In another instance, Cibacron
blue F3GA-attached polyvinylbutyral microbeads have been
shown to be effective in the removal of Cu(II), Cd(II) and Pb(II)
ions (Denizli et al., 1998).
However, one should understand that waste microbial bio-
masses 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 re-
mediation of metal/dye polluted effluents, should be regarded as
having served its purpose.
13. 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 technol-
ogy 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 immo-
bilization 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.
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 mechanismresponsible 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.
Acknowledgements
This work was supported by a grant from the Post-Doc
program, Chonbuk National University (the second half term of
2006) and, in part, by KOSEF through AEBRC at POSTECH.
The authors would like to express sincere thanks to all researchers
of Environmental Biotechnology Laboratory (Chonbuk National
University), for their help. Finally, the authors would like to thank
one anonymous journal reviewer who provided constructive
comments in the improvement of this manuscript.
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