Article

Biosorbents for Heavy Metal Removal and Their Future

Laboratory of Environmental Technology, INET, Tsinghua University, Beijing 100084, PR China.
Biotechnology advances (Impact Factor: 9.02). 03/2009; 27(2):195-226. DOI: 10.1016/j.biotechadv.2008.11.002
Source: PubMed
ABSTRACT
A vast array of biological materials, especially bacteria, algae, yeasts and fungi have received increasing attention for heavy metal removal and recovery due to their good performance, low cost and large available quantities. The biosorbent, unlike mono functional ion exchange resins, contains variety of functional sites including carboxyl, imidazole, sulphydryl, amino, phosphate, sulfate, thioether, phenol, carbonyl, amide and hydroxyl moieties. Biosorbents are cheaper, more effective alternatives for the removal of metallic elements, especially heavy metals from aqueous solution. In this paper, based on the literatures and our research results, the biosorbents widely used for heavy metal removal were reviewed, mainly focusing on their cellular structure, biosorption performance, their pretreatment, modification, regeneration/reuse, modeling of biosorption (isotherm and kinetic models), the development of novel biosorbents, their evaluation, potential application and future. The pretreatment and modification of biosorbents aiming to improve their sorption capacity was introduced and evaluated. Molecular biotechnology is a potent tool to elucidate the mechanisms at molecular level, and to construct engineered organisms with higher biosorption capacity and selectivity for the objective metal ions. The potential application of biosorption and biosorbents was discussed. Although the biosorption application is facing the great challenge, there are two trends for the development of the biosorption process for metal removal. One trend is to use hybrid technology for pollutants removal, especially using living cells. Another trend is to develop the commercial biosorbents using immobilization technology, and to improve the biosorption process including regeneration/reuse, making the biosorbents just like a kind of ion exchange resin, as well as to exploit the market with great endeavor.

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Available from: Jianlong Wang
Research review paper
Biosorbents for heavy metals removal and their future
Jianlong Wang
, Can Chen
Laboratory of Environmental Technology, INET, Tsinghua University, Beijing 100084, PR China
abstractarticle info
Article history:
Received 8 October 2008
Received in revised form 18 November 2008
Accepted 21 November 2008
Available online 6 December 2008
Keywords:
Biosorbent
Biosorption
Heavy metal ions
Bacteria
Fungi
Algae
Biomass
Kinetics
Immobilization
Application
A vast array of biological materials, especially bacteria, algae, yeasts and fungi have received increasing
attention for heavy metal removal and recovery due to their good performance, low cost and large available
quantities. The biosorbent, unlike mono functional ion exchange resins, contains variety of functional sites
including carboxyl, imidazole, sulphydryl, amino, phosphate, sulfate, thioether, phenol, carbonyl, amide and
hydroxyl moieties. Biosorbents are cheaper, more effective alternatives for the removal of metallic elements,
especially heavy metals from aqueous solution. In this paper, based on the literatures and our research
results, the biosorbents widely used for heavy metal removal were reviewed, mainly focusing on their
cellular structure, biosorption performance, their pretreatment, modication, regeneration/reuse, modeling
of biosorption (isotherm and kinetic models), the development of novel biosorbents, their evaluation,
potential application and future. The pretreatment and modication of biosorbents aiming to improve their
sorption capacity was introduced and evaluated. Molecular biotechnology is a potent tool to elucidate the
mechanisms at molecular level, and to construct engineered organisms with higher biosorption capacity and
selectivity for the objective metal ions. The potential application of biosorption and biosorbents was
discussed. Although the biosorption application is facing the great challenge, there are two trends for the
development of the biosorption process for metal removal. One trend is to use hybrid technology for
pollutants removal, especially using living cells. Another trend is to develop the commercial biosorbents
using immobilization technology, and to improve the biosorption process including regeneration/reuse,
making the biosorbents just like a kind of ion exchange resin, as well as to exploit the market with great
endeavor.
© 2008 Elsevier Inc. All rights reserved.
Contents
1. Introduction .............................................................. 196
2. Cell structure: prokaryotes and eukaryotes................................................ 197
2.1. Bacterial structure ........................................................ 198
2.1.1. Shape and size ..................................................... 198
2.1.2. Cell structure ...................................................... 198
2.2. Fungal structure......................................................... 201
2.2.1. Classication and general characteristics ......................................... 201
2.2.2. Cell wall and its main composite polysaccharide ..................................... 202
2.2.3. Cell membrane ..................................................... 203
2.2.4. Cytoplasm ....................................................... 203
2.3. Algae structure ......................................................... 203
2.3.1. Introduction and its classication ............................................ 203
2.3.2. Cell wall of algae .................................................... 204
2.4. Functional groups related to the biosorption ........................................... 205
3. Bacterial biosorbents .......................................................... 206
4. Fungal biosorbents ........................................................... 207
4.1. Introduction........................................................... 207
4.2. Yeast .............................................................. 207
4.3. Filamentous fungi ........................................................ 208
Biotechnology Advances 27 (2009) 195226
Corresponding author. Tel.: +86 10 62784843; fax: +86 10 62771150.
E-mail address: wangjl@tsinghua.edu.cn (J. Wang).
0734-9750/$ see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2008.11.002
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Page 1
4.3.1. Penicillium ....................................................... 208
4.3.2. Aspergillus ....................................................... 210
4.3.3. Other fungi ....................................................... 211
4.4. Selectivity and competitive biosorption by fungi .......................................... 211
4.5. Comparison of fungi and yeast with other biosorbents ....................................... 211
5. Marine algae as biosorbents ....................................................... 212
5.1. Introduction ........................................................... 212
5.2. Performance........................................................... 212
5.3. Comparison of algae with other biosorbents ............................................ 213
6. Effect of pre-treatment on biosorption .................................................. 214
7. Biosorbentimmobilizationforbioreactorsandregeneration/reuse...................................... 215
8. Modeling of biosorption: isotherm and kinetic models .......................................... 216
8.1. Equilibrium modeling of biosorption ............................................... 216
8.2. Kinetic modeling of biosorption in a batch system ......................................... 217
9. Biosorbent selection and assessment ................................................... 217
10. Development of novel biosorbents .................................................... 218
11. Application of biosorption ........................................................ 219
11.1. Several attempts of the biosorption commercialization ...................................... 219
11.2. Application feasibility and consideration............................................. 220
12. The future of biosorption ........................................................ 221
Acknowledgements .............................................................. 222
References .................................................................. 222
1. Introduction
Biosorption can be de ned as the removal of metal or metalloid
species, compounds and particulates from solution by biological
material (Gadd, 1993). Large quantities of metals can be accumulated
by a variety of processes dependent and independent on metabolism.
Both living and dead biomass as well as cellular products such as
polysaccharides can be used for metal removal.
Heavy metal pollution is one of the most important environmental
problems today. Various industries produce and discharge wastes
containing different heavy metals into the environment, such as
mining and smelting of metalliferous, surface nishing industry,
energy and fuel production, fertilizer and pesticide industry and
application, metallurgy, iron and steel, electroplating, electrolysis,
electro-osmosis, leatherworkin g, photography, electric applia nce
manufacturing, metal surface treating, aerospace and atomic energy
installation etc. Thus, metal as a kind of resource is becoming shortage
and also brings about serious environmental pollution, threatening
human health and ecosystem. Three kinds of heavy metals are of
concern, including toxic metals (such as Hg, Cr, Pb, Zn, Cu, Ni, Cd, As,
Co, Sn, etc.), precious metals (such as Pd, Pt, Ag, Au, Ru etc.) and
radionuclides (such as U, Th, Ra, Am, etc.) (Wang and Chen, 2006).
Methods for removing metal ions from aqueous solution mainly
consist of physical, chemical and biological technologies. Conventional
methods for removing metal ions from aqueous solution have been
suggested, such as chemical precipitation, ltration, ion exchange,
electrochemical treatment, membrane technologies, adsorption on
activated carbon, evaporation etc. However, chemical precipitation
and electrochemical treatment are ineffective, especially when metal
ion concentration in aqueous solution is among 1 to 100 mg L
1
, and
also produce large quantity of sludge required to treat with great
difculty. Ion exchange, membrane technologies and activated carbon
adsorption process are extremely expensive when treating large
amount of water and wastewater containing heavy metal in low
concentration, they cannot be used at large scale. Volesky (2001)
summarized the advantages and disadvantages of those conventional
metal removal technologies.
In recent years, applying biotechnology in controlling and
removing metal pollu tion has been paid much a ttention, and
gradually becomes hot topic in the eld of metal pollution control
because of its potential application. Alternative process is biosorption,
which utilizes various certain natural materials of biological origin,
including bacteria, fungi, yeast, algae, etc. These biosorbents possess
metal -sequestering property and can be used to dec rease the
concentration of heavy metal ions in solution from ppm to ppb
level. It can effectively sequester dissolved metal ions out of dilute
complex solutions with high efciency and quickly, therefore it is an
ideal candidate for the treatment of high volume and low concentra-
tion complex wastewaters (Wang and Chen, 2006).
The capability of some living microorganisms to accumulate metallic
elements have been observed at rst from toxicological point of view
(Volesky, 1990a,b,c). However, further researches have revealed that
inactive/dead microbial biomass can passively bind metal ions via
various physicochemical mechanisms. Therefore researches on biosorp-
tion have become an active eld for the removal of metal ions or organic
compounds. Biosorbent behavior for metallic ions is a function of the
chemical make-up of the microbial cells of which it consists (Volesky
and Holan, 1995). Mechanisms responsible for biosorption, although
understood to a limited extent, may be one or combination of ion
exchange, complexation, coordination, adsorption, electrostatic inter-
action, chelation and microprecipitation (Veglio and Beolchini, 1997;
Vijayaraghavan and Yun, 2008; Wang and Chen, 2006).
A large quantity of materials has been investigated as biosorbents
for the removal of metals or organics extensive ly. The tested
biosorbents can be basically classied into the following categories:
bacteria (e.g. Bacillus subtillis), fungi (e.g. Rhizopus arrhizus), yeast
(e.g., Saccharomyces cerevisiae), algae, industrial wastes (e.g., S.
cerevisiae waste biomass from fermentation and food industry),
agricultural wastes (e.g. corn core) and other polysaccharide materi-
als, etc. (Vijayaraghavan and Yun, 2008). The role of some groups of
microorganisms has been well reviewed, such as bacteria, fungal,
yeast, algae, etc.
These tested biomasses have been reported to bind a variety of
heavy metals to different extents (Gupta et al., 2000). Some potential
biomaterials with high metal binding capacity have been identied in
part. Some types of biosorbents binding and collecting the majority of
heavy metals with no specic priority, while others can even be
specic for certain types of metals (Volesky and Holan, 1995).
The biosorbent materials among easily available include three
groups: algae, fungi, and bacteria, the former two perhaps giving
broader choices. Waste materials or by-product biomass from large-
scale fermentation processes are the source of new family of
biosorbents conveniently. In particular, some waste mycelia are
available in large quantities for the removal of heavy metals (Kapoor
196 J.L. Wang, C. Chen / Biotechnology Advances 27 (2009) 195226
Page 2
and Viraraghavan, 1995; Wang and Chen, 2006). Seaweeds from the
oceans produced in copious quantities are another inexpensive source
of biomass. Marine algae, especially brown algae such Sargasso
seaweed was investigated for metal removal (Davis et al., 2003c).
Abundant natural materials, particularly cellulosic nature, have been
suggested as potential biosorbents for the removal of heavy metals.
For economical reasons, other low-cost biosorbents are of interest
recently, such as agricultural wastes (Bailey et al., 1999).
The rst major challenge for the biosorption eld was to select the
most promising types of biomass from an extremely large pool of readily
av ailable and inexpensive biomaterials (Kratochvil and Volesky, 1998).
Although many biological materials can bind heavy metals, only those
with sufciently high metal-binding capacity and selectivity for heavy
metals are suitable for use in a full-scale biosorption process. A large
number of biomass types have been investigated for their metal binding
capability under various conditions. Volesky and Holan (1995) have
presented an exhaustive list of microbes and their metal-binding
capacities. The published work on testing and evaluating the perfor-
mance of biosorbents offered a good basis for looking for new and
potentially feasible metal biosorbents. Another challenge is that the
application of biosorption is facing up with great difculty (Tsezos,
2001). Great efforts have to be made to improve biosorption process,
including immobilization of biomaterials, improvement of regeneration
and re-use, optimization of biosorption process etc.
In recent 10 years, our lab has attempted to carry out the relevant
researches on biosorption phenomena, especially for the removal of
metal ions (Chen and Wang, 2007a,b,c,d,e, 2008a,b,c; Liu et al., 2002;
Wang et al., 2000, 2001). In this review, an extensive list of biosorbent
literature including our research results has been compiled to provide
a summary of available information on a wide range of biosorbents for
metal removal. The cell structure was introduced rst. Then biosorp-
tion performances of various biosorbents, including bacteria, lamen-
tous fungi, and marine algae were summarized. Because the different
criteria were used by the various authors in searching for suitable
material, the results were reported in different units and in different
ways, which often make quantitative comparison impossible. As for
the biosorbents, it can be easily available biomass, or specially isolated
microorganisms, or modied raw biomass to improve its biosorption
application properties. It should be noted that comparing results from
different sources involve in standardizing the different ways that the
sorption capacity may be expressed.
The aim of this work is to present the state of the art of biosorbent
investigation and to compare results found in the literature. The
pretreatment, immobilization, and regeneration/reuse of biosorbents,
modeling of biosorption process, biosorbent assessment, as well as the
development of novel biosorbents were presented and discussed,
their potential application and future were predicted.
2. Cell structure: prokaryotes and eukaryotes
A variety of reviews and books of microbiology were devoted to the
microbial structure and function (Baron, 1996; Madigan et al., 2000;
Moat et al., 2002; Prescott et al., 20 02; Remacle, 1990; Talaro and
Talaro, 2002; Tortora et al., 20 04; Urrutia, 1997). Here we only simply
introduce the basic structure necessary for understanding the
mechanisms of biosorption.
Microbial cells have two fundamentally different types of cells
procaryotic and eukaryoticand are distributed among several king-
doms or domains. Procaryotic cells have a much simpler and smaller
structure than eukaryotic cells and lack a true membrane-delimited
nucleus. It generally lacks extensive, complex, internal membrane
systems although with a plasma membrane. In contrast, eukaryotic
cell have a membrane-enclosed nucleus and many membranous
organelles. They are more complex morphologically and are usually
larger than procaryotes. Algae, fungi, protozoa, higher plants, and
animals are eukaryotic (Prescott et al., 20 02). Prokaryotes are
represented by bacteria and archaea. Most bacteria can be divided
into Gram-positive and Gram-negative groups based on their cell wall
structure and response to the Gram staining. Most bacteria and yeast
are unicellular. Typical bacterial cells range in diameter from 0.5 to
1.0 μm,
some wider than 50 μm.
Typical eukaryotic cells may be 2 μm
to more than 20 0 μm in diameter. Apart from the above-mentioned
differences, procaryotes are simpler functionally in several ways than
eukaryotic cells. Eukaryotic cells have mitosis and meiosis, and many
complex eukaryotic processes which are absent in procaryotes:
phagocytosis and pinocytosis, intracellular digestion, directed cyto-
plasmic streaming, ameboid movement, and others. The plasma
membrane in prokaryotes performs most functions carried out by
membranous organelles in eukaryotes. Despite the profound struc-
tural and functional differences between prokaryotes and eucaryotes,
both cells are similar on the biochemical level. A typical cell of
prokaryotes or eukaryotes includes four major components: cell wall,
cell membrane, cytoplasm, and nuclear area. Cell wall is a rigid outer
layer of the cell membrane, which provides support and protection
from osmotic lysis. The chemical composition of the cell wall differs
from group to another cell. All fungi, and most bacteria and algae have
cell walls. The cell membrane, or plasma membrane, or cytoplasmic
membrane, is the critical permeability barrier, with a lipid and protein
layer surrounding cytoplasm. It is the boundary between the cell and
its environment when lacking cell walls. The membrane is the chief
point of contact with the cell's environment and thus is responsible for
communication with the outside world. The exact proportions of
protein and lipid in the cell membrane vary widely in different group
of microorganisms. Eukaryotic plasma membranes usually have a
lower proportion of protein than bacterial membranes. Cell mem-
branes are about 5 to 10 nm thickness, and can be only viewed under
electron microscope. Lipids in membrane are structurally asymmetric
with polar ends (hydrophilic) and nonpolar ends (hydrophobic),
usually these asymmetri c lipids are phospholipids. One major
difference in chemical composition of membrane between eukaryotic
and prokaryotic cells is that bacterial membranes, unlike eukaryotic
membranes, lack sterols such as cholesterol. Sterols can make up from
5 to 20% of the total lipids of eukaryotic membranes. Sterols are rigid,
planar molecules, whereas fatty acids are exible serving to stabilized
its structure and make it less exible. However, bacterial membranes
contain pentacyclic sterol-like molecules called hopanoids. A most
widely accepted model for membrane structure is the uid mosaic
model, proposed by S. Jonathan Singer and Garth Nicholson. There are
two types of membrane proteins: peripheral proteins and integral
proteins. The former are loosely connected to the membrane and can
be easily removed, they are soluble in aqueous solutions and make up
about 20 to 30% of total membrane protein. The later compose about
70 to 80% of membrane proteins. They are not easily extracted from
membranes and are insoluble in aqueous solutions when free of lipids.
The integral proteins are also asymmetric (Prescott et al., 2002).
Cytoplasm, aqueous uid of the cell, contains organelles, enzymes,
chemicals, in which most cellular metabolic activities occur, e.g.
ribosomes. Bacteria do not contain internal membrane-bound
organelles, their interior appears morphologically simple. Ribosomes
are small particles composed of protein and ribonucleic acid (RNA).
Ribosomes are part of the translation apparatus, and the synthesis of
cell proteins takes place on these structures. Procaryotic cel ls
occasionally cont ain inclusions consisting of storage m aterials,
compounds made up of carbon, nitrogen, sulfur, or phosphorus,
formed when these nutrients are in excess. Algae have an additional
type of organelle: chloroplast. Eukaryotic cells differ most obviously
from procaryotic cells, they have a variety of complex membranous
organelles in the cytoplasmic matrix and the majority of their genetic
materials are within membrane-delimited nuclei. Each organelle has a
distinctive structure directly related to specic functions. Nuclear area
includes hereditary materials, deoxyribonucleic acid (DNA). For most
of cells, but not bacteria, DNA existed within nuclear area. In bacteria,
197J.L. Wang, C. Chen / Biotechnology Advances 27 (2009) 195226
Page 3
the genetic materials are localized in a discrete region, the nucleoid,
and are not separated from the surrounding cytoplasm by mem-
branes. Prokaryotic cells do not possess true nucleus, the function of
the nucleus is performed by a single molecule of DNA. The key
difference between eukaryotic and prokaryotic cells is that eukaryotes
contain true nuclei.
In this review, we will mainly discuss three groups of biomass
materials related to metal biosorpion: bacteria (Gram-positive and
Gram-negative cells), fungi (lamentous fungi and yeast) and algae. The
interface between the microbial cells and its externalenvironment is cell
surface. The structure and composition of different cell surfaces can vary
considerably, depending on the organism. Due to the importance of the
cell surface, especially the cell wall for metal biosorption, the cell surface
structures of three groups of microorganisms will be described in detail
in term of their metal biosorption performance.
2.1. Bacterial structure
2.1.1. Shape and size
Bacteria have simple morphology. The most commonly bacteria
present in three basic shapes: spherical or ovoid (coccus), rod (bacillus,
with a cylindrical shape), and spiral (spirillum), although there are a
great variety of shapes due to differences in genetics and ecology.
Bacteria vary in size as much as in shape. For many prokaryotes, the cells
remain together in groups or clusters after division (pairs, chains,
tetrads, clusters, etc.). Cocci or rods may occur in long chains. The gram-
negative organism, Escherichia coli often as typical size of bacteria cell, is
about 1.1 to 1.5 μmwideby2.0to6.0μm long. The smallest bacteria are
about 0.3 μm, and a few bacteria become fairly large, e.g. some
spirochetes occasionally reach 500 μm in length, and cyanobacterium
Oscillatoria is about 7 μm in diameter. Cell size is an important
characteristic for an organism. Small size of bacteria is very important
because size affects a number of cell biological properties. Small size of
bacteria ensures rapid metabolic processes.
2.1.2. Cell structure
A typical bacterial cell (e.g., E. coli), contains cell wall, cell
membrane, cytoplasmic matrix consisting of several constituents,
which are not membrane-enclosed: inclusion bodies, ribosomes, and
the nucleoid with its genetic material. Some bacteria have special
structure, such as agella, S-layer.
2.1.2.1. Cell wall and Gram-negative cell envelope. Main function of
cell wall include: (1) The cell wall gives cell shape and protect it from
osmotic lysis; (2) The wall can protects cell from toxic substances (3)
The cell wall offers the site of action for several antibiotics. (4) The cell
wall is necessary for normal cell division.
The major classes of chemical constituents in the walls and
envelopes of Gram-positive and Gram-negative bacteria are summar-
ized by Salton and Kwan (http://gsbs.utmb.edu/microbook/ch002.
htm), shown in Table 1.
By Gram staining technique, the Gram-positive bacteria stained
purple, whereas Gram-negative bacteria were colored pink or red. The
surface of Gram-negative cells is much more complex chemically and
structurally than that of Gram-positive cells. Because of the thicker
peptidoglycan layer, the walls of Gram-positive cells are stronger than
those of Gram-negative bacteria.
Cellular wall shape and strength is primarily due to peptidoglycan,
which is a rigid, porous, and amorphous material, the core of which is
very similar in all bacteria. Unique features of almost all prokaryotic
cells are cell wall peptidoglycan and the specic enzymes involved in
its biosynthesis. The amount and exact composition of peptidoglycan
only found in cell walls vary among the major bacterial groups.
Peptidoglycan is a linear polymer of alternating units of two sugar
derivatives, N-acetylglucosamine (NAG) and N-acetylmuramic acid
(NAM) (Fig. 1). Peptidoglycan also contain several different amino
acids, three of which
D-glu tamic acid, D-alanine, and meso-
Table 1
The major classes of chemical constituents in the walls and envelopes of Gram-positive
and Gram-negative bacteria
Gram-positive cell walls Examples
Peptidoglycan All species
Polysaccharides Streptococcus group A, B, C substances
Teichoic acids
Ribitol S. aureus
B. subtilis
Lactobacillus spp
Glycerol B. licheniformis
M. lysodeikticus
Teichuronic acids (aminogalacluronic or
aminomannuronic acid polymers)
Peptidoglycolipids (muramylpeptide
polysaccharidemycolates)
Corynebacterium spp
Mycobacterium spp
Nocardia spp
Glycolipids (Waxes)
(polysaccharidemycolates)
Gram-negative envelopes
LPS (Lipoteichoic acids) All species
Lipoprotein E. coli and many enteric bacteria
Pseudomonas aeruginosa
Porins (maj or outer membr ane prot ein s) E. coli, Salmonella typhimurium
Phospholipids and proteins All species
Peptidoglycan Almost all species
Source: Salton and Kwan 1996, http://gsbs.utmb.edu/microbook/ch002.htm.
Fig. 1. Peptidoglycan subunit composition. The peptidoglycan subunit of Escherichia coli,
most other Gram-negative bacteria, and many Gram-positive bacteria, NAG is N-
acetylglucosamine; NAM is N-acetylmuramic acid (NAG with lactic acid attached by an
ether linkage). The tetrapeptide side chain is composed of alternating
D- and L-amino
acids since meso-diaminopimelic acid is connected through its
L-carbon. NAM and the
tetrapeptide chain attached to it are shown in different shades of color for clarity.
Source: Prescott et al., 2002:56.
198 J.L. Wang, C. Chen / Biotechnology Advances 27 (2009) 195226
Page 4
diaminopimelic acidare not found in proteins. N-acetylglucosamine is
also the main constituent of chitin. However, the three-dimensional
structure differs from the crystalline structure of the chitin. A peptide
chain of four or ve alternating
D-andL-amino acids is connected to the
carboxyl group of N-acetylmuramic acid. The disaccharide-peptide units
are joined by direct peptide bonds or by short peptides. The carboxyl
group of the terminal
D-alanine is often connected directly to the amino
group of diaminopimelic acid. A common feature of bacterial cell walls is
cross-bridging between the peptide chains. There are several types of
peptidoglycan, depending on the nature and the localization of the
peptide bridge. In a Gram-positive cell, the cross-bridging between
adjacent peptides may be close to 100%, such as Staphylococcus aureus.
By contrast, the frequency of cross-bridging in E. coli (a Gram-negative
organism) may be as low as 30%. The peptidoglycan layer of a Gram-
negative cell is generally a single monolayer, composed of phospholi-
pids, lipopolysaccharides, enzymes, and other proteins, including
lipoproteins. Fig. 1 showed the peptidoglycan cross-links in a Gram-
negative and a Gram-positive cell. Most Gram-negative cell wall
peptidoglycans lack the peptide interbridge. This cross-linking results
in an enormous peptidoglycan sac which is actually a dense,
interconnected network. These sacs are elastic and porous, molecules
can penetrate them (Prescott et al., 2002).
The Gram-positive cell wall consists of a single 20 to 80 nm thick
homogeneous peptidoglycan or murein layer lying outside the plasma
membrane. It also contains large amounts of teichoic acids, polymers
of glycerol or ribitol joined by phosphate groups (Fig. 2). Peptidogly-
can of a Gram-positive cell wall accounts for 40 to 90% of the cell wall
materials, containing a peptide interbridge. This peptidoglycan core is
usually between 20 and 40 layers thick, and adjacent glycan chains are
cross linked through the amino acid stems forming a highly resilient,
three-dimensional macromolecule that surrounds the cells. Amino
acids such as
D-alanine or sugars like glucose are attached to the
glycerol and ribitol groups. The teichoic acids are connected to either
the peptidoglycan itself by a covalent bond with the six hydroxyl of N-
acetylmuramic acid or to plasma membrane lipids (called lipoteichoic
acids) (Prescott et al., 2002). Lipoteichoic acids, only present in Gram-
positive organismsare synthesized at the membrane surface and
may extend through the peptidoglycan layer to the outer surface, are
polymers of amphiphitic glycophosphates with the lipophilic glyco-
lipid and anchored in the cytoplasmic membrane. They are antigenic,
cytotoxic and adhesins (e.g., Streptococcus pyogenes).
Teichoic acids appear to extend to the surface of the peptidoglycan,
and, because they are negatively charged, they are helpful to give the
Gram-positive cell wall negative charge. The teichuronic acids are free
of phosphate and made up of hexuronic acid linear chains. The
proportion of teichoic acids and teichuronic acids depends on the
cultural conditions, especially on the phosphate supply. The functions
of these molecules are still unclear, but they may be important in
maintaining the structure of the wall. Teichoic acids are not present in
Gram-negative bacteria. It is proved that the teichoic acids and
teichuronic acids participate in metal tripping. Both the phosphoryl
groups of the secondary polymers and the carboxyl groups of the
peptide chains provide negatively charged sites in the Gram-positive
cell wall (Moat et al., 2002; Prescott et al., 2002; Remacle, 1990;
Urrutia, 1997).
Cell wall teichoic acids are found only in certain Gram-positive
bacteria (such as Bacillus spp.), and their structures are illustrated in
Fig. 3. Structures of cell wall teichoic acids. Teichoic acid is a polymer of chemically
modied ribitol (A) or glycerol phosphate (B). The nature of the modication (e.g.,
sugars, amino acids) can dene the serotype of the bacteria. Teichoic acid may be
covalently attached to the peptidoglycan. Lipoteichoic acid is anchored in the cytoplasm
membrane by a covalently attached fatty acid. Source: http://micro.digitalproteus.com/
morphology3.php.
Fig. 2. Composition of the cell surfaces of Gram-positive and Gram-negative bacteria. Not all structures shown are found in all organisms. For example, M protein is only used to
describe a structure in some of the streptococci. Also, not all organisms have agella. Source: Moat et al., 2002:3.
199J.L. Wang, C. Chen / Biotechnology Advances 27 (2009) 195226
Page 5
Fig. 3. Teichoic acids are polyol phosphate polymers, with either ribitol
or glycerol linked by phosphodiester bonds. Substituent groups on the
polyol chains include
D-alanine (ester linked), N-acetylglucosamine,
N-acetylgalactosamine, and glucose. They are strongly antigenic.
These highly negatively charged polymers of the bacterial cell wall
can serve as a cation-sequestering mechanism.
The Gram-negative cell wall is much more complex than the Gram-
positive cell, about 30 to 80 nm thick. It is a multilayered structure. It
has a 2 to 7 nm peptidoglycan layer surrounded by a 7 to 8 nm thick
outer membrane. The peptidoglycan is sandwiched between the
plasma membrane and the outer membrane, which is composed of
phospholipids, lipopolysaccharides, enzymes, and other proteins,
including lipoproteins. The thin peptidoglycan layer next to the
plasma membrane may constitute not more than 5 to 10% of the cell
wall weight. In E. coli it is about 2 nm thick and contains only one or
two layers or sheets of peptidoglycan. Only one type of the peptide
bridge occurs between the glycan chains. The space between the outer
membrane and the inner membrane is referred to as the periplasmic
space, which is the translucent region where various enzymes and
proteins located. The peptidoglycan is covalently bound to the outer
membrane by lipoproteins. The outer membrane is composed of
lipopolysaccharide (LPSs), phospholipids and proteins.
The Gra m-negative bacteria have various types of complex
macromolecular lipopolysaccharide (LPS). LPSs are probably the
most unusual constituents of the outer membrane. LPSs structure
was illustrated in Figs. 4 and 5. LPSs contain both lipids and
carbohydrates, and consist of three parts: (1) lipid A, (2) the core
polysaccharide, and (3) the O side chain. The structure of lipid A
required for insertion in the outer leaet of the outer membrane
bilayer; a covalently attached core composed of 2-keto-3deoxyoctonic
acid (KDO), heptose, ethanolamine, N-acetylglucosamine, glucose,
and galactose; and polysaccharide chains linked to the core. The
polysaccharide chains constitute the O-antigens of the Gram-negative
bacteria, and the individual monosaccharide constituents confer
serologic specicity on these components. LPS and phospholipids
help confer asymmetry to the outer membrane of the Gram-negative
bacteria, with the hydrophilic polysaccharide chains outermost. Each
LPS is held in the outer membrane by relatively weak cohesive forces
(ionic and hydrophobic interactions) and can be dissociated from the
cell surface with surface-active agents (http://gsbs.utmb.edu/micro-
book/ch002.htm). The net negative charge of LPSs attributes to the
negative surface charge of Gram-negative bacteria. The phosphate
groups within LPSs and phospholipids have been proved to be the
primary sites for metal interaction. However, only one of the carboxyl
groups in LPSs is free to interact with metals (Moat et al., 2002;
Prescott et al., 2002; Remacle, 1990; Urrutia, 1997).
2.1.2.2. Capsules and loose slime. Some bacterial cells can produce
capsules or slime layer above the bacterial cell wall. They are highly
hydrated (N 95% water) and loosely arranged polymers of carbohy-
drates and proteins. Capsules are composed of polysaccharides (high
molecular-weight polymers of carbohydrates), and a few consist of
proteins or polymers of amino acids called polypeptides (often formed
from the
D- rather than the L-isomer of an amino acid). The capsule of
Streptococcus pneumoniae type III is composed of glucose and
glucuronic acid in alternating β-1, 3- and β-1, 4- linkages (Moat
et al., 2002):
Bacillus anthracis, the anthrax bacillus, can produce polypeptide
capsules composed of
D-glutamic acid subunits. Capsule may be thick
or thin, rigid or exible, depending on specic organism. Several
different terms can be found to describe the capsule layer, such as
slime layer, glycocalyx (dened as the polysaccharide-containing
material lying outside the cell), extracellular polysaccharide (EPS).
Capsule polymers are usually acidic in nature although capsules can
Fig. 4. Lipopolysaccharide Structure. (A) The lipopolysaccharide from Salmonella. This slightly simplied diagram illustrates one form of the LPS. Abbreviations: Abe, abequose; Gal,
galactose; Glc, glucose; GlcN, glucosamine; Hep, heptulose; KDO, 2-keto-3-deoxyoctonate; Man, mannose; NAG, N-acetylglucosamine; P, phosphate; Rha,
L-rhamnose. Lipid A is
buried in the outer membrane. (B) Molecular model of an Escherichia coli lipopolysaccharide. The lipid A and core polysaccharide are straight; the O side chain is bent at an angle in
this model. Source: Prescott et al., 2002:60.
200 J.L. Wang, C. Chen / Biotechnology Advances 27 (2009) 195226
Page 6
consist of neutral polysaccharide, charged polysaccharide or charged
polypeptide. Capsule arrangement is important to metal binding
(Madigan et al., 2000; Moat et al., 2002; Urrutia, 1997).
Many prokaryotes contain a cell surface layer composted of a two-
dimensional array of proteins, or glycoproteins, called S-layers or
paracrystalline surface layer. S-layers have a crystalline appearance in
p1, p2, p4, p6 symmetry, such as hexagonal (p6) and tetragonal (p4),
depending on the number and structure of proteins or glycoproteins
subunits of which they are composted. Non-covalent interactions, such
as hydrogen bonding, electrostatic attraction, and salt-bridging, are
involved in the attachment between neighbouring subunits and the
underlying wall. Commonly, divalent metal cations contribute to the
correct assembly of the structure. Metals can also be bound after
assembly. S-layers are associated with LPSs of Gram-negative or
peptidoglycan of a Gram-positive cell (Madigan et al., 2000; Urrutia,
1997).
2.2. Fungal structure
2.2.1. Classication and general characteristics
Microscopic fungi include yeasts with spherical budding cells and
molds with elongate lamentous hyphae in mycelia. The molds are
lamentous fungi, such as Penicillium, Aspergillus, etc. The body or
vegetative structure of a fungus is called thallus (pl., thalli), which
varies in complexity and size from single cell microscopic yeasts to
multicellular molds. A single lament is called a hypha. Hyphae
usually grow together, collectively called a mycelium. Classication of
fungi was showed in Table 2. Apart from Oomycetes, which are
phylogenetically distinct, the other groups of fungi are closely related.
Yeasts are unicellula r fungi mainly ascomycetes. Fungi may be
grouped into molds or yeasts based on the development of the
thallus, which is the body or vegetative structure of a fungus. Yeasts
are unicellular fungi. Yeasts reproduce either asexually by budding
and transverse division or sexually through spore formation. A mold
consists of long, branched, thread-like laments of cells, the hyphae,
which form a tangled mass called a mycelium. Hyphae may be either
septate or coenocytic (nonseptate). The mycelium can produce
reproductive structures (Prescott et al., 2002).
Most fungi are lamentous. The hyphae are typically 510 μm wide
but may vary from 0.5 μm to 1.00 mm, depending on the various
species (Lester and Birkettn, 1999). The mycelium is composed of a
complex mass of laments or hyphae. The hyphae have walls which
are composed of cellulose or chitin or both of them. A common
cytoplasm exists throughout the hyphae. Thus fungi cellular organiza-
tion has three types: (1) coenocytic, where the hypha contains a mass
of multi-nucleate cytoplasm, also called as aseptate; (2) septate with
uni-nucleate protoplasts, where the hypha is divided by crosswalls or
septa, each compartment containing a single nucleus; (3) septate with
multi-nucleate protoplasts between the septa. In septate species there
is a central pore in the septum connecting the cytoplasm of
neighbouring cells and permitting the migration of both cytoplasm
and nuclei (Lester and Birkettn, 1999).
The yeasts provide an example of a unicellular fungus. Generally
yeast cells are larger than bacteria, vary considerably in size. Typical
yeast cell is about 2.5 to 10 μmwideby4.5to21
μm
long. Yeast cell
mor
phology is commonly spherical to oval shaped and varies, depend-
ing on the yeast species, nutrition level, cultural condition. The cells of
most microscopic fungi grow in loose associations or colonies. Most
yeasts reproduce only as single cell, however some yeasts can form
laments under certain conditions. Some yeasts exhibit sexual repro-
duction by a process called mating. The colonies of yeasts are much like
those of bacteria because they have a soft, uniform texture and
appearance. The most important commercial yeasts are the baker's
and brewer's yeasts, which are member of the genus Saccharomyces
(Madigan et al., 2000). Baker's and brewer's yeasts are eukaryotic cells.
They are easily manipulable thus are excellent models for the study of
many important problems in eukaryotic biology. S. cerevisiae is a famous
model eukaryote for scienticstudy,andwastherst eukaryote to have
its genome completely sequenced.
S. cerevisiae is a species of budding yeast. Saccharomyces derives
from Greek, and means sugar mold; cerevisiae comes from Latin,
and means of beer. It is perhaps the most useful yeast owing to its
use since ancient times in baking and brewing. It is believed that it was
originally isolated from the skins of grapes (one can see the yeast as a
component of the thin white lm on the skins of some dark-colored
fruits such as plums; it exists among the waxes of the cuticle). It is one
of the most intensively studied eukaryotic model organisms in
molecular and cell biology, much like E. coli as the model prokaryote.
Fig. 5. The three major, covalently linked regions that form the typical LPS. Source: Salton and Kwan 1996, http://gsbs.utmb.edu/microbook/ch002.htm.
Table 2
The classication of fungi
Group Common name Hyphae Typical representative
Ascomycetes Sac fungi Septate Neurospora
Saccharomyces
Morchella
Basidiomycetes Club fungi, mushroom Septate Amanita
Agaricus
Zygomycetes Bread molds Coenocytic Mucor
Rhizopus
Oomycetes Water molds Coenocytic Allomyces
Deuteromycetes Fungi imperfecti Septate Penicillium
Aspergillus
Candida
Adapted from Madigan et al., 2000:729.
201J.L. Wang, C. Chen / Biotechnology Advances 27 (2009) 195226
Page 7
It is the microorganism behind the most common type of fermenta-
tion. S. cerevisiae cells are round to ovoid, 510 μm in diameter. It
reproduces by a division process known as budding. Our lab has
selected the cells of S. cerevisiae to explore the characteristics of metal
biosorption and interaction of metal-microbe, and conducted a series
of experiments, published some meaningful results (Chen and Wang,
2006, 2007a,b,c,d,e, 2008a,b,c; Wang, 2002a; Wang and Chen, 2006).
In general, yeast cells have a cell wall, cytoplasmic membrane,
cytoplasm and inclusions, a single nucleus, mitochondria, endoplas-
mic reticulum, Golgi apparatus, vacuoles, cytoskeleton, and glycoca-
lyx. The yeast has no agella but do possess most of the other
eukaryotic organelles.
2.2.2. Cell wall and its main composite polysaccharide
The cell walls of the fungi and algae are rigid and provide structural
support and shape, but they are different in chemical composition
from procaryotic cell walls. Fungal cell walls are mainly 8090%
polysaccharide, with proteins, lipids, polyphosphates, and inorganic
ions, making up the wall-cementing matrix. Chitin is a common
constituent of fungal cell walls. Chitin is a strong but exible nitrogen-
containing polysaccharide, consisting of N-acetylglucosamine resi-
dues. Two layers were observed in ultrastructural studies of the fungal
cell walls (Fig. 6): a thin outer layer consisting of mixed glycans (such
as glucans, mannans, or galactans), and a thick inner mcirobrillar
layer of polysaccharide bers composed of chitin or cellulose with
chitin chains in parallel arrangement, sometimes of cellulose chains or
Fig. 6. Glycocalyx structure. Cross section through the tip of a fungal cell shows the
general structure of the cell wall and other features. Top: photomicrograph. (S, growing
tip; CV, coated vesicles; G, Golgi apparatus; M, mitochondrion) Bottom: the cell wall is a
thick, rigid structure composed of complex layers of polysaccharides and proteins. From
Talaro and Talaro, (2002):127.
Fig. 7. Structures of cellulose, chitin, glucan and manna. Source for Cellulose: http://www.scienticpsychic.com/tness/carbohydrates2.html. Source for Chitin: http://www.
scienticpsychic.com/tness/carbohydrates2.html. Source for β-Glucan: http://www.scienticpsychic.com/tness/glucan.gif. Source for Manna: Davis et al., 2003.
202 J.L. Wang, C. Chen / Biotechnology Advances 27 (2009) 195226
Page 8
in certain yeasts, noncellulosic glucan (Remacle, 1990; Talaro and
Talaro, 2002).
The structures of cellulose, chitin, glucan, manna were shown in
Fig. 7.
2.2.3. Cell membrane
The cell membrane of eukaryotic cells is a thin, double-layered sheet
composed of lipids, such as phospholipids and sterols (averaging about
40% of membrane content) and protein molecules (averaging about
60%). Sterols are different from phospholipids in both structure and
behavior. Fluid mosaic model for membrane structure are widely
accepted. Cell membrane is a continuous bilayer formed by lipids that
are oriented with the polar lipid heads toward outside and the nonpolar
heads toward the center of the membrane. Embedded at numerous sites
in this bilayer are various sized globular proteins. Some cell membranes
are so thinon the average, just 7 nm thick. Cytoplasmic membranes
served as selectively permeable barriers in transport. Unlike procar-
yotes, eukaryotic cells also contain a number of individual membrane-
bound organelles that are extensive enough to account for 60% to 80% (in
volume) (Prescott et al., 2002; Talaro and Talaro, 2002).
2.2.4. Cytoplasm
The cytoplasm contains the organelles characteristic of eukaryotic
organism s including mitochondria, rib osomes and an extensive
endoplasmic reticulum. Vacuoles containing storage materials such
as glycogen, lipids and volutin are also present. In a uni-cellular fungus
such as Saccharomyces spp., the protoplast is enclosed in a semiperme-
able membrane, the plasma membrane, which is contained within a
rigid cell wall. In lamentous species the protoplasm is concentrated
in the tips of the young growing hyphae. The older hyphae are usually
metabolically inactive and contain large vacuoles in their cytoplasm.
The fungi all lack chlorophyll and are heterotrophic. A mycelium
normally develops from the germination of a single reproductive cell
or spore. Germination initially results in the production of a single
long hypha which subsequently branches and ramies to form a mass
of hyphae which constitutes the mycelium.
Cytoplasm is important for living cells to interact with metal ions.
After entering into the cell, the metal ions are compartmentalized into
different subcellular organelles (e.g. mitochondr ia, vacuole etc.).
Vijver et al. (2004) summarized the metal ion accumulation strategies
especially internal compartmentalization strategies. Metal accumula-
tion strategies for essential and non-essential metal ions may be
different. For essential metals, limiting metal uptake or strategies with
active excretion, storage in an inert form or excretion of stored metal
are the main strategies. For non-essential metals, excretion from the
metal excess pool and internal storage without elimination are the
major strategies and the metal concentration in the cells will increase
with elevating external concentration. They pointed out that the
cellular sequestration mechanisms mainly have two types: the
formation of distinct inclusion bodies and the binding of metals to
heat-stable proteins. The former includes three types of granules: type
A, amorphous deposits of calcium phosphates, e.g. Zn; type B, mainly
containing acid phosphatase, accumulating e.g. Cd, Cu, Hg and Ag; and
type C, excess iron stored in granules as haemosiderin. The latter
mechanism mainly relates to a specic metal-binding protein,
metallothioneins (MT), which are low molecular weight and
cysteine-rich, usually occurring in the anima l k ingdom, plants,
eukaryotic microorganisms or some prokaryotes. MT can be induced
by many substances, including heavy metal ions, such as Cd, Cu, Hg,
Co, Zn etc. (Vijver et al., 2004).
The researches on the role of vacuole detoxication of metal ions
showed that vacuole-decient strain displayed much higher sensitiv-
ity and decreased large uptake of Zn, Mn, Co and Ni (Ramsay and Gadd,
1997). However no signicant difference in Cd and Cu uptake and the
sensitivity to both the metal ions between wild type and mutant of S.
cerevisiae was observed. Gharieb and Gadd (1998) found that
vacuolar-lacking and -defective mutants of S. cerevisiae display higher
sensitivity to chromate and tellurite with the decrease on the cellular
content of the each metal, whereas the tolerance to selenite with the
increase on the cellular content of Se. Avery and Tobin (1992) also
conrmed
that
Sr
2+
accumulated mainly stay in the vacuole of the
living yeast cell of S. cerevisiae.
2.3. Algae structure
2.3.1. Introduction and its classication
Algae abound in nature in aquatic habitats, freshwater, marine and
moist soil. Algae contai n chlorophyll and carry out oxygenic
photosynthesis.
Algae are eukaryotic microorganisms that carry out the process of
photosynthesis. In these organisms, as well as in green plants, an
additional type of organelle is found: the chloroplase. The chloroplast
Table 3
The properties of major groups of algae
Group Common name Morphology Pigments Typical
representative
Carbon reserve materials Cell walls Major
habitats
Kingdom
Chrysophyta Yellowgreen and
golden-brown
algae; diatoms)
Unicellular Chlorophylls a
and c
Navacul Lipids Many have two
overlapping
components made
of silica
Freshwater,
marine,
soil
Protista (single
cell or colonial;
eukaryotic)
Euglenophyta Euglenoids Unicellular,
photosynthetic
euglenoid
agellates
Chlorophylls a
and b
Euglena Pramylon (β-1,2-glucan) No wall present Freshwater,
a few
marine
Protista
Pyrrhophyta Dinoagellates Protista
Charophyta Stoneworts Protista
Chlorophyta Green algae Unicellular to leafy Chlorophylls a
and b
Chlamydomonas Starch (α-1,4-glucan) Cellulose Freshwater,
soil, a few
marine
Protista
Phaeophyta Brown algae Filamentousto leafy,
occasionally
massive and
plantlike
Chlorophylls a
and c,
xanthophylls
Laminaria Laninarin (β-1,3-glucan),
mannitol
Cellulose marine Plantae
(multicellular;
eukaryotic)
Rhodophyta Red algae Unicellular,
lamentous to leafy
Chlorophylls a
and d,
phycocyanin,
phycoerythrin
Polysiphonia Floridean starch (α-1,4- and
α-1,6-glucan), uoridoside
(glycerol-galactoside)
Cellulose Marine Plantae
Adapted from Madigan et al., 2000:736 and Prescott et al., 2002:572.
203J.L. Wang, C. Chen / Biotechnology Advances 27 (2009) 195226
Page 9
is green and is the site where chlorophyll is localized and where the
light-gathering functions involved in photosynthesis occur.
Algae have been extensively studied due to their ubiquitous
occurrence in nature. The term algae refer to a large and diverse
assemblage of eukaryotic organisms that contain chlorophyll and
carry out oxygenic photosynthesis. It should be noticed that algae are
distinct from cyanobacteria, which are also oxygenic phototrophs, but
are eubacteria (true bacteria), and are therefore evolutionarily distinct
from algae. Although most algae are of microscopic size and hence are
clearly microorganisms, a number of forms are macroscopic, some
seaweeds growing to over 100 ft in length (Madigan et al., 1997).
Algae are unicellular of colonial, the latter occurring as aggregates of
cells. When the cells are arranged end to end, the algae is said to be
lamentous. Among the lamentous forms, both unbranched laments
and more intricate branched lamentous forms occur. Most algae
contain chlorophyll and are thus green in color. However, a few kinds of
common algae are note green but appear brown or red because in
addition to chlorophyll, other pigments such as carotenoids are present
that mask the green color. Algae cells contain one or more chloroplasts,
membranous structures that house the photosynthetic pigments.
Several characteristics are used to classify algae, including the
nature of the chlorophyll(s) present, the carbon reserve polymers
produced, the cell wall structure, and the type of motility. All algae
contain chlorophyll a. Some, however, also contain other chlorophylls
that differ in minor ways from chlorophylls a. The presence of these
additional chlorophylls is characteristic of particular algal groups. The
distribution of chlorophylls and other photosynthetic pigments in
algae was summarized by Madigan et al. (1997). The algal groups
include Chlorophyta (green algae), Euglenophyta (euglenoids, also
considered with the protozoa), Chrysophyta (golden-brown algae,
diatoms), Phaeophyta (brown algae), Pyrrophyta (dino-agellates) and
Rhodophyta (red algae). One of the key characteristics used in the
classication of algal groups is the nature of the reserve polymer
synthesized as a result of photosynthesis. Algae of the division
Chlorophyta produce starch in a form very similar to that of higher
plants. By contrast, algae of other groups produce a variety of reserve
substances, some polymeric and some as free monomers.
In biosorption, various algae were used and investigated as
biosorbents for metal removal. The major groups of algae were listed
in Table 3 based on their type of pigments, cell wall, stored food
materials, and body plan (Talaro and Talaro, 2002).Thenatureofthe
chlorophyll(s), the cell wall chemistry, agellation, form in which food or
assimilatory products of photosynthesis are stored, cell morphology,
habitat; reproductive structures; life history patterns, etc., these
characteristics can be used for the classication of algae. The important
differences between brown algae and other algae are in the storage
products they utilize as well as in their cell wall chemistry, shown in
Table 3 (Davis et al., 2003c; Madigan et al., 2000; Prescott et al., 2002).
The algal cell is surrounded by a thin, rigid cell wall. Some algae
have an outer matrix lying outside the cell wall, similar to bacterial
capsules. The nucleus has a typical nuclear envelope with pores;
within the nucleus there are nucleolus, chromatin, and karyolymph.
The chloroplasts have membrane-bound sacs called thylakoids that
carry out the light reactions of photosynthesis. These organelles are
embedded in the stroma where the dark reactions of carbon dioxide
xation take place. A dense proteinaceous area, the pyrenoid that is
associated with synthesis and storage of starch may be present in the
chloroplasts. Mitochondrial structure varies greatly in the algae. Some
algae (euglenoids) have discoid cristae; some, lamellar cristae (green
and red algae); and the remaining, (golden-brown and yellow
gr
een,
bro
wn, and diatoms) have tubular cristae (Prescott et al., 2002).
2.3.2. Cell wall of algae
Algae show considerable diversity in the structure and chemistry
of their cell walls. In many cases the cell wall is composed of a network
of cellulose brils, but it is usually modied by the addition of other
polysaccharides such as pectin (highly hydrated polygalacturonic acid
containing small amounts of the hexose rhamnose), xylans, mannans,
alginic acids or fucinic acid. In some algae, the wall is additionally
strengthened by the deposition of calcium carbonate; these forms are
often called calcareous or coralline (corallike) algae. Sometimes
chitin, a polymer of N-acetylglucosamine, is also present in the cell
wall. In euglenoids cell wall is absent. In diatioms, the cell wall is
composed of silica, to which protein and polysaccharide are added.
Even after the diatom dies and the organic materials have dis-
appeared, the external structure remains, showing that the siliceous
component is indeed responsible for the rigidity of the cell. Because of
the extreme resistance to decay of these diatom frustules, they remain
intact for long periods of time and constitute some of the best algal
fossils ever found. Algal cell walls are freely permeable to low
molecular-weight constituents such as water, ions, gases, and other
nutrients. Their cell walls are essentially impermeable, however, to
larger molecules or to macromolecules. Algae cell walls contain pores
about 35 nm wide to all ow pass only low-molecular-weight
substances such as water, inorganic ions, gases and other small
nutrient substances for metabolism and growth. It is usually made of a
multilayered microbrillar framework generally consisting of cellu-
lose and intersperse with amorphous material (Madigan et al., 2000).
The cellulose can be replaced by xylan in the Chlorophyta and Rho-
dophyta in addition to mannan in the Chlorophyta. The Phaeophyta
algal mainly contain alginic acid or alginate (the salt of alginic acid)
with a smaller amount of sulfated polysaccharide (fucoidan). The
Rhodophyta contains a number of sulfated galactans. Both the Phaeo-
phyta and Rhodophyta are potentially excellent heavy metal biosor-
bents because two divisions contain the largest amount of amorphous
embedding matrix polysaccharides and their well known metal
binding ability (Davis et al., 2003c).
Cell walls are more complex in algae than in fungi or bacteria, and
three groups of algae, i.e., brown, red and green algae, are of interest,
and need to be differentiated in those three evolutionary pathways
(Kuyicak and Volesky, 1990; Rincon et al., 2005). The cell walls of
brown alg ae (Phaeophyta) generally contain three components:
cellulose, the structural support; alginic acid, a polymer of mannuro-
nic and guluronic acids (M and G) and the corresponding salts of
sodium, potassium, magnesium and calcium; and sulphated poly-
saccharides (fucoidan matrix). Red algae (Rhodophyta) also contain
cellulose, but their interest in connection with biosorption lies in the
presence of sulphated polysaccharides made of galactanes (agar and
carragenates). Green algae (Chlorophyta) are mainly cellulose, and a
high percentage of the cell wall is proteins bonded to polysaccharides
to form glycoproteins (Romera et al., 2006).
The Chlorophyta or green algae are an extremely varied division.
They have chlorophylls
a and b along
with
specic carotenoids, and
store carbohydrates as starch. Many of them have cell walls of
cellulose. They can present in unicellul ar, colonial, lamentous,
membranous or sheet-like, and tubular types. Green algae are
associated with the land plants and have mitochondria with lamellar
cristae (Prescott et al., 2002).
Most Rhodophyta or red algae are lamentous and multicellular.
The stored food is the carbohydrate called oridean starch (composed
of α-1,4 and α-1,6 linked glucose residues). The cell walls of most red
algae include a rigid inner part composed of microbrils and a
mucilaginous matrix. The matrix is composed of sulfated polymers of
galactose called agar, funori, porphysan, and carrageenan, which are
responsible for exible, slippery texture of the red algae. Agar is used
extensively in the laboratory as a culture medium component. Many
red algae also deposit calcium carbonate in their cell walls and play an
important role in building coral reefs (Prescott et al., 2002).
The Phaeophyta or brown algae have been proved to be most
effective biosobent for metal removal, based on statistical review
among those algae tested in biosorption (Romera et al., 2006). Davis
et al. (2003c) summarized the characteristics of brown algae and other
204 J.L. Wang, C. Chen / Biotechnology Advances 27 (2009) 195226
Page 10
algae. The cellular structure and biochemistry were introduced in
detail, including cellular structure, storage polysaccharides, cell wall
and extracellular polysaccharides (fucoidan and alginic acid). Among
thirteen orders in the Phaeophyta; however, only Laminariales and
Fucales are important from the viewpoint of biosorption. Laminariales,
also called as kelps, have many commercial uses (e.g. water holding
property for frozen foods, syrups, and frozen deserts; gelling property
for instant puddings and dessert gels, or even explosives; emulsifying
properties for polishes; stabilizing properties in ceramics, welding
rods and cleaners). The well-known algal genus Sargassum belongs to
the order Fucales which and have shown good capacity for metal
binding. Brown algae are multicellular and occur almost exclusively in
the sea. Most of the conspicuous seaweeds that are brown to olive
green in color are assigned to this division. The main storage product is
laminarin, similar to chrysolaminarin in structure.
The algal cell wall, similar to the fungal cell wall in structure, is
made of multi-layered microbrillar framework generally consisting
cellulose and interspersed with amorphous material. The algal cell
wall is complex, and even more than ten layers can be found in certain
kind of algal cell wall. The microbrils can be organized in parallel or
randomly. The amorphous embedding matrix consists of glycopro-
teins. The cellulous composted 90% of the algal cell wall. The algal cells
covered by mucilaginous layers bind metal due to the presence of
uronic acids (Remacle, 1990). The schematic cell wall structure of
brown algae, and its composition could referees to the review written
by Davis et al. (2003c). The cell wall of algae is composed of at least
two different layers. The innermost layer consists of a microbrillar
skeleton, and the outer layer is an amorphous embedding matrix,
which does not penetrate the bers, but rather is attached to this layer
via hydrogen bonds. The inner layer of brown algae is mainly
comprisedoftheunchargedcellulosepolymer(β-1,4-linked
unbranched glucan). Two other brillar molecules, xylan (principally
β-1,3-linked
D-xylose) and mannan (β-1,4-linked -linked D-mannose)
occur in the red and green algae. Alginate contributes to the strength
and exibility to the cell wall of brown algae. Cellulose remains the
principal structural component even if alginate occurs in the inner
layer. Fucoidan is present not only in the matrix but also within the
inner cell wall.
Structures of algal cellulose, xylan, manna, fucoidan and alginate
were illustrated in the review (Davis et al., 2003c). The molecular
structure of cellulose as a ca rbohydrate polymer comprises of
repeating β-
D-glucopyranose units which are covalently linked
through acetal functions between the OH group of the C4 and C1
carbon atoms (β-1,4-glucan). Cellulose is a large, linear-chain polymer
with a large number of hydroxyl groups (three per anhydroglucose
(AGU) unit) and present in the preferred 4C1 conformation. To
accommodate the preferred bond angles, every second AGU unit is
rotated 180° in the plane. The length of the polymeric cellulose chain
depends on the number of constituent AGU units (degree of
polymerisation, DP) and varies with the origin and treatment of the
cellulose raw material. Cellulose has a ribbon shape allowing it to
twist and bend in the direction out of the plane, thus making the
molecule moderately exible. There is a relatively strong interaction
between neighbouring cellulose molecules in dry bres due to the
presence of the hydroxyl (OH) groups, which stick out from the chain
and form intermolecular hydrogen bonds. Regenerated bres from
cellulose contain 250500 repeating units per chain. Cellulose is
hydrophilicity, chirality and degradability. Chemical reactivity is
largely due to the high donor reactivity of the OH groups (O'Connell
et al., 2008).
2.4. Functional groups related to the biosorption
According to the metal classication by Pearson (1963) as well as
by Nieboer and Richardson (1980), metal afnity for ligands is
supposed and illustrated in Table 4 (Remacle, 1990). The symbol R
represents an alkyl radical such as CH
2
,CH
3
CH
2
, etc. Class A metal
ions preferred to bind the ligands of I through oxygen. Class B metal
ions show high afnity for III types of ligands, but also form strong
binding with the ligands with II types of ligands. Borderline metal ions
could bind these three types of ligands with different preferences.
Table 4
The ligands present in biological systems and three classes of metals
Ligand class Ligands Metal classes
I: Ligands
preferred to
Class A
F
,O
2
,OH
,H
2
O, CO
3
2
,SO
4
,
ROSO
3
,NO
3
, HPO
4
2
,PO
4
3
, ROH,
RCOO
,CfO, ROR
Class A: Li, Be, Na, Mg, K, Ca, Sc, Rb,
Sr, Y, Cs, Ba, La, Fr, Ra, Ac, Al,
Lanthanides, Actinides
II: Other
important
ligands
Cl
,Br
,N
3
,NO
2
,SO
3
2
,NH
3
,N
2
,
RNH
2
,R
2
NH, R
3
N, fN, CON
R, O
2
,O
2
,O
2
2
Borderline ions: Ti, V, Cr, Mn, Fe, Co,
Ni, Cu, Zn, Ga, Cd, In, Sn, Sb, As
III: Ligands
preferred to
Class B
H
,I
,R
,CN
, CO, S
2
,RS
,R
2
S,
R
3
As
Class B: Rh, Pd, Ag, Lr, Pt, Au, Hg, Tl,
Pb, Bi
Source: Nieboer and Richardson, 1980; Pearson, 1963; Remacle, 1990.
Table 5
The representative functional groups and classes of organic compounds in biomass
Source: Talaro and Talaro, 2002:38.
205J.L. Wang, C. Chen / Biotechnology Advances 27 (2009) 195226
Page 11
According to the Hard and So ft Acid Base P rinciple (HSAB
principle), hard ions which bind F
strongly, such as Na
+
,Ca
2+
,Mg
2+
could form stable bonds with OH
, HPO
4
2
,CO
3
2+
,RCOO
and fCfO,
which are oxygen-containing ligands. Contrast to hard ions, soft ions,
for example, heavy metal ions such as Hg
2+
and Pb
2+
form strong bond
with CN
.RS
, SH
,NH
2
and imidazol, which are groups containing
nitrogen and sulfur atoms. Borderline or intermediate metal ions such
as Zn
2+
and Co
2+
are less toxic. Hard ions mainly show ionic nature of
binding, whereas soft ions binding exhibit a more covalent degree
(Nieboer and Richardson, 1980; Pearson, 1963; Remacle, 1990).
Metal biosorption by biomass mainly depend on the components
on the cell, especially through cell surface and the spatial structure of
the cell wall. Peptidoglycan, teichoic acids and lipoteichoic acids are all
important chemical components of ba cterial surface structures.
Various polysaccharides, including cellulose, chitin, alginate, glycan,
etc. existed in fungi or algae cell walls, have been proved to play a very
important role in metal binding. Various proteins are also proved to
involve in metal binding for c ertain kinds of biomasses. Some
functional groups have been found to bind metal ions, especially
carboxyl group. There are some evidence to conrm that the O-, N-, S-,
or P-containing groups participate directly in binding a certain metals.
Some active sites involved in the metal uptake are determined by
using techniques of titration, infra-red and Raman spectroscopy,
electron dispersive spectroscopy (EDS), X-ray photoelectron spectros-
copy (XPS), electron microscopy (scanning and/or transmission),
nuclear magnetic resonance (NMR), X-ray diffraction analysis (XRD),
XAFS (X-ray absorption ne structure spectroscopy) etc. The most
import ant of these groups are summarized by Volesky (2007),
including Carbonyl (ketone), Carboxyl, Sulfhydryl (thiol), Sulfonate,
Thioether, Amine, Secondary amine, Amide, Imine, Imidazole, Phos-
phonate, Phosphodiester. The relevant structural formula, pK
a
,HSAB
classication, ligand atom, as well as occurrence in selected
biomolecules were offered.
Table 5 offers a representative functional groups and classes of
organic compounds in biomass. The symbol R is shorthand for residue,
and its placement in a formula indicates that what is attached at that
site varies from one compound to another, according to Talaro and
Talaro (2002).
3. Bacterial biosorbents
Bacteria are the most abundant and versatile of microorganisms
and constitute a signicant fraction of the entire living terrestrial
biomass of ~10
18
g(Mann, 1990). Early 1980, some microorganisms
were found to accumulate metallic elements with high capacity
(Vijayaraghavan and Yun, 2008). Some marine microorganisms
enriched Pb and Cd by factors of 1.7 ×10
5
and 1.0 × 10
5
respectively,
relative to the aqueous solute concentration of these elements in
ocean waters (Mann, 1990). Bacteria were used as biosorbents because
of their small size, their ubiquity, their ability to grow under controlled
conditions, and their resilience to a wide range of environmental
Table 6
Bacterial biomass used for metal removal (mg g
1
)
Metal
ions
Bacteria species Biosorption
capacity
References
Pb Bacillus sp. 92.3 Tunali et al. (2006)
Pb Bacillus rmus 467 Salehizadeh and
Shojaosadati (2003)
Pb Corynebacterium glutamicum 567.7 Choi and Yun (2004)
Pb Enterobacter sp. 50.9 Lu et al. (2006)
Pb Pseudomonas aeruginosa 79.5 Chang et al. (1997)
Pb Pseudomonas aeruginosa 0.7 Lin and Lai (2006)
Pb Pseudomonas putida 270.4 Uslu and Tanyol (2006)
Pb Pseudomonas putida 56.2 Pardo et al. (2003)
Pb Streptomyces rimosus 135.0 Selatnia et al. (2004c)
Zn Streptomyces rimosus 30 Mameri et al. (1999)
Zn Bacillus rmus 418 Salehizadeh and
Shojaosadati (2003)
Zn Aphanothece halophytica 133.0 Incharoensakdi and
Kitjaharn (2002)
Zn Pseudomonas putida 6.9 Pardo et al. (2003)
Zn Pseudomonas putida 17.7 Chen et al. (2005)
Zn Streptomyces rimosus 30.0 Mameri et al. (1999)
Zn Streptomyces rimosus 80.0 Mameri et al. (1999)
Zn Streptoverticillium cinnamoneum 21 .3 Puranik and Paknikar
(1997)
Zn Thiobacillus ferrooxidans 82.6 Celaya et al. (2000)
Zn Thiobacillus ferrooxidans 172.4 Liu et al. (2004)
Cu Bacillus rmus 381 Salehizadeh and
Shojaosadati (2003)
Cu Bacillus sp. 16.3 Tunali et al. (2006)
Cu Bacillus
subtilis 20.8 Naka
jima et al. (2001)
Cu Enterobacter sp. 32.5 Lu et al. (2006)
Cu Micrococcus luteus 33.5 Nakajima et al. (2001)
Cu Pseudomonas aeruginosa 23.1 Chang et al. (1997)
Cu Pseudomonas cepacia 65.3 Savvaidis et al. (2003)
Cu Pseudomonas putida 6.6 Pardo et al. (2003)
Cu Pseudomonas putida 96.9 Uslu and Tanyol (2006)
Cu Pseudomonas putida 15.8 Chen et al. (2005)
Cu Pseudomonas stutzeri 22.9 Nakajima et al. (2001)
Cu Sphaerotilus natans 60 Beolchini et al. (2006)
Cu Sphaerotilus natans 5.4 Beolchini et al. (2006)
Cu Streptomyces coelicolor 66.7 Ozturk et al. (2004)
Cu Thiobacillus ferrooxidans
a
39.8 Liu et al. (2004)
Cd Ochrobactrum anthropi Ozdemir et al. (2003)
Cd Sphingomonas paucimobilis Tangaromsuk et al. (2002)
Cd Aeromonas caviae 155.3 Loukidou et al. (2004)
Cd Enterobacter sp. 46.2 Lu et al. (2006)
Cd Pseudomonas aeruginosa 42.4 Chang et al. (1997)
Cd Pseudomonas putida 8.0 Pardo et al. (2003)
Cd Pseudomonas sp. 278.0 Ziagova et al. (2007)
Cd Staphylococcus xylosus 250.0 Ziagova et al. (2007)
Cd Streptomyces pimprina 30.4 Puranik et al. (1995)
Cd Streptomyces rimosus 64.9 Selatnia et al. (2004a)
Fe(III) Streptomyces rimosus 122.0 Selatnia et al. (2004b)
Cr(IV) Bacillus coagulans 39.9 Srinath et al. (2002)
Cr(IV) Bacillus megaterium 30.7 Srinath et al. (2002)
Cr(IV) Zoogloea ramigera 2 Nourbakhsh et al. (1994)
Cr(IV) Aeromonas caviae 284.4 Loukidou et al. (2004)
Cr(IV) Bacillus coagulans 39.9 Srinath et al. (2002)
Cr(IV) Bacillus licheniformis 69.4 Zhou et al. (2007)
Cr(IV) Bacillus megaterium 30.7 Srinath et al. (2002)
Cr(IV) Bacillus thuringiensis 83.3 Sahin and Ozturk (2005)
Cr(IV) Pseudomonas sp. 95.0 Ziagova et al. (2007)
Cr(IV) Staphylococcus xylosus 143.0 Ziagova et al. (2007)
Fe Bacillus sp. Volesky and Holan (1995)
Ni Bacillus thuringiensis 45.9 Ozturk (2007)
Streptomyces rimosus 32.6 Selatnia et al. (2004d)
Pd Desulfovibrio desulfuricans 128.2 de Vargas et al. (2004)
Desulfovibrio fructosivorans 1
1
9.8 de Vargas et al. (2004)
Desulfovibrio vulgaris 106.3 de Vargas et al. (2004)
Pt Desulfovibrio desulfuricans 62.5 de Vargas et al. (2004)
Desulfovibrio fructosivorans 32.3 de Vargas et al. (2004)
Desulfovibrio vulgaris 40.1 de Vargas et al. (2004)
U Arthrobacter nicotianae 68.8 Nakajima and Tsuruta (2004)
U Bacillus licheniformis 45.9 Nakajima and Tsuruta (2004)
U Bacillus megaterium 37 .8 Nakajima and Tsuruta (2004)
U Bacillus subtilis 52.4 Nakajima and Tsuruta (2004)
U Corynebacterium equi 21 .4 Nakajima and Tsuruta (20 04)
U Corynebacterium glutamicum 5.9 Nakajima and Tsuruta (2004)
Table 6 (continued)
Metal
ions
Bacteria species Biosorption
capacity
References
U Micrococcus luteus 38.8 Nakajima and Tsuruta (2004)
U Nocardia erythropolis 51.2 Nakajima and Tsuruta (2004)
U Zoogloea ramigera 49.7 Nakajima and Tsuruta (2004)
Th Arthrobacter nicotianae 75.9 Nakajima and Tsuruta (2004)
Th Bacillus licheniformis 66.1 Nakajima and Tsuruta (2004)
Th Bacillus megaterium 74.0 Nakajima and Tsuruta (2004)
Th Bacillus subtilis 71.9 Nakajima and Tsuruta (2004)
Th Corynebacterium equi 46.9 Nakajima and Tsuruta (2004)
Th Corynebacterium glutamicum 36.2 Nakajima and Tsuruta (2004)
Th Micrococcus luteus 77.0 Nakajima and Tsuruta (20 04)
Th Zoogloea ramigera 67.8 Nakajima and Tsuruta (2004)
206 J.L. Wang, C. Chen / Biotechnology Advances 27 (2009) 195226
Page 12
situations (Urrutia, 1997). Bacteria species such as Bacillus, Pseudo-
monas, Streptomyces, Escherichia, Micrococcus, etc, have been tested
for uptake metals or organics. Table 6 summarizes some of the
important results of metal biosorption using bacterial biomasses,
according to some published references (Ahluwalia and Goyal, 2007;
Vijayaraghavan and Yun, 2008). Metal uptake capacity i s not
necessarily to reach the maximum values in the application. Some
uptake values were experimental uptake, and some were predicted by
the Langmuir model. Table 6 also provides the basic information to
evaluate the possibility of using bacterial biomass for the removal of
metal ions.
Bacteria may either possess the capacity for biosorption of many
elements or, alternatively, depending on the species, may be element
specic. It is likely that, in the future, microorganisms will be tailored
for a specic element or a group of elements, using recombinant DNA
technology which is based on genetic modication using endores-
trictive nucleases (Mann, 1990).
4. Fungal biosorbents
4.1. Introduction
Although fungi are a large and diverse group of eukaryotic
microorganisms, three groups of fungi have major practical impor-
tance: the molds, yeasts and mushrooms. Filamentous fungi and
yeasts have been observed in many instances to bind metallic
elements.
Fungi are ubiquitous in natural environments and important in
industrial processes. A range of morphologies are found, from
unicellular yeasts to polymorphic and lamentous fungi, many of
which have complex macroscopic fruiting bodies. Their most
important roles are as decomposers of organic mater ials, with
concomitant nutrients cycling, as pathogens and symbionts of animals
and plants, and as spoilage organisms of natural and synthetic
materials, e.g. wood, paint, leather, food and fabrics. They are also
utilized as producers of economically important substances, e.g.
ethanol, citric acid, antibiotics, polysaccharides, enzymes and vita-
mins (Gadd, 1993).
The importance of metallic ions to fungal and yeast metabolism
has been known for a long time (Gadd, 1993). The presence of heavy
metals affects the metabolic activities of fungal and yeast cultures, and
can affect commercial fermentation processes, which created interest
in relating the behavior of fungi to the presence of heavy metals. The
results from such studies led to a concept of using fungi and yeasts for
the removal of toxic metals (such as lead and cadmium) from
wastewater and recovery of precious metals (such as gold and silver)
from process waters (Kapoor and Viraraghavan, 1997a). Both living
and dead fungal cells possess a remarkable ability for taking up toxic
and precious metals.
In the eld of biosorption, the molds and yeast are of interests and
many researches are reported and reviewed. The molds are lamentous
fungi. The yeasts are unicellular fungi and most of them are classied
with the Ascomycetes. The most important commercial yeasts are the
baker's and brewer's yeasts, which are members of the genus Sacchar-
omyces. The original habitats of these yeasts were undoubtedly fruits
and fruit juices, but the commercial yeasts of today are probably quite
different from wild strains because they have been greatly improved
through the years by careful selection and genetic manipulation
eukaryotic cells, and they are thus excellent models for the study of
many important problems in eukaryotic biology. Yeast cells are much
larger than bacterial cells and can be distinguished microscopically from
bacteria by their size and by the obvious presence of internal cell
structures, such as the nucleus (M
ad
igan et al., 1997).
Fungi and yeasts are easy to grow, produce high yields of biomass
and can be manipulated genetically and morphologically. The fungal
organisms are widely used in a variety of large-scale industrial
fermentation processes. For example, strains of Aspergillus are used in
the production of ferrichrome, kojic acid, gallic acid, itaconic acid,
citric acid and enzymes like amylases, glucose isomerase, pectinase,
lipases and glucanases; while S. cerevisiae is used in the food and
beverage industries. The biomass can be cheaply and easily procured
in rather substantial quantities, also as a by-product from the
established industrial fermentation processes, for the biosorption of
heavy metals and radio nuclides, which made the fungi of primary
interest as a raw material serving as a basis for formulating suitable
biosorbents. The use of biomass as an adsorbent for heavy-metal
pollution control can generate revenue for industries presently
wasting the biomass and at same time ease the burden of disposal
costs associated with the waste biomass produced. Alternatively, the
biomass can also be grown using unsophisticated fermentation
techniques and inexpensive growth media (Kapoor and Viraraghavan,
1995). It is not a priority from the economical point of view to use the
waste biomass, but the fungal cultures are also amenable to genetic
and morpholocial manipulations which may result in better raw
biosorbents material (Volesky, 1990a).
This section will review and summarize the removal of heavy
metals and radio nuclides by lamentous fungi (such as Penicillium
sp., Aspergillus sp., Mucor sp., Rhizopus sp.) and yeast (Saccharomyces
spp.) from aqueous solutions.
4.2. Yeast
The yeast biomass has been successfully used as biosorbent for
removal of Ag, Au, Cd, Co, Cr, Cu, Ni, Pb, U, Th and Zn from aqueous
solution. Yeasts of genera Saccharomyces, Candida, Pichia are efcient
biosorbents for heavy metal ions. Most of yeasts can sorb a wide range
of metal ions or be strictly specic in respect of only one metal ion. S.
cerevisiae as biosorbents is of special interest (Podgorskii et al., 2004).
A number of literatures have proved that S. cerevisiae can remove toxic
metals, recover precious metals and clean radionuclides from aqueous
solutions to various extents . The advantages of S. cerevisiae as
biosorbents in metal biosorption, the forms of S. cerevisiae in
biosorption research, biosorptive capacity of S. cerevisiae,the
selectivity and competitive biosorption by S. cerevisiae were depicted
in detail by Wang and Chen (2006).
Table 7 presents some data on the biosorptive capacities of the
yeast (in various forms) for different metal ions reported in literatures.
Based on data presented in Table 7, the magnitude order of metal
uptake capacity by S. cerevisiae can be estimated as the followings: for
Lead, biosorptive capacity by S. cerevisiae is in the order of 23, above
tenth and less than 300 mg Pb/g dry weight biomass; for copper, in the
order of 1
2, less than 20 mg Cu/g dry weight yeast; for zinc, in the
or
der
of 12, usually less than 30 mg Zn/g dry weight; for cadmium, in
the order of 23, usually above 10 but less than 100 mg Cd/g dry mass;
for mercury, in the order of 2; for chromium and nickel, usually in the
order of 1, seldom more than 40 mg/g dry mass; for precious metals,
such as Ag, Pt, Pd, in the order of 2, around 50 mg/g dry weight yeast.
Biosorptive capacity of radionuclide uranium by S. cerevisiae is usually
between 150 and 300 mg U/g dry weight biomass. It should be noted
that comparing results from different literatures involves in standar-
dizing the different ways the sorption capacity may be expressed. At
same time, metal uptake, q, should be compared in almost the same
equilibrium concentration of metals in solution for the purpose of
evaluating performance of the biomaterial (Kratochvil and Volesky,
1998). In particular, there is no standard measurement of dry weight
of biomass, i.e. no standard of dry temperature and dry hours when
drying biomass. Park et al. (2003) obtained the dry-cell weight by
drying cells at 70 °C until the weight of the cells became constant. Ozer
and Ozer (2003) dried the yeast at 100 °C for 24 h. Obviously, the
numeric value of dry weight of biomass obtained in different drying
conditions is sure to be different. Hence, attention should be paid to
these conditions when comparing the different results.
207J.L. Wang, C. Chen / Biotechnology Advances 27 (2009) 195226
Page 13
4.3. Filamentous fungi
This section will review the removal of heavy metal ions and
radionuclides by lamentous fungi (such as Penicillium spp., Asper-
gillus spp., Rhizopus spp. and white rot fungi) from aqueous solutions.
Different species of Penicillium, under some circumstances, also As-
pergillus, have been reported as good biosorbents of metal ions. The
genus Rhizopus, such as R. arrhizus and Rhizopus javanicus, has been
discovered to owe the relatively good-sequestering properties
(Volesky, 1990a). Table 8 summarizes some of the important results
of metal biosorption using fungal biomasses.
4.3.1. Penicillium
Penicillium can remove a variety of heavy metal ions from aqueous
solutions, such as Cu, Au, Zn, Cd, Mn, U and Th, see Table 9. Penicillium
italicum (Mendil et al., 2008), Penicillium spinulosum, Penicillium
oxalicum (Svecova et al., 2006) Penicillium austurianum (Awofolu et al.,
200 6), Penic illium verrucosum (Cabuk et al., 2005), Penicillium
purpurogenum (Say et al., 2003a), Penicillium canescens (Say et al.,
2003b), Penicillium griseofulvum (Shah et al., 1999), P. austurianum
(Rostami and Joodaki, 2002), Penicillium chrysogenum, etc. were
Table 7
Biosorption by Saccharomyces cerevisiae (mg g
1
)
Metal ions Source or form of biosorbents Biosorption capacity
a
References
Pb Free cells 79.2 Al-Saraj et al. (1999)
Pb Immobilized cells in a solgel matrix 41.9 Al-Saraj et al. (1999)
Pb Whiskey distillery spent wash, lyophilized 189 Bustard and McHale (1998)
Pb Lab cultivated, then dried at 100 °C 270.3 Ozer and Ozer (2003)
Pb Ethanol treated waste baker's yeast 17.5 Goksungur et al. (2005)
Cu Adapted and growing cells 2.049.05 Donmez and Aksu, (1999)
Cu Waste yeast from fermentation industry and then autoclaved at 120 °C 4.93 Bakkaloglu et al. (1998)
Cu Free cells 6.4 Al-Saraj et al. (1999)
Cu Whiskey distillery spent wash lyophilized 5.7 Bustard and McHale (1998)
Cu immobilized cells on sepiolite 4.7 Bag et al. (1999a)
Cu Waste yeast from brewery, formaldehyde cross-linked cells in column bioreactors 8.1 Zhao and Duncan (1997)
Zn Waste yeast from fermentation industry and then autoclaved at 120 °C 3.451.95 Bakkaloglu et al. (1998)
Zn Free cells 23.4 Al-Saraj et al. (1999)
Zn Immobilized cells in a solgel matrix 35.3 Al-Saraj et al. (1999)
Zn Whiskey distillery spent wash, lyophilized 16.9 Bustard and McHale (1998)
Zn Immobilized cells on sepiolite 8.37 Bag et al. (1999a)
Zn Formaldehyde cross-linked cells in column bioreactors 7.1 Zhao and Duncan (1997)
Cd Deactivated protonated yeast from yeast co. 9.9186.3 Vasudevan et al. (2003)
Cd Free cell suspended in solution Lab culture 35.558.4 Park et al. (2003)
Cd Free cell suspended in solution Lab culture 14.320.0 Park et al. (2003)
Cd Immobilized cells on sepiolite 10.9 Bag et al. (1999a)
Cd Waste yeast from brewery, formaldehyde cross-linked cells in column bioreactors 14 Zhao and Duncan (1997)
Cd Ethanol treated waste baker's yeast 15.6 Goksungur et al. (2005)
Cd Non-living and resting cells from aerobic culture 70 Volesky et al. (1993)
Hg Free cells 64.2 Al-Saraj et al. (1999)
Co Free cells 9.9 Al-Saraj et al. (1999)
Ni Waste yeast from fermentation industry and then autoclaved at 120 °C 1.47 Bakkaloglu et al. (1998)
Ni Free cells 8 Al-Saraj et al. (1999)
Ni Lab cultivated, then dried at 100 °C 46.3 Ozer and Ozer (2003)
Ni Deactivated protonated yeast from yeast co. oven at 80 °C for 24 h 11.4 Padmavathy et al. (2003)
Cr(VI) Lab cultivated, dehydrated at 30 °C, 15% of cell humidity; 80.5% of the viability About 5.5 Rapoport and Muter (1995)
Cr(VI) As a by-product from brewery, formaldehyde cross-linked cells in xed-bed column 6.3 Zhao and Duncan (1998)
Cr(VI) Lab cultivated, then dried at 100 °C 32.6 Ozer and Ozer (2003)
Fe Whiskey distillery spent wash, lyophilized 16.8 Bustard and McHale (1998)
Pd Immobilized cells of waste yeast 40.6 Xie et al. (2003a)
Pt Immobilized cells of waste yeast 44 Xie et al. (2003b)
Ag Whiskey distillery spent wash lyophilized 59 Bustard and McHale (1998)
Ag Industrial strain, then lab cultivated and freeze-dried 41.7 Simmons and Singleton (1996)
241
Am Lab cultivated, free cell 7.451880.0
b
Liu et al. (2002)
U Whiskey distillery spent wash lyophilized 180 Bustard and McHale (1998)
U Beer yeast, 8.75 mmol UO
2
2+
/g yeast 2082.5
c
Popa et al. (20 03)
Washed and unwashed non-viable spent yeast from a company in Greece 360150 Riordan et al. (1997)
U 150 Tsezos (1997)
Th 63 Tsezos (1997)
a
Metal sorption is not necessarily maximum.
b
Unit: μgUg
1
.
c
The value calculated by converting the data 8.75 mmol UO
2
2+
g
1
yeast.
Table 8
Biosorption by fungal biomass (mg g
1
)
Species of fungi Metal
ions
References
Aspergillus niger, Mucor rouxii, Rhizopus arrhizus
(living cells)
Au Kapoor and
Viraraghavan (1997a)
Penicillium spp. (living cells) Ag
Cu Kapoor and
Viraraghavan (1997a)
Cd
Pb
Penicillium, Aspergillus, Trichoderma, Rhizopus, Mucor,
Saccharomyces, Fusarium (living cells)
Pb Kapoor and
Viraraghavan (1997a)
Cu
Cd
Zn
Aspergillus, Pen i c i lliu m , Rhizopus , Saccharomyces,
Trichoderma, Mucor, Rhizopus (living cells)
Th Kapoor and
Viraraghavan (1997a)
U
Sr
Cs
La
Phanerochaete chryosporium (living cells) Cd Day et al. (2001)
Pb
Cu
208 J.L. Wang, C. Chen / Biotechnology Advances 27 (2009) 195226
Page 14
reported to adsorb various metals. For example, P. chrysogenum can
extract gold from a cyanide solution. However, the biosorption
capacity was not encouraging (Vieira and Volesky, 2000). P. spinulo-
sum was reported to be capable of removing Cu, Au, Zn, Cd, Mn
(Kapoor and Viraraghavan, 1995). Among these Penicillium sp., P.
chrysogenum was studied most. The P. chrysogenum, a semi-known
strains Hyphomycetes gang door Hyphomycetes Head (Cong stems
Species) CONG stems Branch Penicillium spores of fungi. Classied
asymmetry Penicillium group, cashmere-like Penicillium Asian group,
the middle P. chrysogenum and it is typical of penicillin-producing
bacteria. Fungi have smooth surface, 150350 μm long, 3 to 3.5 μm
wide, 23 branches, they are brush sticks asymmetry, ranging from
the length of the vice sticks, based on the growing stems and small
stems. Conidia is oval, (24) μm ×(2.83.5) μm. They grow faster,
round, tight cashmere-like or slightly tempered, blue and green
(white edge), a radial grooving on the surface, often yellow-colored
droplets exudative. Bright yellow colony back to a dark brown and
yellow-soluble spread to the medium. They are widely distributed in
the air, soil and organic matters on biodeterioration. Penicillinum is
famous for organics production, which can produce organic acids,
such as glucose acid, citric acid, as well as glucose oxidase (http://
www.chemyq.com/En/xz/xz4/39623ajqia.htm). P. chrysogenum exhib-
ited preferential sorption orders: Pb
2+
N Cu
2+
N Zn
2+
N Cd
2+
N Ni
2+
N Co
2+
(Puranik and Paknikar, 1999). For non-living P. chrysogenum:Pb
2+
N
Cd
2+
N Cu
2+
N Zn
2+
N As
3+
. P. canescens exhibited the same sorption
orders: Pb(II) N Cd(II) N Hg(II) N As(III) at non-competitive conditions
or competitive conditions. At non-competitive conditions, metal
uptake capacity of P. canescens were 26.4 mg/g for As(III), 54.8 mg/g
for Hg(II), 102.7 mg/g for Cd(II) and 213.2 mg/g for Pb(II), respectively.
However, competitive adsorption capacity for the heavy metal ions
were 2.0 mg/g for As(III), 5.8 mg/g for Hg(II), 11.7 mg/g for Cd(II) and
Table 9
Biosorption by Penicillium sp. (mg g
1
)
Species Metal ions Biosorption
capacity
References
Penicillium canescens Cd 102.7 Say et al. (2003b)
Penicillium canescens Pb 213.2 Say et al. (2003b)
Penicillium canescens Hg 54.8 Say et al. (2003b)
Penicillium canescens As(III) 26.4 Say et al. (2003b)
Penicillium chrysogenum Cd 11 Niu et al. (1993)
Penicillium chrysogenum Cu 9 Niu et al. (1993)
Penicillium chrysogenum Pb 116 Niu et al. (1993)
Penicillium chrysogenum Cd 56 Holan and Volesky (1995)
Penicillium chrysogenum Cd 39 Fourest et al. (1994)
Penicillium chrysogenum Th Gadd and White (1992)
Penicillium chrysogenum Zn 6.5 Niu et al. (1993)
Penicillium chrysogenum
(surface imprinted)
Ni 82.5 Su et al. (2006)
Penicillium chrysogenum
(waste biomass)
Ni 56.2 Su et al. (2006)
Penicillium chrysogenum Cr(VI) Park et al. (2005)
Penicillium chrysogenum
(modied)
Cd 210.2 Deng and Ting (2005b)
Penicillium chrysogenum
(modied)
Cu 108.3 Deng and Ting (2005b)
Penicillium chrysogenum
(modied)
Cu 92 Deng and Ting (2005a)
Penicillium chrysogenum
(modied)
Pb 204 Deng and Ting (2005a)
Penicillium chrysogenum
(modied)
Ni 55 Deng and Ting (2005a)
Penicillium chrysogenum
(modied)
Ni 260 Tan et al. (2004)
Penicillium chrysogenum (raw) Cr(III) 18.6 Tan and Cheng (2003)
Penicillium chrysogenum (raw) Ni 13.2 Tan and Cheng (2003)
Penicillium chrysogenum (raw) Zn 6.8 Tan and Cheng (2003)
Penicillium chrysogenum
(Alkaline pretreatment)
Cr(III) 27.2 Tan and Cheng (2003)
Penicillium chrysogenum
(Alkaline pretreatment)
Ni 19.2 T
an
and Cheng (2003)
Penicillium chrysogenum
(Alkaline pretreatment)
Zn 25.5 Tan and Cheng (2003)
Penicillium chrysogenum Cd 56 Holan and Volesky (1995)
Penicillium chrysogenum Pb 96 Skowronski et al. (2001)
Penicillium chrysogenum Cd 21.5 Skowronski et al. (2001)
Penicillium chrysogenum Zn 13 Skowronski et al. (2001)
Penicillium chrysogenum Cu 11.7 Skowronski et al. (2001)
Penicillium chrysogenum Pb 116 Niu et al. (1993)
Penicillium chrysogenum Th 150 Veglio and Beolchini
(1997)
Penicillium chrysogenum Th 142 Kapoor and
Viraraghavan (1995)
Penicillium chrysogenum Pb 116 Kapoor and
Viraraghavan (1995)
Penicillium chrysogenum U70Kapoor and
Viraraghavan (1995)
Penicillium digitatum Ni, Zn, Cd, Pb Kapoor and
Viraraghavan (1995)
Penicillium digitatum Cd 3.5 Veglio and Beolchini
(1997)
Pb 5.5
Penicillium griseofulvum
(immobilized)
Cu 20.47 Shah et al. (1999)
Penicillium griseofulvum (free) Cu 1.51 Shah et al. (1999)
Penicillium italicum Cu, Th, Zn Ahluwalia and Goyal
(2007)
Penicillium italicum Cu 0.42 Ahluwalia and Goyal
(2007)
Penicillium italicum Zn 0.2 Ahluwalia and Goyal
(2007)
Penicillium notatum Cu 80 Kapoor and
Viraraghavan (1995)
Penicillium notatum Zn 23 Kapoor and
Viraraghavan (1995)
Penicillium notatum Cd 5.0 Kapoor and
Viraraghavan (1995)
Penicillium janthinellum U 52.7 Kapoor and
Viraraghavan (1995)
Penicillium purpurogenum Cr(VI) 36.5 Say et al. (2004)
Penicillium purpurogenum Cd 110.4 Say et al. (2003a)
(continued on next page)
Table 9 (continued)
Species Metal ions Biosorption
capacity
References
Penicillium purpurogenum Pb 252.8 Say et al. (2003a)
Penicillium purpurogenum Hg 70.4 Say et al. (2003a)
Penicillium purpurogenum As 35.6 Say