Bioremediation

BIOSORPTION MECHANISMS
The complex structure of microorganisms implies that there are many ways for the metal to be taken
up by the microbial cell. The biosorption mechanisms are various and are not fully understood. They
may be classified according to various criteria.
According to the dependence on the cell's metabolism, biosorption mechanisms can be divided into:
1. Metabolism dependent and
2. Non -metabolism dependent.
According to the location where the metal removed from solution is found, biosorption can be classified
as
1. Extra cellular accumulation/ precipitation
2. Cell surface sorption/ precipitation and
3. Intracellular accumulation.
Transport of the metal across the cell membrane yields intracellular accumulation, which is dependent
on the cell's metabolism. This means that this kind of biosorption may take place only with viable cells. It
is often associated with an active defense system of the microorganism, which reacts in the presence of
toxic metal.
During non-metabolism dependent biosorption, metal uptake is by physico-chemical interaction
between the metal and the functional groups present on the microbial cell surface. This is based on
physical adsorption, ion exchange and chemical sorption, which is not dependent on the cells'
metabolism. Cell walls of microbial biomass, mainly composed of polysaccharides, proteins and lipids
have abundant metal binding groups such as carboxyl, sulphate, phosphate and amino groups. This type
of biosorption, i.e., non-metabolism dependent is relatively rapid and can be reversible.
In the case of precipitation, the metal uptake may take place both in the solution and on the cell surface
. Further, it may be dependent on the cell's' metabolism if, in the presence of toxic metals, the
microorganism produces compounds that favour the precipitation process. Precipitation may not be
dependent on the cells' metabolism, if it occurs after a chemical interaction between the metal and cell
surface.
TRANSPORT ACROSS CELL MEMBRANE
Heavy metal transport across microbial cell membranes may be mediated by the same mechanism used
to convey metabolically important ions such as potassium, magnesium and sodium. The metal transport
systems may become confused by the presence of heavy metal ions of the same charge and ionic radius
associated with essential ions. This kind of mechanism is not associated with metabolic activity. Basically
biosorption by living organisms comprises of two steps. First, a metabolism independent binding where
the metals are bound to the cell walls and second, metabolism dependent intracellular uptake, whereby
metal ions are transported across the cell membrane.
PHYSICAL ADSORPTION
In this category, physical adsorption takes place with the help of van der Waals' forces. Kuyucak and
Volesky 1988, hypothesized that uranium, cadmium, zinc, copper and cobalt biosorption by dead
biomasses of algae, fungi and yeasts takes place through electrostatic interactions between the metal
ions in solutions and cell walls of microbial cells. Electrostatic interactions have been demonstrated to
be responsible for copper biosorption by bacterium Zoogloea ramigera and alga Chiarella vulgaris , for
chromium biosorption by fungi Ganoderma lucidum and Aspergillus niger .
ION EXCHANGE
Cell walls of microorganisms contain polysaccharides and bivalent metal ions exchange with the counter
ions of the polysaccharides. For example, the alginates of marine algae occur as salts of K+, Na+, Ca
2
+,
and Mg
2
+. These ions can exchange with counter ions such as CO
2
+, Cu
2
+, Cd
2
+ and Zn
2
+ resulting in the
biosorptive uptake of heavy metals. The biosorption of copper by fungi Ganoderma lucidium and
Aspergillus niger was also up taken by ion exchange mechanism.
COMPLEXATION
The metal removal from solution may also take place by complex formation on the cell surface after the
interaction between the metal and the active groups. Aksu et al. 1992 hypothesized that biosorption of
copper by C. vulgaris and Z. ramigera takes place through both adsorption and formation of
coordination bonds between metals and amino and carboxyl groups of cell wall polysaccharides.
Complexation was found to be the only mechanism responsible for calcium, magnesium, cadmium, zinc,
copper and mercury accumulation by Pseudomonas syringae. Microorganisms may also produce organic
acids (e.g., citric, oxalic, gluonic, fumaric, lactic and malic acids), which may chelate toxic metals
resulting in the formation of metallo-organic molecules. These organic acids help in the solubilisation of
metal compounds and their leaching from their surfaces. Metals may be biosorbed or complexed by
carboxyl groups found in microbial polysaccharides and other polymers.
PRECIPITATION
Precipitation may be either dependent on the cellular metabolism or independent of it. In the former
case, the metal removal from solution is often associated with active defense system of the
microorganisms. They react in the presence of a toxic metal producing compounds, which favour the
precipitation process. In the case of precipitation not dependent on the cellular metabolism, it may be a
consequence of the chemical interaction between the metal and the cell surface. The various
biosorption mechanisms mentioned above can take place simultaneously.
USE OF RECOMBINANT BACTERIA FOR METAL REMOVAL
Metal removal by adsorbents from water and wastewater is strongly influenced by physico-chemical
parameters such as ionic strength, pH and the concentration of competing organic and inorganic
compounds. Recombinant bacteria are being investigated for removing specific metals from
contaminated water. For example a genetically engineered E.coli, which expresses Hg
2+
transport system
and metallothionin (a metal binding protein) was able to selectively accumulate 8mole Hg
2+
/g cell dry
weight. The presence of chelating agents Na
+
, Mg
2+
and Ca
2+
did not affect bioaccumulation.
The following factors affect the biosorption process:
1. Temperature seems not to influence the biosorption performances in the range of 20-35
0
C.
2. pH seems to be the most important parameter in the biosorptive process: it affects the solution
chemistry of the metals, the activity of the functional groups in the biomass and the competition of
metallic ions.
3. Biomass concentration in solution seems to influence the specific uptake: for lower values of biomass
concentrations there is an increase in the specific uptake . Gadd et al. 1988 suggested that an increase in
biomass concentration leads to interference between the binding sites. Fourest and Roux, 1992
invalidated this hypothesis attributing the responsibility of the specific uptake decrease to metal
concentration shortage in solution. Hence this factor needs to be taken into consideration in any
application of microbial biomass as biosorbent.
4. Biosorption is mainly used to treat wastewater where more than one type of metal ions would be
present; the removal of one metal ion may be influenced by the presence of other metal ions. For
example: Uranium uptake by biomass of bacteria, fungi and yeasts was not affected by the presence of
manganese, cobalt, copper, cadmium, mercury and lead in solution . In contrast, the presence of Fe
2+

and Zn
2+
was found to influence uranium uptake by Rhizopus arrhizus and cobalt uptake by different
microorganisms seemed to be completely inhibited by the presence of uranium, lead, mercury and
copper .
yield the metals in a concentrated form;
restore the biosorbent to close to the original condition for effective reuse with undiminished metal
uptake and
no physical changes or damage to the biosorbent.
While the regeneration of the biosorbent may be accomplished by washing the metal- laden biosorbent
with an appropriate solution, the type and strength of this solution would depend on the extent of
binding of the deposited metal. Dilute solutions of mineral acids like hydrochloric acid, sulphuric acid,
acetic acid and nitric acid can be used for metal desorption from the biomass .

BIOSORPTION OF HEAVY METALS BY MICROORGANISMS
A large number of microorganisms belonging to various groups, viz. bacteria, fungi, yeasts,
cyanobacteria and algae have been reported to bind a variety of heavy metals to different extents. Most
of the biosorption studies reported in literatures have been carried out with living microorganisms.
However due to certain inherent disadvantages, use of living microorganisms for metal removal and
recovery is not generally feasible in all situations. For example, industrial effluents contain high
concentrations of toxic metals under widely varying pH conditions. These conditions are not always
conducive to the growth and maintenance of an active microbial population.
There are several advantages of biosorption of using non living biomass and they are as follows:
1. Growth independent nonliving biomass is not subject to toxicity limitation by cells.
2. The biomass from an existing fermentation industry, which essentially is a waste after
fermentation, can be a cheap source of biomass.
3. The process is not governed by physiological constraints of microbial cells.
4. Because nonliving biomass behaves as an ion exchanger, the process is very rapid, requiring
anywhere between few minutes to few hours. Metal loading is very high on the surface of the
biomass leading to very efficient metal uptake.
5. Because cells are non-living processing conditions are not restricted to those conducive for the
growth of the cells. Hence, a wider range of operating conditions such as pH, temperature and
metal concentrations are possible. Also aseptic operating conditions are not essential.
6. Metals can be desorbed readily and then recovered. If the value and the amount of metal
recovered are insignificant and if the biomass is plentiful, the metal loaded biomass can be
incinerated, eliminating further treatment.
Biosorption essentially involves adsorption processes such as ionic, chemical and physical adsorption. A
variety of ligands located on the fungal cell walls are known to be involved in metal chelation. These
include carboxyl, amine, hydroxyl, phosphate and sulphydryl groups. Metal ions could be adsorbed by
complexing with negatively charged reactions sites on the cell surface. This Table mentioned below
presents an exhaustive list of microrganisms used for the uptake of heavy metals.

BIOSORBENT UPTAKE OF METALS BY MICROBIAL BIOMASS
Metal Biomass Type Biomass class Metal uptake (mg/g)
Ag Freshwater alga Biosorbent 86-94
Fungal biomass Biosorbent 65
Rhizopus arrhizus Fungus 54
Streptomyces noursei Filamentous bacter 38.4
Sacchromyces cerevisiae Yeast 4.7
Au Sargassum natans Brown alga 400
Aspergillus niger Fungus 176
15
Palmaria tevera Marine alga 164
Palmaria palmata Marine alga 124
Chlorella pyrenoidosa Freshwater alga 98
Cyanidium caldarium Alga 84
Chlorella vulgaris Freshwater alga 80
Bacillus subtilis Bacteria Cell wall 79
Chondrus crispus Marine alga 76
Bacillus subtilis Bacterium 70
Spirulina platensis Freshwater alga 71
58
Rhodymenia palmata Marine alga 40
Ascophyllum nodosum Brown marine alga 24
Cd Ascophyllum nodosum Brown markertman
ine alga
215
Sargassum natans Brown marine alga 135
Fucus vesiculosus Brown marine alga 73
Candida tropicalis Yeast 60
Pencillium chrysogenum Fungus 56
11
Sacchromyces cervisiae Yeast 20-40
Rhizopus nigricans Fungus 19
Pencillium spinulosum Fungus 0.4
Pantoea sp. TEM 18 Bacteria 204.1
Chlamydomonas reinhardtii Alga 42.6
Spirulina sp. Blue green algae 1.77 meq/g
Enterobacter cloaceae
(Exopolysaccharide)
Marine bacterium 16
Padina sp. Brown seaweed 0.75
Sargassum sp. Brown seaweed 0.76
Ulva sp. Green seaweed 0.58
Gracillaria sp. Red seaweed 0.30
Gloeothece magna Cyanobacteria 115425 g mg1
Co Ascophyllum nodosum Brown marine algae 100
Sacchromyces cerevisiae Yeast 4.7
Ulva reticulata Marine green algae 46.1
Enterobacter cloaceae Marine bacterium 4.38
Cr Bacillus biomass Bacterium 118 Cr3+
60 Cr 6+
Candida tropicalis Yeast 4.6
Streptomyces nouresei Bacteria 1.8
Spirulina sp. Cyanobacteria 10.7 meq/g
Spirogyra sp. Filamentous algae 4.7
Cu Bacillus subtilis Biosorbent 152
Manganese oxidising bacteria MK-2 50
Cladosporium resinae Fungus 18
Saccharomyces crevisae Yeast 17-40; 10; 6.3
Pichia guilliermondii Yeast 11
Scenedesmus obliquus Freshwater algae 10
Pencillium chrysogenum Fungus 9
Streptomyces noursei sp. Filamentous bacteria 5
Bacillus sp Bacterium 5
Pencillium spinulosum Fungus 0.4-2
Aspergillus niger Fungus 1.7
Trichoderma viride Fungus 1.2
Pencillium chrysogenum Fungus 0.75
Ulva reticulata Marine green alga 56.3
Spirulina sp. Blue green algae 6.17 meq/g
Enterobacter cloaceae
(Exopolysaccharide)
Marine bacterium 6.60
Thiobacillus thiooxidans Bacteria 38.54
Ulothrix zonata Algae 176.20
Fe Bacillus subtillis Bacterial cell wall
preparation
201
Bacillus biomass Bacterium 107
Sargassum fluitans Brown alga 60
Hg Rhizopus arrhizus Fungus 54
Pencillium chrysogenum
(biomass not necessarily in its
natural state)
Fungus 20
Cystoseira baccata Marine alga 178
Chlamydomonas reinhardtii Algae 72.2
Ni Fucus vesiculosus Brown marine algae 40
Ascophylum nodosum Brown marine algae 30
Sargassum natans Brown marine algae 24-44
Bacillus licheniformis Bacterial cell wall
preparation
29
Bacillus subtillis Bacterial cell wall
preparation
6
Absidia orchidis Fungus 5
Ulva reticulata Marine green algae 46.5
Polyporous versicolor White rot fungus 57
Pb Bacillus subtilis (biomass not
necessarily in its natural state)
Biosorbent 601
Absidia orchidis Fungus 351
Fucus vesiculosus Brown marine algae 220-370
Ascophyllum nodosum Brown marine algae 270-360
Sargassum natans Brown marine algae 220-270
Bacillis subtilis (biomass not
Biosorbent 189
Pencillium chrysogenum Fungus 122; 93
Streptomyces longwoodensis Filamentous bacteria 100
Rhizopus arrhizus Fungus 91; 55
Streptomyces noursei Filamentous bacteria 55
Chlamydomonas reinhardtii Algae 96.3
Ecklonia radiata Marine alga 282
Pd Freshwater alga(biomass not
Biosorbent 436
Fungal biomass Biosorbent 65
Pt Freshwater alga (biomass not
Biosorbent 53
U Sargassum fluitans Brown algae 520
Streptomyces longwoodensis Filamentous bacteria 440
Sacchromyces crevisae Yeast 55-140
Bacillus sp. Bacterium 38
Chaetomium distortum Fungus 27
Trichoderma harzianum Fungus 26
Pencillium chrysogenum
(biomass not necessarily in its
natural state)
Fungus 25
Alternaria tenulis
Th Rhizopus arrhizus Fungus 160; 93
Sacchromyces cerevisae Yeast 70
Zn Bacillus subtilis (biomass not
Biosorbent 137
Sargassa sp. Brown algae 70
Manganese oxidising bacteria (MK-2) 39
Sacchromyces cerevisae Yeast 14-40
Pencillium chrysogenum Fungus 6.5
Bacillus sp. Bacterium 3.4
Pencillium spinulosum Fungus 0.2
Thiobacillus thiooxidans Bacteria 43.29

REPORTED ADSORPTION CAPACITIES (MG/G) FOR SEVERAL MISCELLANEOUS SORBENTS
Material Source Cd Cr Hg Pb Ni Zn Cu
Dry pine needles Masri et al.,
1974

175

Dry redwood
leaves
Masri et al.,
1974

175

Dyed bamboo
pulp (C.I. Reactive
orange 13)
Shukla and
Sakhardand
e, 1992

15.6 15

Undyed bamboo
pulp
Shukla and
Sakhardand
e, 1992

9.2 8.4

Dyed jute (C.I.
Reactive orange
13
Shukla and
Sakhardand
e, 1992

13.7 14.1

Undyed jute Shukla and
Sakhardand
e, 1992

7.6 7.9

Dyed sawdust (C.I.
Reactive orange
13)
Shukla and
Sakhardand
e, 1992

18.0 24.0

Undyed sawdust Shukla and
Sakhardand
e, 1992

8.5 7.3

Milogranite
(activated sewage
sludge)
Masri et al.,
1974

460 95.3

Modified wool Masri and
Friedman,
1974
87 17 632 135

Moss Low and
Lee, 1991
46.5

Orange peel
(white inner skin)
Masri et al.,
1974

125

Orange peel
(outer skin)
Masri et al.,
1974

275

PEI wool Freeland et
al., 1974

330.9
7

Senna leaves Masri et al.,

250

1974
Unmodified jute Shukla and
Pai, 2005

3.37 3.55 4.23
Modified jute Shukla and
Pai, 2005

5.57 8.02 7.73
Papaya wood Saeed et al.,
2005
17.35

14.44 19.99
Activated carbon
from apricot
stone
Kobya et al.,
2005
3.08 34.70

6.69 2.50

4.86
Lignocellulosic
fibres
unmodified
Shukla et al.,
2005

7.49 7.88

Lignocellulosic
fibres oxidised
with hydrogen
peroxide
Shukla et al.,
2005

2.51 1.83

Carbon aerogel Meena et
al., 2005
400.8

45.62 0.70 12.85 1.84 561.7
1
Dye loaded
groundnut shells
Shukla and
Pai, 2005

9.87 17.09 8.07
Unloaded
sawdust
Shukla and
Pai, 2005

8.05 10.96 4.94
Siderite Erdem and
zverdi,
2005

14.06

Diatomite Khraisheh,
2004
16.08

24.94

27.55
Manganese
treated diatomite
Khraisheh,
2004
27.08

99.00

55.56
Wheat shell Basci et al.,
2004

10.84
Wheat bran Farajzadeh
et al., 2004
21 93 70 62 12

15
Tea industry
waste
Cay et al.,
2004
11.29

8.64
Sawdust of P.
sylvestris
Taty-
Costodes, et
al., 2003
19.08

22.22

Cork biomass Chubar et
al., 2003

0.34
meq./
g
0.76
meq/
g
0.63
meq/
g
Cocoa shells Meunier et
al., 2003

6.2

Vermicompost Matos and
Arruda,
33.01

92.94

28.43 32.63
2003
Peanut hulls Johnson et
al., 2002

9
Peanut pellets Johnson et
al., 2002

12
poly(ethyleneglyc
ol dimethacrylate-
co-acrylamide)
beads
Kesenci et
al., 2002
0.370mmol/
g

0.270
mmol/
g
1.825
mmol/
g

Activated carbon
derived from
bagasse
Dinesh
Mohan and
Kunwar P.
Singh, 2002
49.07

14.0

Polyacrylamide-
grafted iron(III)
oxide
Manju et al.,
2002
151.47

163.21 218.53

Carboxylated
alginic acid
Jeon et al.,
2002

3.09
mmol/
g

Petiolar felt
sheath of palm
Iqbal et al.,
2002
10.8 5.32

11.4 6.89 5.99 8.09
Sheep manure
waste
Munther
Kandah,
2001

13.8

Peanut husk
carbon
Ricordel et
al., 2001
0.45

0.55 0.28 0.20

Kudzu (Pueraria
lobata ohwi)
Brown et al.,
2001
15

35 32
Turkish coal Arpa et al.,
2000
0.008
mmol/g

0.039
mmol/
g
0.041
mmol/
g

Peanut hulls Brown et al.,
2000
6

30

9 8
Peanut hull
pellets
Brown et al.,
2000
6

30

10 10
Commercial grade
ion exchange
Resin
Brown et al.,
2000
50

90 85
Carrot residue Nasernejad
et al., 2005

45.09

29.61 32.74
The results of many biosorption studies vary widely because of the different criteria used by the authors
in searching for suitable materials. Some researchers have used easily available biomass types, others
specially isolated strains, and some processed the raw biomass to different extents to improve its
biosorption properties. In the absence of uniform technology, results have been reported in different
units and in many different ways, making quantitative comparison impossible.
BIOSORPTION OF DYES BY MICRORGANISMS
A wide variety of microorganisms including bacteria, fungi and yeasts are used for the biosorption of a
broad range of dyes. Textile dyes vary greatly in their chemistries, and therefore their interactions with
microorganisms depend on the chemical structure of a particular dye, the specific chemistry of the
microbial biomass and characteristics of the dye solution or wastewater. Depending on the dye and the
species of microorganism used different binding capacities have been observed in the following table.
BIOSORBENT UPTAKE OF DYES BY MICROORGANISMS
Biosorbent Dye Biosorption capacity
qeq (mg-l)
Activated sludge Basic Red 29 113.2
Basic Yellow 24 105.6
Basic Blue 54 86.6
Basic Red 18 133.9
Basic Violet 3 113.6
Basic Blue 4 157.5
Basic Blue 3 36.5
Activated sludge Reactive Blue 2 102.0
Reactive Yellow 2 119.4
Activated sludge Maxilon Red BL-N (123.2)
Aeromonas sp. Reactive Blue 5 124.8
Reactive Red 22 116.5
Reactive Violet 2 114.5
Aspergillus niger Basic Blue 9 18.5 (1.2)
Acid Blue 29 13.8 (6.6)
Congo Red 14.7
Disperse Red I 5.6
Aspergillus niger Reactive Brilliant Red 14.2
Botrytis cinerea Reactive Blue 19 42 (13.0)
Sulphur Black I 360 (49.7)
Candida sp.. Remazol Blue 169
Candida lipolytica Remazol Blue 230
Candida membranaefaciens Remazol. Blue 149
Candida quilliermendii Remazol Blue 152
Candida tropicalis Remazol Blue 180
Candida utilis Remazol Blue 113
Candida rugosa Reactive Blue 19 8 (8)
Reactive Black 5 31 (31)
Sulphur Black I 407 (308)
Cryptococcuss heveanensis Reactive Blue 19 23 (22)
Reactive Black 5' 76 (60)
Dekkera bruxellensis Reactive Blue 19 19 (36)
Reactive Black 5 36 (38).
Endothiella aggregata Reactive Black 5 44
Sulphur Black I 307
Escherichia coli Reactive Blue 5 89.4
Fomitopsis carnea Orlamar Red BG 503.1
Orlamar Blue G 545.2
Orlamar Red GTL 643.9
Geotrichum fici Reactive Blue 19 17 (60)
Kluyveromyces marxianus Remazol Black B 37
Rem. Turquoise Blue 98
Remazol Red 68
Rem. Golden Yellow 33
Cibacron Orange 8.5
Kluyveromyces marxianus Remazol Blue 161
Kluyveromyces waltii Reactive Blue 19 14 (20)
Sulphur Black 1 549 (445)
Laminaria digitata Reactive Brilliant Red 20.5
Myrothecum verrucaria Orange II 70%
lOB (Blue) 86%
RS (Red) 95%
Phanerochaete
chrysosporium
Congo red 90%
Pichia carsonii Reactive Blue 19 5 (3)
Pseudomonas luteola Reactive Blue 5 102.5
Rhizopus arrhizus Humic acid 91.9
Rhizopus arrhizus Reactive Orange 16 190
Reactive Blue 19 90
Reactive Red 4 150

Rhizopus arrhizus Remazol Black B 500.7
Rhizopus oryzae (26668) Reactive Brilliant Red 102.6
Rhizopus oryzae (57412) Reactive Brilliant Red 37.2
Rhizopus oryzae Reactive Black 5 452 (99)
Saccharomyces cerevisiae Remazol Blue 162
Saccharomyces cerevisiae Reactive Blue 19 69 (52)
Saccharomyces pombe Remazol Blue 152
Streptomycetes BW130 Anthraquinone Blue 114 27.0%

Azo-copper Red
171
73.0%

Azo-reactive Red 147 29.0%
Formazan Blue 209 70.0%
Phytalocyanine Blue 116 39.0%
Tremella uciformis Reactive Blue 19 35 (41)
Xeromyces bisporus Reactive Blue 19 60 (0)

LOW COST ADSORBENTS FOR DYE/S REMOVAL
In recent times, attention has been focused on various natural solid supports, which are able to remove
pollutants from contaminated water at low cost. Cost is actually an important parameter for comparing
the adsorbent materials. Certain waste products from industrial and agricultural operations, natural
materials and biosorbents represent potentially economical alternative sorbents. Many of them have
been tested and proposed for dye removal.
WASTE MATERIALS FROM AGRICULTURE AND INDUSTRY
The by-products from the agriculture and industries could be assumed to be low-cost adsorbents since
they are abundant in nature, inexpensive, require little processing and are effective materials.
ACTIVATED CARBONS FROM SOLID WASTES
Commercially available activated carbons (AC) are usually derived from natural materials such as wood,
coconut shell, lignite or coal, but almost any carbonaceous material may be used as precursor for the
preparation of carbon adsorbents. Due to its availability and cheapness, coal is the most commonly used
precursor for AC production. Coal is a mixture of carbonaceous materials and mineral matter, resulting
from the degradation of plants. The sorption properties of each individual coal are determined by the
nature of the original vegetation and the extent of the physicalchemical changes occurring after
deposition. Coal adsorption capacities are reported in Table mentioned below.. However, since coal is
not a pure material, it has a variety of surface properties and thus different sorption properties.
Activated carbons from solid wastes
Raw material Dye Qmax
Pinewood Acid blue 264 1176
Pinewood Basic blue 69 1119
Corncob Acid blue 25 1060
Bagasse Basic red 22 942
Cane pith Basic red 22 941.7
Corncob Basic red 22 790
Bagasse Acid blue 25 674
Cane pith Acid blue 25 673.6
Pinewood Basic blue 9 556
Rice husk Basic green 4 511
Bagasse Acid blue 80 391
Waste newspaper Basic blue 9 390
Coal Basic blue 9 250
Waste carbon slurries Acid blue 113 219
Waste carbon slurries Acid yellow 36 211
Waste carbon slurries Ethyl orange 198
Sewage sludge Basic red 46 188
Mahogany sawdust Acid yellow 36 183.8
Coal Basic red 2 120
Sewage sludge Basic blue 9 114.94
Charcoal Acid red 114 101
Rice husk Acid yellow 36 86.9
Rice husk Acid blue 50
Charfines Acid red 88 33.3
Lignite coal Basic blue 9 32
Lignite coal Acid red 88 30.8
Bituminous coal Acid red 88 26.1
Rice husk Basic blue 9 19.83
Straw Basic blue 9 19.82
Date pits Basic blue 9 17.3
Hazelnut shell Basic blue 9 8.82
Coir pith Acid violet 8.06
Charfines Direct brown 1 6.4
Coir pith Direct red 28 6.72
Sugarcane bagasse Acid orange 10 5.78
Coir pith Basic violet 10 2.56
AGRICULTURAL SOLID WASTES
Raw agricultural solid wastes and waste materials from forest industries such as sawdust and bark have
been used as adsorbents. These materials are available in large quantities and may have potential as
sorbents due to their physico-chemical characteristics and low-cost. Sawdust is an abundant by-product
of the wood industry that is either used as cooking fuel or as packing material. Sawdust is easily
available in the countryside at zero or negligible price. It contains various organic compounds (lignin,
cellulose and hemicellulose) with polyphenolic groups that might be useful for binding dyes through
different mechanisms mentioned in the following table.
ADSORPTION CAPACITY OF AGRICULTURAL SOLID WASTES FOR THE REMOVAL OF DYES
Adsorbent Dye Qmax
Bark Basic red 2 1119
Bark Basic blue 9 914
Rice husk Basic red 2 838
Sugar-industry-mud Basic red 22 519
Tree fern Basic red 13 408
Pine sawdust Acid yellow 132 398.8
Palm-fruit bunch Basic yellow 327
Rice husk Basic blue 9 312
Pine sawdust Acid blue 256 280.3
Vine Basic red 22 210
Rice hull ash Direct red 28 171
Egyptian bagasse pith Basic blue 69 168
Vine Basic yellow 21 160
Egyptian bagasse pith Basic blue 69 152
Coir pith Basic blue 9 120.43
Coir pith Basic violet 10 94.73
Eucalyptus bark Remazol BB 90
Raw date pits Basic blue 9 80.3
Fly ash Basic blue 9 75.52
Egyptian bagasse pith Basic red 22 75
Treated sawdust Basic green 4 74.5
Wood sawdust Basic blue 69 74.4
Metal hydroxide sludge Reactive red 2 62.5
Treated sawdust Basic green 4 26.9
Fe(III)/Cr(III) hydroxide Basic blue 9 22.8
Banana peel Methyl orange 21
Banana peel Basic blue 9 20.8
Banana peel Basic violet 10 20.6
Orange peel Methyl orange 20.5
Egyptian bagasse pith Acid red 114 20
Orange peel Acid violet 19.88
Orange peel Basic blue 9 18.6
Egyptian bagasse pith Acid blue 25 17.5
Egyptian bagasse pith Acid blue 25 14.4
Orange peel Basic violet 10 14.3
Fly ash Alizarin sulfonic 11.21
Coir pith Acid violet 7.34
Wood sawdust Acid blue 25 5.99
Sugar cane dust Basic green 4 4.88
Banana pith Direct red 5.92
Red mud Direct red 28 4.05
Neem sawdust Basic violet 3 3.78
Neem sawdust Basic green 4 3.42

CHITIN AND CHITOSAN
The sorption of dyes using biopolymers such as chitin and chitosan is one of the reported emerging
biosorption methods for the removal of dyes, even at low concentration (ppm or ppb levels). Chitin and
chitosan are abundant, renewable and biodegradable resources. Chitin, a naturally occurring
mucopolysaccharide, has been found in a wide range of natural sources such as crustaceans, fungi,
insects, annelids and molluscs. However, chitin and chitosan are only commercially extracted from
crustaceans (crab, krill, and crayfish) primarily because a large amount of the crustaceans exoskeleton is
available as a by-product of food processing. The annual worldwide crustacean shells production has
been estimated to be 1.2 10
6
tonnes, and the recovery of chitin and protein from this waste is an
additional source of revenue. These studies demonstrated that chitosan-based biosorbents are efficient
materials and have an extremely high affinity for many classes of dyes in the following table. They are
also versatile materials. This versatility allows the sorbent to be used in different forms, from flake-types
to gels, bead-types or fibers.
CHITOSAN BIOSORENTS FOR REMOVAL OF DYES
Biosorbent Dye qmax
Crosslinked chitosan bead Reactive blue 2 2498
Crosslinked chitosan bead Reactive red 2 2422
Crosslinked chitosan bead Direct red 81 2383
Crosslinked chitosan bead Reactive red 189 1936
Crosslinked chitosan bead Reactive yellow 86 1911
Chitosan bead Reactive red 189 1189
Chitosan (bead, crab) Reactive red 222 1106
Chitosan (bead, lobster) Reactive red 222 1037
Chitosan Acid orange 12 973.3
Chitosan Acid orange 10 922.9
Chitosan Acid red 73 728.2
Chitosan Acid red 18 693.2
Chitosan Acid green 25 645.1
Chitosan (flake, lobster) Reactive red 222 398
Chitosan (flake, crab) Reactive red 222 293
The traditional and commercial source of chitin is from shells of crab, shrimp and krill that are wastes
from the processing of marine food products. However, this traditional method of extraction of chitin
creates its own environmental problems as it generates large quantities of waste and the production of
chitosan also involves a chemical deacetylation process. These problems can explain why it is difficult to
develop chitosan-based materials as adsorbents at an industrial scale.
DISADVANTAGES OF BIOSORPTION USING MICRORGANISMS
There are certain inherent disadvantages of using microorganisms for the biosorption of heavy
metals/dyes and they are as follows: the protein rich algal and fungal biomass projected as metal/dye
biosorbents have limitations as proteinious materials are likely to putrefy under moist conditions.
Further, most metal/dye sorption reported in literature is based on algal and fungal biomass, which
must be cultured, collected from their natural habitats and pre-processed, if available as discards and
transported under special conditions, thus introducing the factor of additional costs.
MICROBIAL METAL TRANSFORMATIONS
Microorganisms can transform metals and metalloids by oxidation, reduction, methylation and
dealkylation. Continuous cultures of Hg
+2
resistant bacteria which can reduce Hg
+2
to Hg
0
with mercuric
reductase and can volatilize it at a rate of 2.5mg/L/hour. Many bacteria, algae, fungi and yeasts can
reduce Au (III) to Au (0) and Ag (I) to Ag(0). Microbial transformations of Arsenic and Chromium are
also associated with a decrease in the toxicity and have relevance to water treatment.
Organomercurials are detoxified by organomercurial lyase which converts the dissolved mercury to Hg
+2

and then mercuric reductase converts it to Hg
0
.
A taxonomically diverse group of heterotrophic bacteria utilize metallic cations as terminal electron
acceptors under anaerobic conditions. In this process, the metal is reduced to a lower valency state.
There are several metallic elements which possess multiple valencies which can potentially be utilized in
this way by the micro organisms. Fe (III) and Mn (IV) appear to be the most commonly utilized metals as
terminal electron acceptors. In this process, Molybdenum (Vl) is reduced to molybdenum blue by a
strain of Enterobacter cloacea which was isolated from the molybdenum polluted waters. Another
strain of Enterobacter can reduce Cr(VI) to Cr(III) uder similar conditions. A strain of E.coli can perform
the similar function of chromium reduction under aerobic conditions as well but at a very slower rate.
A strain of Shewanella (Alteromonas) puterifaciens which reduces Fe(III) an Mn(IV) can also reduce U(VI)
to U(IV) but due to the low solubility of U(VI), the reaction was accomplished by formation of a black
precipitate of Uranium IV carbonate. The bacteria cannot use it further and it is separated from them by
dialysis tubing.
The sulphates reducing bacteria Desulfovibrio desulfuricans utilizing a mechanism that is involved in the
electron transport chain although the organism cannot utilize U(VI) for its growth.
Many bacteria belonging to genra Pseudomonas, Aeromonas, Enterobacter, Escherichia, Bacillus,
Streptomyces can reduce the Cr in two steps but complete and qualitative transformation of chromium
is shown by Escherichia coli and Agrobacterium radiobacter.
Lab experiments showed that the process of chromium reduction is increased by increasing the cell
density as experimented on specific strains of E.coli ATCC 33456, Pseudomonas fluroescens LB300,
Bacillus spp, A. radiobacter and Enterobacter cloacea HO1. A variety of organic compounds work as
electron donors during the process of Chromium reduction , these include natural aliphatic compounds,
low molecular weight carbohydrates, amino acids and carbohydrates. Biological reduction of chromium
may occur both aerobically and anaerobically. In aerobic conditions, chromium reduction is generally
associated with the soluble proteins with NADH as an electron donor either for requirement or
enhanced activity. Under anaerobic conditions, cytochrome c in E. cloaceae and cytochrome b and d in
E.coli , and cytochrome c3 in D. vulgaris serve as terminal electron acceptors. Strains of A. radiobacter,
E.coli, Aerococcus spp. Aeromonas spp. Aerococcus spp. And Micrococcus spp. Are capable of reducing
chromium in liquid medium in both aerobic and anaerobic conditions.
Later studies showed that a diversity of Pyrobaculum islandicum and Pyrobaculum aerophilum can work
at even 100
o
C and can reduce the chromium, iron, uranium, tellurium, cobalt and molybdenum by
utilizing yeast extract as terminal electron acceptor.
In addition to heavy metals, phenolic compounds are also present in chromium conataminated waters
and sites. Phenol and para-cerol at 4 mM and 2-chlormphenicol at 2mM concentrations severly inhibit
chromium reduction and cell growth of p. fluorescens LB300. Therefore anaerobic cultures of E.coli
ATCC33456 are used instead.
MICROBIAL METALLOID TRANSFORMATIONS
Two major transformation processes have been described for the metalloids.
1) Reduction of metalloid oxyanions to elemental forms eg. SeO
4
-2
and SeO
3
-2
.
2) Methylation of metalloids, metalloid oxyanions and organometalloids to methyl derivatives e.g.
AsO
4
-3
, AsO
2
and methyloarsonic acid trimethyarsine.
Transformation proessess have geological significance since they may modify the mobility and
toxicity of metalloids as well as being of biotechnological potential in bioremediation. Reduction of
SeO
4
-2
and SeO
3
-2
to elemental selenium (Se
o
) results in immobilization and detoxification.
Methylation of arsenic and selenium compounds results in volatilization.
Microbially mediated reduction of selenate and selenite to elemental selenium involve several
bacterial species like Wellinola succinogenes and Pseudomonas maltophila which were isolated from
the selenium rich soils and waters. They are so efficient in the removal of selenium and they isolate
it in the form of red precipitate which is further treated to yield elemental selenium.
4CH
3
COOH
-
+ 3 SeO
4
3SE
0
+ 8 CO
2
+ 4 H
2
0 +4H
+
Recently another very efficient species Thaurea selenatis was isolated which was capable of
reducing SeO
4
-2
and SeO
3
-2
to elemental selenium. This strain utilizes periplasmic nitrite reductase
enzyme and cytochrome C551 for the reduction.
Reduction of TeO32
-
to elemental tellurium is performed by a genetically modified bacteria named
P. maltophila. Besides that the photosynthetic bacterium Rhodobacter spheroids is also proved very
helpful and its efficiency can be increased by adding significant levels of FADH
2.
In contrast to bacterial systems, fungal reduction of metalloids has received less attention.
Reduction of SeO
4
-2
and SeO
3
-2
to Se
o
has been observed by Fusarium spp. Mortierella spp.
Saccharomyces cervisiae, Candidia albicans, Aspergillus funiculosus. Both intracellular and
extracellular depositions of Se
0
have been observed. Numerous fungal colonies show red colour
after the reduction however, Schizosaccromyces pombe showed black and grey colour of colonies.
The color is appeared due to deposition of the selenium in their bodies.
METHYLATION OF METALLOIDS
Microbial methylation of metalloids to yield volatile derivatives such as dimethylselenide or
trimehtylarsine is a well-known phenomenon. Bacterial species which are known to produce methyl
derivatives of selenium from SeO
4
-2
SeO
3
-2
include Aeromonas sp., Bacillus sp. and Pseudomonas sp.
Dimethylselenide is the most common methylated product. There has been much research into
selenium methylation by soil fungi. Alternaria alternata performed the methylation of the compound
more rapidly as compared to the organic forms. Both dimethylselenide and dimethyldiselenide have
been detected as volatile products of selenium methylation. Since both bacteria and fungi are thought
to play an important role in the methylation of selenium compounds but bacteria have been proved to
be more efficient in the selenium contaminated waters.
MECHANISM
The mechanism for selenium methylation involves the transfer of methyl groups as carbonium ions via
the S-adenosyl methionine system.
Much less work is done on the methylation of tellurium but it is thought to be converted into
dimethyltelluride and dimethylditelluride by pencillium sp.
Methylation of arsenic is performed by Methanobacterium sp. which by subsequent methylation,
convert the arsenic to methylarsonic acid, dimethylarsenic acid and finally dimethylarsine. In this case,
vitamin B
12
is thought to be methyl group donor.
SULPHIDE PRECIPITATION
Hydrogen sulfide is produced by sulphate reducing bacteria.e.g. Desulfovibrio and Desulfotomaculum.
The solubility products of most metal sulphides are extremely low and they are easily precipitated as
sulphides.e.g. ZnS, CdS, CuS, and FeS. Sulphate reducing activity can occur as a useful auxiliary metal
removing mechanism.
MECHANISM
For this purpose, a 9m
3
stain less steel sludge blanket reactor using sulphide reducing bactria was
plotted for the first time by shell research company. This plant successfully removed the toxic metals
and sulphates from the ground water at a long standing smelter site precipitation as metal sulphites.
Now a days, many large concrete reactors with capacity upto 7000m
3
have been prepared which use
spent mushroom compost as a substrate and can remove 95% of the metals and 20% of the sulphated
efficiently.
CELL WALL COMPONENTS
Microbial exopolymers which are considered the most important in the metal binding are those which
form slime layers and capsules. Most of these are composed of polysaccharide, glycoproteins and
lipopolysaccharides.
IN CASE OF BACTERIA
The cell walls of bacteria also have several metal binding components which contribute to the
biosorption of the processes. The carboxyl groups of the peptidoglycans are the main binding sites in
gram positive cell walls with the phosphate groups contributing significantly in the gram negative cell
walls.
IN CASE OF FUNGI
Many fungi have high contents of chitin in their cell walls and this polymer of N-acetylglucosamine is an
effective metal and radionuclide binder. Insoluble chitosan-glucans and glucan possessing sugar and acid
sugar groups from Aspergillus niger exhibit biosorptive properties and performes the efficient removal
of the transitional metals from the solutions. Also, some fungi possess melanin granules which help in
the absorption of thr metallic compounds from the solution.
METAL BINDING PROTEINS AND PEPTIDES
Virtually all biological material has a high affinity for the toxic metals and the radionuclides.
Metellothinines play a significant role in this aspect. These are low molecular weight cysteine rich
protein found in animals, higher plants, eukaryotic organisms and also in some prokaryotes. Basically
their function is to bind essential metals like Cu and Zn but they can also bind non essential metals such
as Cd. They form complexes with metals for their detoxification. They mediate copper ressistance in
Saccromyces cervisiae .
Metal -glutamyl peptides are short peptides involved in the heavy metal detoxification in algae, plants,
some fungi and yeasts.
POTENTIAL FOR THE FUTURE DEVELOPMENT
Several biotechnological approaches seem to have established as a means of combating toxic metal
pollution from industrial and other resources. Although none is in widespread use. Several design
preferences are beginning to encourage within the field with the processes currently in our near to
practical operation, mainly utilization biosorption, and bioprecipitation as these require the least
modification of the biological material and can be established in those processes which are well
established utilizing the ion exchange treatment or biological treatment of the organic wastes. Also,
attention is also being given to the possibilities of integrating the biological removal with sewage and
other waste treatment.
A technically simpler approach utilizing artificial wetlands with defined biota has been found useful for
the cost effective treatment of high volume, low concentration wastes. In longer term, the use of
purified biopolymers as specific metal binding agents hold out considerable potential.
The application of both genetic and protein engineering could possibly lead to peptides and other
biopolymers with enhanced metal specificity, stability and other useful properties.
CONCLUSION
Many of the microbial metal interaction have potential application for the bioremediation of the metal
contaminated environments and waste streams. However, potential bioremediation strategies for
metal have not been applied in large scale environmental restoration efforts. Collaboration between
microbiologists, environmental engineers and geochemists is required in order to bring this promising
technology to frutition.

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